CN1896238A - Genic engineering strain of expression recombinant prawn peptide Pen24 and its use - Google Patents

Abstract

The invention discloses a genetic engineering strain expressing recombinant prawn peptide Pen24 and its application, encoding the nucleotide and amino acid sequence of the Pen24, and expressing the genetic engineering strain E.coliOrigamiB(DE3) pLysS(pET-pen) of recombinant prawn peptide Pen24, CCTCC No: M206003, using engineering strains to express recombinant prawn peptide Pen24. Pen24 has good antibacterial activity against Gram-positive bacteria, negative bacteria; ampicillin-resistant Escherichia coli and strong pathogenic bacteria, such as Bacillus subtilis, Bacillus thuringiensis and Pseudomonas aeruginosa. Pen24 has obvious antiviral activity against viral infection. Use of Pen24 in the treatment or prevention of bacterial, fungal and viral diseases of animals and plants, as a preservative in cosmetics, food and animal feed.

Description

A Genetic Engineering Strain Expressing Recombinant Prawn Peptide Pen24 and Its Application

Technical field

The invention belongs to the technical field of genetic engineering, and more specifically relates to a genetically engineered strain expressing a recombinant prawn peptide Pen24, and more specifically relates to a genetically engineered strain E.coli OrigamiB(DE3)pLysS(pET-pen) containing a prawn peptide gene, which is related to The recombinant prawn peptide Pen24 has obvious antibiological activity against Gram-positive bacteria, Gram-negative bacteria, fungi, and viruses. It also relates to the preparation method of genetically engineered bacterial strains, and at the same time, also relates to the application of recombinant genetically engineered protein Pen24 in the prevention and treatment of bacteria, fungi and viral diseases of animals and plants, and its application as a preservative in the fields of cosmetics, food and feed, etc. use.

Background technique

Antimicrobial peptides are an important part of the host immune defense system and an ideal first line of defense for the body. Boman G et al. first induced the chrysalis chrysalis to produce antibacterial polypeptides, cecropins, by injecting Bacillus cloacae and Escherichia coli [Boman H G et al., 1987]. Since then, scientists have discovered and isolated about 300 endogenous antimicrobial peptides in various organisms such as mammals, tunicates, amphibians, insects, fish, birds and plants.

Antimicrobial peptides are a type of small molecular polypeptides produced by organisms to resist the invasion of exogenous pathogenic microorganisms, and can resist the infection of external pathogens. Antimicrobial peptides are a class of small molecule polypeptides produced by specific bacterial genes, which have broad-spectrum antibacterial properties, especially for drug-resistant strains, and do not destroy biological cells, and are non-immunogenic[Michael C et al ., 2003]. Andropin and drosocin in fruit flies, apidaecin and abaecin in bees, acanthoscurrin in tarantulas, etc. The skin of amphibians can secrete a large amount of antimicrobial peptides, and its content is very high. Such as the most widely studied magaininsm, dermaseptin from South American frogs, bombininh from tree frogs, and melittin-related peptides from Japanese frogs. A peptide related to the antimicrobial peptide found in marine organisms, dolabellanin of the sea hare. In mammals, antimicrobial peptides are expressed in phagocytes and mucosal epithelial cells, and a large number of antimicrobial active peptides represented by defensins and cathelicidins families have been found.

In addition to antibacterial activity, antimicrobial peptides also have a variety of other regulatory functions. Mammalian defensins mainly include α-defensins and β-defensins, which have a wide range of activities against Gram-negative and Gram-positive bacteria, fungi and some enveloped viruses. A new gene specifically expressed was successfully cloned from epithelial cells of rat epididymis head, and it was observed that the polypeptide encoded by this gene has antibacterial function and may be related to fertility [Zhang Yonglian et al., 2002].

Antimicrobial peptides are broad-spectrum antibacterial, and have inhibitory effects on viruses, parasites, and cancer cells. Mammalian antimicrobial peptides have multiple conservative cysteines, can form several stable disulfide bonds, and have a very broad antibacterial spectrum. In addition to anti-bacteria, fungi, and viruses, they are also active against mycoplasma, chlamydia, spirochetes, and some Cells (such as tumor cells) have a killing effect. Cecropin P1 is a mammalian antimicrobial peptide, which can inhibit and kill G-bacteria and some G+ bacteria. The highly conserved amino acid residues of antimicrobial peptides are indispensable for some antimicrobial peptide molecules to have antibacterial activity, and the C-terminus of some natural antimicrobial peptides is often amidated, which is related to the broad-spectrum antibacterial activity of antimicrobial peptides.

Generally, most antimicrobial peptides are composed of more than 30 amino acid residues. The C-terminal is rich in non-polar amino acids such as alanine, glycine, and valine, and the N-terminal is rich in cationic amino acids such as arginine and lysine. , the middle part is rich in proline [Daniel M L et al., 2003; Harder J et al., 2001]. There are some more conserved amino acid residues in many specific positions. Most antimicrobial peptides have either an α-helix or a β-sheet structure, and some contain both. Ceceopin P1 is a polypeptide composed of 31 amino acids with a molecular weight of about 3ku. It does not contain cysteine and cannot form intramolecular disulfide bonds. Its molecule contains an amphipathic α-helix, and the middle is glutamine that forms a flexible bend. The acid-glycine (Glu-Gly) coiled sequence has 64% similarity to insect cecropin IA and 75% similarity to cecropin B.

Antimicrobial peptides are considered to be one of the main components of the defense system of fish, shrimp, and shellfish. Studies by Chisholm et al. have proved that the blood cell lysate supernatant of crustaceans contains factors that can kill bacteria [Chisholm J R S et al., 1995]; 1997]; Schnapp et al. isolated three peptides with bactericidal effect from blood cells of blue crabs [Schnapp D et al., 1996]. So far, a variety of antimicrobial peptides have been found in Penaeus vannamei, Penaeus vannamei, Penaeus indica, Penaeus sinensis and Penaeus monodon.

The Penaeidin family is a cationic antimicrobial peptide, the COOH-terminal contains 6 cysteines, forming 3 disulfide bonds, and the NH2-terminal is rich in proline. The N-terminus of the precursor of Penaeidin has a highly conserved signal peptide, which is processed to obtain a mature peptide. The mature peptide of Penaeidin has the characteristics of post-translational modification, either amidation at the C-terminus, or cyclization at the N-terminus by pyroglutamic acid.

The natural production of antimicrobial peptides is limited, and domestic and foreign scholars obtain antimicrobial peptides through chemical synthesis. However, the chemical synthesis method has complex steps, low yield and high cost. At present, scholars at home and abroad, on the basis of studying the primary structure of antimicrobial peptides, use molecular biology and genetic engineering techniques to express antimicrobial peptides in prokaryotic cells, eukaryotic cells or some algae, and obtain antimicrobial peptide transgenic animals and plants. Escherichia coli is a Gram-negative bacterium with a clear genetic background. There are a large number of strains that can be used to clone and express exogenous genes, and it is easy to be cultured on a large scale. However, due to the toxicity or bactericidal effect of antimicrobial peptides on E. coli, E. coli is not suitable for expressing antimicrobial peptides, but there are a few successful examples. Qiu Dinghong selected the cecropin antibacterial peptide B (CB) cDNA with the strongest activity in nature to be fused with human interferon A1 (h IFN A1) cDNA to construct the fusion expression plasmid pBV-20-hIFN A1-CB of the prokaryotic expression vector pBV-20 ( pHC), transfected into Escherichia coli E.coli JMB strain, and expressed the purified fusion protein hIFN A1-CB after temperature-controlled induction [Qiu Dinghong et al., 2002]. It is of great significance to produce antimicrobial peptides through genetic engineering technology and realize the mass production of antimicrobial peptides. It is hoped that antimicrobial peptides will become new drugs that inhibit and kill major pathogens, especially drug-resistant bacteria.

Contents of Invention

The purpose of the present invention is to provide a genetic engineering strain expressing the recombinant prawn peptide Pen24. Recombinant prawn peptide Pen24 prawn peptide has broad-spectrum antibacterial effect, and has obvious antibacterial activity against Gram-positive bacteria and Gram-negative bacteria; strong pathogenic bacteria that are not sensitive to ampicillin, such as Bacillus subtilis, thuringiensis Bacillus and Pseudomonas aeruginosa have good antibacterial activity. At the same time, the recombinant prawn peptide Pen24 has antibacterial activity against ampicillin-resistant E. coli. Recombinant prawn peptide Pen24 is effective against insect nuclear polyhedrosis virus (including silkworm nuclear polyhedrosis virus, Autographa californica nuclear polyhedrosis virus, cotton bollworm nuclear polyhedrosis virus, beet armyworm nuclear polyhedrosis virus, etc.) , Shrimp White Spot Syndrome Virus (WSSV) infection have obvious antiviral activity. The genetically engineered recombinant prawn peptide Pen24 can be widely used in the prevention and treatment of bacterial, fungal and viral diseases in animals and plants, as well as in cosmetics, food and feed as a preservative.

Due to the problems of drug resistance and drug residues brought about by the application of traditional antibiotics, the research and development of antibiotic peptides has become a frontier topic in the research of new antibiotic products in the world. Compared with traditional antibiotics, antimicrobial peptides have the advantages of small molecular weight, broad antibacterial spectrum, good thermal stability, and unique antibacterial mechanism. Medicinal properties. The use of antimicrobial peptides to replace traditional antibiotics, as a new generation of antibacterial drugs, is of great significance and broad application prospects for improving the quality of animal products and promoting the development of green biotechnology.

The purpose of the present invention is to provide a recombinant genetically engineered Escherichia coli strain containing the Penaeidin-2 gene of Penaeus vannamei, characterized in that: the bacterial strain is a genetically engineered strain E.coli OrigamiB (DE3 )pLysS(pET-pen), the strain was preserved in the China Center for Type Culture Collection on January 15, 2006, with the preservation number CCTCC No: M206003, containing a recombinant expression plasmid inserted with the Penaeidin-2 expression cassette of Penaeus vannamei , or comprise a DNA fragment encoding the genetically engineered recombinant prawn peptide Pen24 of the present invention. The genetic engineering recombinant prawn peptide Pen24 can be expressed by using the genetic engineering strain E. coliOrigamiB(DE3)pLysS(pET-pen).

The purpose of the present invention is to provide a method for constructing an Escherichia coli host cell, which is simple and easy to operate. The host cell contains the nucleotide sequence of the recombinant prawn peptide Pen24. The expression level is high, and the expression level of the genetically engineered protein can reach up to 20% of the total bacterial protein. Fusion expression has high safety and is conducive to industrial production.

One of the purposes of the present invention is to provide a genetic engineering strain expressing recombinant prawn peptide Pen24 in the prevention or treatment of bacteria, fungi and viral diseases of animals and plants, and can also be used as a preservative in cosmetics, food and animal feed in the application.

The invention provides a genetic engineering recombinant prawn peptide Pen24, the molecular weight of which is about 24kDa. The genetically engineered recombinant prawn peptide Pen24 has biological activity against bacterial, fungal and viral infections.

One of the objectives of the present invention is to provide an amino acid sequence encoding the genetically engineered recombinant prawn peptide Pen24, which has at least 70% homology with the amino acid sequence in Sequence Table 2.

One of the objectives of the present invention is to provide an amino acid sequence encoding the genetically engineered recombinant prawn peptide Pen24, which has at least 80% homology with the amino acid sequence in Sequence Table 2.

One of the objectives of the present invention is to provide an amino acid sequence encoding the genetically engineered recombinant prawn peptide Pen24, which has at least 90% homology with the amino acid sequence in Sequence Table 2.

One of the objectives of the present invention is to provide a nucleotide sequence corresponding to the gene engineering recombinant prawn peptide Pen24, which has at least 50% homology with the nucleotide sequence in Sequence List 1.

One of the objectives of the present invention is to provide a nucleotide sequence corresponding to the gene engineering recombinant prawn peptide Pen24, which has at least 80% homology with the nucleotide sequence in Sequence List 1.

One of the objectives of the present invention is to provide a host cell comprising a nucleotide sequence encoding the genetically engineered recombinant prawn peptide Pen24 of the present invention, or comprising a nucleotide sequence encoding the genetically engineered recombinant prawn peptide Pen24 of the present invention DNA fragments.

One of the objectives of the present invention is to provide a method for constructing an Escherichia coli host cell, which contains the nucleotide fragment of the genetically engineered recombinant prawn peptide Pen24 of the present invention.

The invention also relates to the application of the recombinant prawn peptide Pen24 in antibacterial infection.

The invention also relates to the application of the recombinant prawn peptide Pen24 in antifungal infection.

The invention also relates to the application of the recombinant prawn peptide Pen24 in antiviral infection.

In order to achieve the above object, the present invention adopts the following technical measures:

The invention provides a method for preparing a DNA fragment, which comprises the nucleotide sequence encoding the genetically engineered recombinant prawn peptide Pen24.

In one embodiment of the present invention, Pen24, a genetically engineered recombinant prawn peptide provided by the present invention, has a sequence of at least 70% homology with the amino acids shown in Sequence Table 2, and its molecular weight is about 24 kDa. Recombinant prawn peptide Pen24 has biological activity against bacterial, fungal and viral infections.

In one embodiment of the present invention, there is provided a genetically engineered recombinant prawn peptide Pen24, an isolated protein having a sequence at least 80% homologous to the amino acids shown in Sequence Table 2.

In one embodiment of the present invention, there is provided a genetically engineered recombinant prawn peptide Pen24, an isolated protein having a sequence at least 90% homologous to the amino acids shown in Sequence Table 2.

In one embodiment of the present invention, the amino acid sequence of the genetically engineered recombinant prawn peptide Pen24 of the present invention is listed in Sequence List 2.

The amino acid sequence of the genetically engineered recombinant prawn peptide Pen24 of the present invention can be modified and changed within a certain range, and the obtained modified protein or protein fragment has the same biological function as the genetically engineered recombinant prawn peptide Pen24. Recombinant prawn peptide Pen24 has a broad-spectrum antibacterial effect, and has obvious antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria; strong pathogenic bacteria that are not sensitive to ampicillin, such as Bacillus subtilis and Bacillus thuringiensis and Pseudomonas aeruginosa have good antibacterial activity. At the same time, the recombinant prawn peptide Pen24 has antibacterial activity against ampicillin-resistant E. coli. Recombinant prawn peptide Pen24 is effective against insect nuclear polyhedrosis virus (including silkworm nuclear polyhedrosis virus, Autographa californica nuclear polyhedrosis virus, cotton bollworm nuclear polyhedrosis virus, beet armyworm nuclear polyhedrosis virus, etc.) , Shrimp White Spot Syndrome Virus (WSSV) infection have obvious antiviral activity.

The method for modifying and changing proteins is a conventional method [Joe Sambrook, DavidRussell et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab (CSHL) Press, 2001], which is familiar to those skilled in the art. According to the theory of modern life science, proteins with more than 70% amino acid sequence homology are often interpreted as proteins with the same biological function in biology. Modifications and mutations to the protein amino acid sequence within this range are considered not to change the biological properties of the protein, and these changes and modifications include:

(1) Mutation of individual amino acids, especially amino acids at non-functional decision sites.

(2) Deletion or insertion of individual amino acids, especially amino acids at non-functional decision sites. If such a change does not change the spatial conformation of the protein, it will not affect the biological function of the protein, nor will it change the immunogenicity of the protein.

(3) Insertion of special amino acid residues. In order to increase or change the solubility of the protein and increase its stability, some amino acid residues are often added to the N and C terminals of the protein during the genetic engineering production process to avoid the formation of inclusion bodies and reduce the degradation of host cell endoproteinases; Or add some special amino acid functional sites at the N and C terminals to facilitate protein expression and purification.

The above methods for modifying and changing proteins are conventional methods (conventional methods are the same as above), and are familiar to those skilled in the art. The proteins or fragments with 70% homology with the genetic engineering recombinant prawn peptide Pen24 of the present invention obtained by the above method all have the same biological function as the genetic engineering recombinant prawn peptide Pen24 of the present invention, that is, they have anti- The biological activity of bacterial, fungal and viral infections can be used to achieve one or more of the objects of the present invention.

According to the amino acid composition of the protein, combined with the analysis results of SDS-PAGE, the molecular weight of the genetically engineered recombinant prawn peptide Pen24 involved in the present invention is about 24kDa. If the amino acid sequence of the protein is partially or locally changed, the molecular weight of the protein will change. Different host strains and expression vectors are used, and the molecular weight of the protein will also change due to post-translational modifications. Depending on the detection method used, there will be some differences in the molecular weight of the protein, but these changes and differences will not exceed 10% of the molecular weight of the protein in principle.

The present invention uses Penaeus vannamei as a material, extracts total RNA, designs Penaeidin-2 specific primers, and obtains its mature peptide coding region sequence through RT-PCR and PCR amplification. The nucleotide sequence is:

tacaggggcggttacacaggcccgatacccaggccaccaccattggaagaccaccgttcagacctgtttgcaatgcatgctacagactttccgtctcagatgctcgcaattgctgcatcaagttcggaagctgttgtcacttagtaaaaggataa

In one embodiment of the present invention, a nucleotide sequence encoding a genetically engineered recombinant prawn peptide Pen24 is provided as Sequence List 1.

In one embodiment of the present invention, a genetically engineered recombinant prawn peptide Pen24 is provided. The protein is a genetically engineered fusion protein, and its N amino acid is encoded by the vector, and the N-terminal amino acid is:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEF

The present invention provides a nucleotide sequence encoding the genetically engineered recombinant prawn peptide Pen24, which has at least 50% homology with the nucleotides shown in Sequence Table 1.

In one embodiment of the present invention, a nucleotide sequence encoding the genetically engineered recombinant prawn peptide Pen24 is provided, and its nucleotide sequence has at least 80% homology with the nucleotide sequence in Sequence Table 1.

Any change in the amino acid sequence of a genetically engineered protein may change the encoded amino acid, thereby changing the structure and function of the encoded protein, but there is degeneracy in the amino acid codon of organisms. The nucleotide sequence of the genetically engineered recombinant prawn peptide Pen24 of the present invention can be mutated, but the encoded protein has the same biological function. These mutations include missense mutations, synonymous mutations and frameshift mutations. These methods are conventional methods, and can be realized by methods such as point mutation. These methods are familiar to those skilled in the art and can be obtained without creative effort. The nucleotide sequences obtained by this method with 50% homology to the nucleotide sequences of the present invention have the same biological function as the nucleotide sequences of the present invention, and can be used to realize the nucleotide sequences of the present invention one or more purposes. According to the concept of modern biology, especially the theory of bioinformatics, nucleotide sequences with a homology of more than 70% can be judged to have significant similarity, and nucleotide sequences with a homology of more than 80% can be judged have the same biological function.

The nucleotide sequence encoding the genetic engineering recombinant prawn peptide Pen24 described in the present invention can be changed within a certain range, and the obtained nucleotide sequence has the same biological identity as the nucleotide sequence encoding the genetic engineering recombinant prawn peptide Pen24. Function.

In one embodiment of the present invention, the pET-32a (+) plasmid used is the product of Novagen Company, also has His. Enzyme cleavage site, the expressed recombinant prawn peptide Pen24 is a fusion protein with a molecular mass of about 24kDa.

In one embodiment of the present invention, a Penaeidin-2 DNA fragment and its preparation method are provided. The fragment contains the nucleotide sequence encoding the genetically engineered recombinant prawn peptide Pen24 of the present invention.

The DNA fragments involved in the invention can be obtained in the following ways:

(1) Artificial synthesis, the DNA fragments described in the present invention can be synthesized directly with a DNA synthesizer, or the DNA fragments described in the present invention can be synthesized in segments, and these synthetic products have the same biological characteristics as the DNA fragments described in the present invention. function, one or more purposes of the present invention can be achieved.

(2) PCR amplification, with the DNA fragments of the present invention as templates, or with plasmids, vectors, and host cells containing the DNA fragments of the present invention as templates, DNA fragments are obtained by PCR amplification, and these PCR products have With the same biological function as the DNA fragment described in the present invention, one or more purposes described in the present invention can be achieved.

The above methods are commonly used methods in molecular biology, are familiar to those skilled in the art, and can be obtained without creative labor. The obtained DNA fragments are considered to have the same biological identity as the nucleotide sequences involved in the present invention. It can further realize one or more objectives of the present invention by means of genetic engineering.

In one embodiment of the present invention, a host cell—Escherichia coli E.coli is provided, the host cell contains the nucleotide sequence encoding the genetically engineered recombinant prawn peptide Pen24 of the present invention, or contains the nucleotide sequence encoding the recombinant prawn peptide of the present invention. The DNA fragment of the genetically engineered recombinant prawn peptide Pen24.

In one embodiment of the present invention, a kind of Escherichia coli recombinant genetic engineering strain is provided, and this bacterial strain is classified and named as E.coli OrigamiB (DE3) pLysS (pET-pen), depository unit: China Type Culture Collection Center, preservation Address: China. Wuhan. Wuhan University, date of deposit: January 10, 2006, deposit number: CCTCC No: M206003, the host cell contains the nucleotide sequence of the genetically engineered recombinant prawn peptide Pen24 of the present invention.

In one embodiment of the present invention, a method for constructing Escherichia coli recombinant genetic engineering strain E. coliOrigamiB(DE3)pLysS(pET-pen) is provided.

(1) Extraction of total RNA from Penaeus vannamei and synthesis of the first strand of cDNA

The hemolymph of healthy Penaeus vannamei was collected to obtain blood cells, and the total RNA was extracted according to the instructions of Qiagen's RNeasy Mini Kit, and the first strand of cDNA was synthesized by reverse transcription according to the operation manual of Promega's Universal Riboclone cDNA Synthesis System Kit.

(2) PCR amplification of Penaeidin-2 gene

Using the first strand of cDNA synthesized by reverse transcription as a template, upstream primer P1: 5' GAATTC TACAGGGGCGGTTACACA 3' (underlined is EcoR I site), downstream primer P2: 3'GTGAATCATTTTCCTATT TTCGAA 5' (underlined is Hind III site). The 168bp target gene was amplified by PCR instrument.

(3) Construction and identification of recombinant plasmid pGEM-T/Pen

Recover the PCR product, connect the PCR product to the pGEM-T carrier, transform E.coli JM109 (purchased from Promega, USA) competent cells, spread on the LB plate containing X-gal, IPTG and Amp resistance for blue White spot screening, identification by PCR and restriction endonuclease digestion, and DNA sequencing analysis to identify the recombinant plasmid pGEM-T/Pen.

(4) Construction and identification of recombinant expression plasmid pET-pen

The expression vector pET-32a(+) and the recombinant pGEM-T/Pen plasmid DNA were double-digested with EcoR I and Hind III respectively, and the Penaeidin-2 gene was inserted into the expression vector pET-32a(+) to obtain the recombinant plasmid pET- pen. Transform E.coli Origami B(DE3)pLysS competent cells. The recombinant plasmid pET-pen was identified by PCR, restriction endonuclease identification and DNA sequencing analysis. The strain was named Escherichia coli recombinant genetically engineered strain E.coli OrigamiB(DE3)pLysS(pET-pen).

In one embodiment of the present invention, a method for expressing the genetically engineered recombinant prawn peptide Pen24 using the Escherichia coli recombinant genetically engineered strain E.coli OrigamiB(DE3)pLysS(pET-pen) is provided.

(1) Pick a single colony and inoculate it in LB liquid medium containing 200 μg/mL ampicillin (Amp), 34 μg/mL chloramphenicol and 15 μg/mL kanamycin, and culture it with shaking at 250 r/min at 37 °C for 8 -12h. Place overnight at 4°C.

(2) The next day, insert 4% inoculum into fresh 2YT liquid medium, continue shaking culture at 37°C until the OD600 value of the bacterial solution is 0.6-0.8, add inducer IPTG to a final concentration of 0.4mmoI/L, The expression was induced at 37°C for 3-5h, and the cells were collected by centrifugation at 4°C.

(3) 15% SDS-PAGE electrophoresis to detect the expression of the target protein.

In one embodiment of the present invention, a method for purifying the genetically engineered recombinant prawn peptide Pen24 is provided.

(1) ultrasonic crushing and centrifugation to collect the supernatant;

(2) Purify the genetically engineered recombinant prawn peptide Pen24 by using His Bind resin column according to the operation manual;

(3) The genetically engineered recombinant prawn peptide Pen24 purified by dialysis treatment with PBS buffer (pH7.3);

(4) 15% SDS-PAGE electrophoresis, and laser scanning to detect the purity of the recombinant prawn peptide Pen24.

(5) Bradford assay was used to determine the concentration of the purified recombinant prawn peptide Pen24.

Compared with the prior art, the present invention has the following advantages and effects:

In one embodiment of the present invention, the antibacterial biological activity of the genetically engineered recombinant prawn peptide Pen24 was tested.

(1) The disc diffusion method was used to detect the activity units and antibacterial potency of the recombinant prawn peptide Pen24 against Escherichia coli.

(2) The disc diffusion method was used to detect the activity of the recombinant prawn peptide Pen24 against Gram-positive bacteria, Gram-negative bacteria and ampicillin-resistant E. coli strain E.coli BL21 (pET-32a).

(3) The liquid growth inhibition method was used to determine the MIC of the recombinant prawn peptide Pen24.

The different strains cultured overnight were serially diluted, and 10-3 dilutions were taken for MIC determination, and finally the minimum inhibitory concentration was judged by calculating colony forming units (CFU).

Recombinant prawn peptide Pen24 prawn peptide has broad-spectrum antibacterial effect, and has obvious antibacterial activity against Gram-positive bacteria and Gram-negative bacteria; strong pathogenic bacteria that are not sensitive to ampicillin, such as Bacillus subtilis, thuringiensis Bacillus and Pseudomonas aeruginosa have good antibacterial activity. At the same time, the recombinant prawn peptide Pen24 has antibacterial activity against ampicillin-resistant E. coli.

In one embodiment of the present invention, the biological activity of the genetically engineered recombinant prawn peptide Pen24 against Bombyx mori nuclear polyhedrosis virus (BmNPV) was tested.

The effect of the recombinant prawn peptide Pen24 on the infection of silkworm larvae by Bombyx mori nuclear polyhedrosis virus (BmNPV) was detected by licking method. The recombinant prawn peptide Pen24 has obvious anti-infection activity of Bombyx mori nuclear polyhedrosis virus (BmNPV).

In one embodiment of the present invention, the biological activity of the genetically engineered recombinant prawn peptide Pen24 against Autographa californica nuclear polyhedrosis virus (AcNPV) was tested.

The effect of the recombinant prawn peptide Pen24 on the infection of the larvae of Autographa californica nuclear polyhedrosis virus (AcNPV) was detected by licking method. The recombinant prawn peptide Pen24 has obvious activity against the infection of Autographa californica nuclear polyhedrosis virus (AcNPV).

In one embodiment of the present invention, the biological activity of the genetically engineered recombinant prawn peptide Pen24 against cotton bollworm nuclear polyhedrosis virus (HaNPV) was tested.

The effect of the recombinant prawn peptide Pen24 on the infection of cotton bollworm larvae by nuclear polyhedrosis virus (HaNPV) was detected by licking method. Recombinant prawn peptide Pen24 has obvious anti-infection activity of cotton bollworm nuclear polyhedrosis virus (HaNPV).

In one embodiment of the present invention, the biological activity of the genetically engineered recombinant prawn peptide Pen24 against beet armyworm nuclear polyhedrosis virus (SeNPV) was tested.

The effect of the recombinant prawn peptide Pen24 on the infection of beet armyworm larvae by beet armyworm nucleopolyhedrosis virus (SeNPV) was detected by licking method. Recombinant prawn peptide Pen24 has obvious anti-infection activity of beet armyworm nuclear polyhedrosis virus (SeNPV).

In one embodiment of the present invention, the biological activity of the genetically engineered recombinant prawn peptide Pen24 against prawn white spot syndrome virus (WSSV) was tested.

The effect of the recombinant prawn peptide Pen24 on the infection of Procambarus clarkii by shrimp white spot syndrome virus (WSSV) was detected by injection method. The recombinant prawn peptide Pen24 has obvious activity against the infection of prawn white spot syndrome virus (WSSV).

Description of drawings

The above and other objects, features and advantages of the present invention can be clearly obtained from the following detailed description of preferred embodiments of the present invention and the contents shown in the accompanying drawings, where the same reference symbols represent the same parts in different views. The drawings are not necessarily drawn to scale, the emphasis being placed on illustrating the practice and effect of the invention.

Figure 1 RT-PCR identification diagram of gene penaedin-2.

Lane 1.DL2000 Markers; Lane 2.PCR product

Figure 2 PCR and enzyme digestion identification diagram of recombinant plasmid pGEM-T/Pen-2

Lane 1 DL2000; Lane 2 PCR product; Lane 3 pGEM-T/Pen/EcoRI+HindIII; Lane 4pGEM-T/Pen/EcoRI; Lane 5 pGEM-T/Pen plasma; lane6 marker III

Figure 3 PCR and enzyme digestion identification diagram of the recombinant expression plasmid pET-pen

Lane 1 DL2000 Markers; Lane 2.PCR product; Lane 3.pET-pen/EcoR I+Hind III; Lane 4.pET-pen/EcoRI; Lane 5.pET-32a(+)/EcoRI; Lane 6.Marker III .

Figure 4 Construction process diagram of recombinant expression plasmid pET-pen

Figure 5 15% SDS-PAGE detection of the expression of Pen24

Lane 1. Purified Pen24 fusion protein; Lane 2. Molecular weight standard; Lane 3.E.coli OrigamiB(DE3)pLysS(pET-pen) was not induced; Lane 4.E.coli OrigamiB(DE3)pLysS(pET-pen) ) before induction; Lane 5.E.coli Origami B(DE3)pLysS(pET-pen) induced for 4h; Lane 6.E.coliOrigami B(DE3)pLysS(pET-pen) induced for 3h; Lane 7.E.coli Origami B(DE3)pLysS(pET-32) induced for 4h; Lane 8.E.coli Origami B(DE3)pLysS(pET-32) before induction; Lane 9.E.coli Origami B(DE3)pLysS(pET-32) Not induced

Figure 6 Determination of antibacterial potency of recombinant prawn peptide Pen24

Figure 7 Determination of the activity of recombinant prawn peptide Pen24 in inhibiting BmNPV infection of silkworm

Note: TN+BmNPV: BmNPV positive control group; E.coli OrigamiB (DE3) pLysS (pET) broken supernatant + BmNPV: E.coli OrigamiB (DE3) pLysS (pET) broken supernatant + BmNPV group; A group , Group B: E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant + BmNPV group; TN: TN Buffer control group

Figure 8 Determination of the activity of recombinant prawn peptide Pen24 in inhibiting AcNPV infection of Autographa

Note: AcNPV: AcNPV positive control group; AcNPV+E.coli OrigamiB(DE3)pLysS(pET): E.coli OrigamiB(DE3)pLysS(pET) broken supernatant+AcNPV group; AcNPV+Pen-24: E. coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+AcNPV group; PBS: PBS Buffer

control group

Figure 9 Determination of the activity of recombinant prawn peptide Pen24 inhibiting HaNPV infection of Autographa

Note: HaNPV: HaNPV positive control group; HaNPV+Pen-24: E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+HaNPV group; HaNPV+E.coli OrigamiB(DE3)pLysS(pET): E.coli OrigamiB (DE3) pLysS (pET) broken supernatant + HaNPV group; PBS: PBS Buffer

control group

Figure 10 Determination of the activity of recombinant prawn peptide Pen24 inhibiting SeMNPV infection of Autographa

Note: SeMNPV+E.coli OrigamiB(DE3)pLysS(pET): E.coli OrigamiB(DE3)pLysS(pET) broken supernatant+SeMNPV group; SeMNPV+Pen-24: E.coli OrigamiB(DE3)pLysS( pET-pen) broken supernatant + SeMNPV group; SeMNPV: SeMNPV positive control group; PBS: PBS Buffer control group

Figure 11 Determination of the activity of recombinant prawn peptide Pen-24 inhibiting WSSV infection of Procambarus clarkii

Note: WSSV+E.coli OrigamiB(DE3)pLysS(pET): E.coli OrigamiB(DE3)pLysS(pET) broken supernatant+WSSV group; WSSV+PBS: WSSV positive control group; WSSV+Pen-24: Recombinant prawn peptide Pen24+WSSV group; PBS: PBS Buffer control group

Detailed ways

Example 1 Extraction of total RNA from Penaeus vannamei

The vannamei were reared in an oxygenated water tank at 22°C for use. Select healthy shrimp during the moulting period, rinse with DEPC-treated sterilized water, collect 750 μL of hemolymph from the abdominal sinus of the shrimp with a 2.5 mL disposable syringe, add an equal volume of anticoagulant (PH7.0), and examine under the microscope Count, take the hemolymph with a cell content of 1×10 ⁷ and centrifuge at 800 g for 15 min at 4°C, remove the supernatant, and collect blood cells. Total RNA was extracted according to the product manual of Qiagen's RNeasyMini Kit. Store the extracted RNA solution at -80°C for future use.

Agarose gel electrophoresis of the extracted total RNA of Penaeus vannamei showed that two bands of 28S and 18S were clearly visible, and the color intensity of the two bands was approximately 2:1, indicating that the extracted total RNA was basically not degraded , with better integrity.

Example 2 Synthesis of the first strand of reverse-transcribed cDNA from Penaeus vannamei

Using Oligo(dT)15 as a primer, the first strand of cDNA was synthesized according to the instructions of Promega's Reverse Transcription Reaction Kit. The specific operation steps are as follows: Add the following reagents into the PCR reaction tube soaked in DEPC and sterilized: 25 mM MgCl ₂ , 4 μL; 10× reverse transcription buffer, 2 μL; 10 mM dNTP mixture, 2 μL; recombinant RNasin ^® RNase inhibitor, 0.5 μL; 24 U/μL MV reverse transcriptase, 0.8 μL; 0.5 μg/μL Oligo(dT)15 primer, 1 μL; total RNA, 3 μL; nuclease-free water, 2.7 μL. The reaction conditions are: 42°C, 1 hour; 95°C, 5 minutes; 3°C, 5 minutes.

Store the synthesized first-strand cDNA at -80°C for future use.

Example 3 Amplification of the Penaeidin-2 gene of Penaeus vannamei

1. Synthesis of amplification primers

Primers were designed, upstream primer P1: 5'GAATTC TACAGGGGCGGTTACACA 3' (EcoR I site is underlined), downstream primer P2: 3'GTGAATCATTTTCCTATT TTCGAA 5' (Hind III site is underlined). Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. and purified by PAGE.

2. PCR amplification of the target gene

According to the operation manual of the Reverse Transcription Reaction kit, the target gene Penaeidin-2 was amplified by PCR using the first strand of cDNA synthesized by reverse transcription as a template. PCR reaction system: 10×reactionbuffer, 5μL; 1.5mM MgCl2, 3μL; 0.2mM dNTP, 1μL; 20pmol upstream primer P1, 1μL; 20pmol downstream primer P2, 1μL; 5U/μL Taq DNA polymerase 1μL; template 6μL; Bacterial water to a final volume of 50 μL. PCR reaction conditions: 95°C, 5min; 94°C, 30s; 53°C, 45s; 72°C, 30s (35 cycles); 72°C, 5min.

The results of agarose gel electrophoresis are shown in Figure 1. The product of PCR amplified target gene Penaeidin-2 was subjected to 1.2% agarose gel electrophoresis, and there was a DNA band of about 168bp, which was consistent with the predicted result of target gene Penaeidin-2.

Example 4 Cloning of the Penaeidin-2 gene of Penaeus vannamei

1. Recovery of target gene penaedin-2 fragment

The DNA gel recovery kit (product of Omega Company) was used to recover the target gene fragment according to the instructions of the DNA gel recovery kit of Omega Company. The specific operation steps are as follows:

(1) The PCR product is subjected to 1.2% agarose gel electrophoresis (1×TAE), and the electrophoresis is observed with a UV lamp. When the DNA band to be recovered is completely separated from other bands, stop the electrophoresis and cut it off with a blade under the UV lamp. To recover the band, purify it with a PCR product purification kit.

(2) Crush the glue in an Eppendorf tube, add an equal volume of sol solution Binding Buffer, bathe in water at 65°C for 7 minutes, and shake the Eppendorf tube every 2 minutes until the glue is completely melted.

(3) Add the melted sample to the chromatographic column, centrifuge at 12000 rpm for 1 min, and discard the liquid.

(4) Add 300μL Binding Buffer, centrifuge and discard the liquid.

(5) Add 750μL Washing Buffer, centrifuge and discard the liquid.

(6) Repeat step (5).

(7) Centrifuge the empty column at 12000rpm for 1min to dry the liquid.

(8) Put the column in a 1.5mL Eppendorf tube, add 30μL Elution buffer, incubate at 37°C for 2min, centrifuge at 12000rpm for 1min, collect the centrifugate, and store at -20°C.

2. Cloning the fragment of the target gene penaedin-2 into the pGEM-T vector to construct the recombinant plasmid pGEM-T/Pen

The PCR product purified above was cloned into the pGEM-T vector, and the pGEM-T vector was purchased from Promega Company. The reaction system is: 2×Ligation buffer, 5.0 μL; pGEM-T vector, 0.5 μL; PCR product, 4.0 μL; T4DNA ligase, 0.5 μL; add sterile ddH2O to 10 μL. The above liquids were mixed on ice and connected at 16°C for 15h.

3. Transformation of Plasmids

(1) Preparation of competent cells: Escherichia coli competent cells were prepared by the cold calcium chloride method. Pick a single colony of E.coli JM109, inoculate it in 5mL LB medium, and cultivate overnight at 37°C and 220rpm. The next day, take the above bacterial solution and inoculate it in 50mL LB medium at a ratio of 1:100, shake at 37°C and 220rpm, When the OD value of the bacterial solution is 0.6, take 1.5 mL of the bacterial solution and add it to a sterile Eppendorf centrifuge tube, centrifuge at 4,000 rpm for 10 min, and discard the supernatant. Add 800 μL of ice-cooled 0.1M calcium chloride, shake gently to resuspend the bacterial pellet, and ice-bath for 30 minutes. Centrifuge at 4,000rpm for 10min and discard the supernatant. Add 100 μL of ice-cold 0.1M calcium chloride to resuspend the pellet, store at 4°C, and use within 7-10 days.

(2) Transformation of E.coli JM109 competent cells with plasmids: Take 10 μL of the above ligation product and add it to 100 μL competent cells, mix gently, ice bath for 60 minutes, heat shock at 42°C for 90 seconds, then place in ice bath for 2 minutes, add 390 μL of fresh LB liquid medium, shake gently at 150 rpm for 50 min at 37°C. Take 100 μL of the bacterial solution and spread it on the LB plate containing 5-bromo-4 chloro-3 indole-D-galactoside (X-gal), isopropylthiogalactoside (IPTG) and Amp resistance, Cultivate overnight at 37°C, observe the results, and perform blue-white screening. Pick the white colony and quickly extract the plasmid for PCR and enzyme digestion identification.

4. Identification of recombinant plasmid pGEM-T/Pen

Randomly pick 10 monoclonal leukoplakia, expand the culture, and extract the plasmid with a plasmid extraction kit (product of Qiagen). Using the plasmid pGEM-T/Pen as a template, primers P1 and P2 were used to amplify fragments consistent with the predicted results, indicating that the target gene had been cloned into the pGEM-T vector. The results of PCR and enzyme digestion identification are shown in Figure 2. Recombinant plasmid pGEM-T/Pen was digested with EcoRI+HindIII to obtain expected DNA fragments of about 3000bp and 168bp, and pGEM-T/Pen was digested with EcoRI to obtain a single expected DNA fragment of about 3168bp.

5. Determination of the nucleotide sequence of Penaeidin-2

After the plasmid pGEM-T/Pen DNA was identified by PCR and enzyme digestion, positive clones were randomly selected and sent to Beijing Huada Gene Company for DNA sequencing analysis. The pGEM-T/Pen plasmid was analyzed by automatic sequencing, and the nucleotide sequence of the Penaeidin-2 gene is:

tacaggggcggttacacaggcccgatacccaggccaccaccattggaagaccaccgttcagacctgtttgcaatgc

atgctacagactttccgtctcagatgctcgcaattgctgcatcaagttcggaagctgttgtcacttagtaaaaggataa

Example 5 Genetically engineered strains

Construction and Identification of E.coli OrigamiB(DE3)pLysS(pET-pen)

1. Construction of recombinant expression plasmid pET-pen

Use EcoR I and Hind III to double-enzyme-cut the expression vector pET-32a(+) and recombinant pGEM-T/Pen plasmid DNA respectively, recover and purify the target DNA fragments with gel recovery kits, and then combine the Penaeidin-2 DNA fragments with pET -32a(+) DNA fragment ligation, after ligation at 16°C for 15 hours, the product was transformed into E.coli OrigamiB(DE3)pLysS competent cells, spread on LB plate containing 12.5μg/ml Tetracycline, 15μg/ml Kanamycin, 34μg/ml Chloramphenicol Incubate overnight at 37°C.

2. Identification of recombinant expression plasmid pET-pen

Randomly pick 10 positive clones, after expanding and culturing, use plasmid extraction kit to extract plasmid, PCR and EcoR I, Hind III enzyme digestion to identify pET-pen recombinant expression plasmid. Using the plasmid pET-pen as a template, primers P1 and P2 were used to amplify fragments consistent with the predicted results, indicating that the target gene had been cloned into the pET-32a(+) vector. The results of PCR and enzyme digestion identification are shown in Figure 3. The recombinant plasmid pET-pen DNA was digested with EcoRI+HindIII to obtain two DNA fragments of about 5875bp and 168bp, and the pET-pen DNA was digested with EcoRI to obtain a single DNA fragment of about 6043bp.

The construction process of the recombinant expression plasmid pET-pen is shown in Figure 4.

3. Nucleotide sequence determination

After the plasmid pET-pen DNA was identified by PCR and enzyme digestion, positive clones were randomly selected and sent to Beijing Huada Gene Company for DNA sequencing analysis. The pET-pen plasmid was analyzed by automatic sequencing, and the results showed that the six His sequences were successfully fused to the start codon tac of the mature peptide of the Penaeidin-2 gene, and the sequence reading frame did not shift.

The strain constructed above is E.coli OrigamiB(DE3)pLysS(pET-pen).

Example 6 Expression of genetically engineered recombinant prawn peptide Pen24

Pick the strain E.coli OrigamiB(DE3)pLysS(pET-pen) preserved with glycerol and inoculate it in LB liquid medium containing 100μg/mL Ampicillin, 34μg/mL chloramphenicol and 15μg/mL kanamycin(sulfate), 37 Cultivate with shaking at 250r/min for 8-12h. The next day, insert 4% of the inoculum into fresh 2YT liquid medium containing 100 μg/mL Ampicillin, 34 μg/mL chloramphenicol and 15 μg/mL kanamycin (sulfate), cultivate at 37°C until the OD600 value of the bacterial solution is 0.6-0.8, add Inducer IPTG (final concentration: 0.4mM), induced expression at 37°C, sampled at 2h, 3h, and 4h after induction, centrifuged to precipitate, removed supernatant, added 300μL PBS (PH6.9) buffer, sonicated, and centrifuged at 12,000g After 10 minutes, add 5×SDS-PAGE sample buffer to the supernatant, perform SDS-PAGE according to "Molecular Cloning", and use Coomassie Brilliant Blue R250 staining to detect and analyze the expression results. The results are shown in Figure 5. The results of 15% SDS-PAGE electrophoresis showed that the genetically engineered strain could express the recombinant prawn peptide Pen24 after IPTG induction, and the molecular weight of the expression product was 24kDa, which was in line with the expectation.

Example 7 Purification of genetically engineered recombinant prawn peptide Pen24

(1) Pick the strain E.coli OrigamiB(DE3)pLysS(pET-pen) preserved with glycerol and inoculate it in LB liquid medium containing 100μg/mL Ampicillin, 34μg/mL chloramphenicol and 15μg/mL kanamycin(sulfate) Medium, 37°C, shaking culture at 250r/min for 8-12h. The next day, insert 4% of the inoculum into fresh 2YT liquid medium containing 100 μg/mL Ampicillin, 34 μg/mL chloramphenicol and 15 μg/mL kanamycin (sulfate), cultivate at 37°C until the OD600 value of the bacterial solution is 0.6-0.8, add The inducer IPTG (final concentration: 0.4mM) was induced to express at 37°C, and after 4 hours of induced expression, the bacterial cells were collected by centrifugation at 5000g.

(2) Resuspend in Binding Buffer pH7.920mmol/L Tris-HCl, 5mmol/L NaCl, 4°C, and perform ultrasonic treatment (30min, 10s each time, 30s interval). Centrifuge twice for 20 min at 12,000 g to collect the supernatant, which is the extract of the genetically engineered recombinant prawn peptide Pen24.

(3) Ni ⁺⁺ column chromatography sample treatment: the extract of the genetically engineered recombinant prawn peptide Pen24 was filtered through a 0.45 μm filter membrane, and the volume was adjusted to 100 ml Ni ⁺⁺ column chromatography sample with 1×Binding Buffer. Wash the column bed with 10 beds of 1×Binding Buffer and 20 beds of sterile water; use 10 beds of 1×Charge Buffer (5mM NiSO4) to combine Ni ⁺⁺ ; use 10 beds of 1×Binding Buffer to equilibrate the column bed, and set aside (1 The bed is the volume of medium in the column). The sample passes through the Ni ⁺⁺ column in a circular manner through the peristaltic pump, so that the mobile phase of the protein sample with 6×His is fully combined with the Ni ⁺⁺ in the Ni ⁺⁺ column, and after overnight, it is collected from the outlet of the Ni ⁺⁺ column. ⁺⁺ Bound sample (bleed through). Use 50 beds of 1×Binding Buffer; 75 beds of 60mM imidazole; 75 beds of 100mM imidazole; 30 beds of 150mM imidazole to wash the small amount of sample left in the column and the non-target protein bound to the Ni ⁺⁺ column. Use 1×Elute Buffer (1M imidazole+0.5M NaCl+20mM Tris·HCl pH7.9) to elute the target protein bound to the Ni ⁺⁺ column, collect in fractions, 1mL/tube. The Ni ⁺⁺ column was continuously eluted with 1×Elute Buffer to wash all the proteins in the sample column; then the column was washed with 1×Strip Buffer (100mM EDTA+0.5M NaCl+20mM Tris·HCl pH7.9). The distribution of the target protein in the eluate was checked by SDS-PAGE, SDS-PAGE was carried out according to "Molecular Cloning", and the expression results were detected and analyzed by Coomassie brilliant blue R250 staining. The results are shown in Figure 5. The purified sample was detected by 15% SDS-PAGE, and there was a single band with a size of 24.1kDa.

Example 8 In the purified sample of genetically engineered recombinant prawn peptide Pen24

Determination of protein content

Put 15mL purified sample of genetically engineered recombinant prawn peptide Pen24 collected by elution from Ni ⁺⁺ column into a treated dialysis bag, put it into a beaker filled with 1000mL PBS buffer (PH6.9) and dialyze overnight. All the above operations were carried out at 4°C. The Bradford method was used to determine the content of the recombinant prawn peptide Pen24, which was demarcated in mg/mL.

The specific operation of the Bradford method is as follows:

(1) Add 0, 1, 2, 3, 4, 5, 6 μL of 1 mg/ml bovine serum albumin (BSA) standard solution to the enzyme-labeled microwell plate in sequence, and make up 50 μL with PBS.

(2) Add 200 μL Bradford reagent working solution (0.1% Coomassie Brilliant Blue G250, 5% ethanol, 8.5% phosphoric acid) to each well. After shaking and mixing, stand at room temperature for 2 minutes.

(3) Measure the OD570 value (λ=570nm) of each concentration of BSA protein with a microplate reader. Take the BSA protein concentration as the abscissa, and use the OD570 value of each BSA protein concentration as the ordinate to make a standard curve.

(4) Determine the OD570 value of the sample with the same method, and determine the concentration of the sample from the standard curve.

Example 9 Genetic engineering recombinant prawn peptide Pen24

Antibacterial biological activity

(1) Determination of antibacterial potency and activity unit of recombinant prawn peptide Pen24

The activity unit and antibacterial potency of recombinant prawn peptide Pen24 against Escherichia coli were detected by disc diffusion method. The diameter of the filter paper is 0.6 cm. For the experimental strain Escherichia coli, the Escherichia coli cultured to the logarithmic growth phase (CFU=1.5×106 cells/mL) was evenly spread on the LB agar plate at a ratio of 1:200, and after drying, it was pasted with no Bacteria paper, add 20 μL of the recombinant antibacterial peptide Pen24 solution to be tested on the paper, use 20 μL of E.coli Origami B(DE3)pLysS(pET-32a) as a negative control, and use a drug-sensitive paper containing ampicillin as a positive For the control, after incubation at 37°C for 12 hours, measure the diameter of the inhibition zone.

The results are shown in Figure 6. When the prawn peptide Pen24 was 2.80 μg, the diameter of the bacteriostatic zone was 17.44 mm, and the size of the bacteriostatic zone produced by 2.80 μg Pen24 and 10 μg (including 15 IU) of ampicillin sensitive paper was equal, indicating that the bactericidal activity of the recombinant prawn peptide Pen24 was : 1μg antimicrobial peptide is equivalent to 5IU ampicillin bactericidal activity. In the test, the content of recombinant prawn peptide Pen24 in each drug-sensitive paper sheet increased, and the diameter of the inhibition zone gradually increased.

(2) The disc diffusion method was used to detect the activity of the recombinant prawn peptide Pen24 against Gram-positive bacteria, Gram-negative bacteria and ampicillin-resistant E. coli strain E.coli BL21 (pET-32a). The experimental strains include: ampicillin-resistant Escherichia coli BL21 (pET-32a), Escherichia coli, Staphylococcus aureus, Bacillus megaterium, Bacillus thuringiensis, Pseudomonas aeruginosa (provided by China Center for Type Culture Collection). Different bacteria (CFU=1.5×106/mL) cultured to the logarithmic growth phase were evenly spread on the LB agar plate at a ratio of 1:200. 20 μL of Pen24 solution was added to the paper, 20 μL of E.coli Origami B(DE3)pLysS(pET-32a) was used as a negative control, and the drug-sensitive paper containing ampicillin was used as a positive control. After incubation at 37°C for 12 hours, the pH value was measured. Bacterial circle diameter.

The activity results of the recombinant prawn peptide Pen24 against Gram-positive bacteria, Gram-negative bacteria and ampicillin-resistant E. coli strain E. coli BL21 (pET-32a) are shown in Table 1. The size of the inhibition zone also shows that the recombinant prawn peptide Pen24 has different degrees of antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, and has inhibitory effects on a variety of bacteria including ampicillin-resistant bacteria, and With the increase of antimicrobial peptide content, the inhibition zone gradually increased, and the effect was continuously strengthened.

Table 1 Activity determination of recombinant prawn peptide Pen24 Test bacteria Antibacterial zone diameter (mm) Antibacterial effect Pen24 (1μg) Amp (10μg) Pen24 (1μg) Amp (10μg) Staphylococcus aureus 9.10 0 + - Bacillus subtilis 2.99 0 + - Bacillus thuringiensis 3.52 0 + - Escherichia coli 6.23 17.44 + + Pseudomonas aeruginosa 3.40 0 + - Ampicillin-resistant Escherichia coli E.coli BL21(pET-32a) 4.16 0 + -

(3) Determination of minimum inhibitory concentration (MIC) value of recombinant prawn peptide Penaeidin-2

The determination of the minimum inhibitory concentration (MIC) value refers to the liquid growth inhibition method. The different strains cultivated overnight are diluted 10 times, and the ^10-3 dilution is taken for MIC determination, and 5 μL are taken respectively (CFU=1.5×10 mL) into 1.5mL EP tubes, add 0μL, 10μL, 20μL, 30μL, 35μL, 36μL, 38μL, 40μL and other purified recombinant prawn peptides that have been dialyzed and quantified to each tube, and culture at 37°C for 24 hours , spread evenly on the agar plate, and calculate the colony forming units (CFU) after culturing overnight at 37°C. The results are shown in Table 2. It shows that the recombinant prawn peptide Pen-24 has a good antibacterial effect on a variety of strong pathogenic bacteria, and the antibacterial effect of Pen-24 on Gram-positive bacteria is more significant than that of Gram-negative bacteria.

Table 2 Determination of minimum inhibitory concentration (MIC) of recombinant prawn peptide Pen24 Test bacteria Minimum Inhibitory Concentration MIC (μg/mL) Staphylococcus aureus Bacillus megaterium Bacillus thuringiensis Escherichia coli Pseudomonas aeruginosa 3.68-4.114.12-5.184.47-5.584.90-5.105.23-5.74

Example 10 Genetic engineering recombinant prawn peptide Pen24

Biological activity against silkworm nucleopolyhedrosis virus infection

1. Sample Preparation

(1) Preparation of Bombyx mori nuclear polyhedrosis virus (BmNPV) samples:

The crude extract of Bombyx mori nuclear polyhedrosis virus (BmNPV) was purified by sucrose gradient, counted under a microscope, and the BmNPV content was 1.6×10 ⁹ PIB/mL.

(2) E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant sample preparation:

The strain E.coli OrigamiB(DE3)pLysS(pET-pen) was fermented and the cells were collected. 100 grams of wet bacteria were resuspended in 500 mL NT Buffer, ultrasonically disrupted, centrifuged, and the supernatant was taken.

(3) E.coli OrigamiB(DE3)pLysS(pET-32a(+)) broken supernatant sample preparation:

The strain E.coli OrigamiB(DE3)pLysS(pET-32a(+)) was fermented and the cells were collected. 100 grams of wet bacteria were resuspended in 500 mL NT Buffer, ultrasonically disrupted, centrifuged, and the supernatant was taken.

2. Determination of biological activity of Pen24 against Bombyx mori nuclear polyhedrosis virus (BmNPV) infection

Silkworms were purchased from Cong Sericulture Research Institute in Wuhan and raised at room temperature. The fifth instar silkworm larvae were divided into 5 groups, and 8×10 ⁸ PIB/mL BmNPV was selected as the virus infection dose. The following 4 kinds of samples were used to feed the infection respectively:

①E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant + BmNPV group:

E.coli OrigamiB(DE3)pLysS(pET-pen) crushed supernatant sample was mixed with BmNPV suspension, and after 1h at 26°C, it was applied to 4 pieces of medium-sized fresh and clean mulberry leaves, 300 μL each, and waited for After drying, feed silkworms.

②E.coli OrigamiB(DE3)pLysS(pET-32a(+)) broken supernatant+BmNPV group:

E.coli OrigamiB(DE3)pLysS(pET-32a(+)) crushed supernatant sample was mixed with BmNPV suspension, treated at 26°C for 1 hour, and then applied to 4 medium-sized fresh and clean mulberry leaves, 300 μL each , and feed silkworms after drying.

③BmNPV positive control group:

BmNPV suspension was applied to four medium-sized fresh and clean mulberry leaves, 300 μL each, and fed to silkworms after drying.

④TN Buffer control group:

TN Buffer was applied to four medium-sized fresh and clean mulberry leaves, 300 μL each, and fed to silkworms after drying.

Bombyx mori larvae were reared at 20-25°C. After being infected with BmNPV virus for 3 days, they were fed with fresh and clean mulberry leaves twice a day, and the death of silkworm larvae was recorded twice a day.

The results are shown in Figure 7. The results showed that on the 6th day, the mortality rate of E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant)+BmNPV group was 10%, 20% respectively; the mortality rate of BmNPV positive control group was 40%; E. The mortality rate of coli OrigamiB(DE3)pLysS(pET-32a(+)) supernatant + BmNPV control group was 40%. On the 7th day, the mortality rate of E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant)+BmNPV group was 60% and 70% respectively; the mortality rate of BmNPV positive control group was 100%; E.coliOrigamiB(DE3) The mortality rate of pLysS(pET-32a(+)) broken supernatant+BmNPV control group was 100%. The antibacterial peptide Pen24 can effectively inhibit the infection of fifth instar silkworm larvae by BmNPV, and has biological activity against BmNPV virus infection.

Example 11 Genetic engineering recombinant prawn peptide Pen24

Biological Activity Against Infection by Autographa californica Nuclear Polyhedrosis Virus (AcNPV)

The licking method was used to detect the effect of genetically engineered recombinant shrimp peptide Pen24 against the larvae of Autographa californica nuclear polyhedrosis virus (AcNPV) infection.

1. Sample Preparation

(1) Preparation of Autographa californica nuclear polyhedrosis virus (AcNPV) samples:

The crude extract of Autographa californica nuclear polyhedrosis virus (AcNPV) was purified by sucrose gradient and counted under a microscope. The content of AcNPV was 1.1×10 ⁹ PIB/mL.

(2) E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant sample preparation:

The strain E.coli OrigamiB(DE3)pLysS(pET-pen) was fermented and the cells were collected. 100 grams of wet bacteria were resuspended in 500 mL PBS Buffer, ultrasonically disrupted, centrifuged, and the supernatant was taken.

(3) E.coli OrigamiB(DE3)pLysS(pET-32a(+)) broken supernatant sample preparation:

The strain E.coli OrigamiB(DE3)pLysS(pET-32a(+)) was fermented and the cells were collected. 100 grams of wet bacteria were resuspended in 500 mL PBS Buffer, ultrasonically disrupted, centrifuged, and the supernatant was taken.

2. Determination of biological activity of Pen24 against Autographa californica nuclear polyhedrosis virus (AcNPV) infection

1.1×10 ⁴ PIB/mL AcNPV was selected as the virus infection dose. The silver prints were raised at 26°C and fed with artificial diet. The third-instar silver-leafed larvae were divided into 4 groups, and the following 4 samples were added to feed the infection:

①E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+AcNPV group:

E.coli OrigamiB(DE3)pLysS(pET-pen) crushed supernatant sample was mixed with AcNPV suspension, and after acting for 1 hour at 26°C, 300μL was applied to the surface of 1cm ³ artificial feed to feed the larvae of silver print.

②E.coli OrigamiB(DE3)pLysS(pET-32a(+)) broken supernatant+AcNPV group:

E.coli OrigamiB(DE3)pLysS(pET-32a(+)) crushed supernatant sample was mixed with AcNPV suspension, and after acting for 1 hour at 26°C, apply 300μL on the surface of 1cm ³ artificial feed to feed the larvae of silver print .

③AcNPV positive control group:

Apply 300 μL of the AcNPV suspension on the surface of 1 cm ³ artificial feed to feed the larvae of the silver-leaved armyworm.

④PBS Buffer control group:

Apply 300 μL of the PBS Buffer suspension on the surface of 1 cm ³ artificial feed to feed the larvae of the silver-leaved armyworm.

The larvae of Autographa larvae were reared at 20-25°C. After being infected with AcNPV virus for three days, they were fed with fresh artificial feed every day, and the death of the larvae of Autographa larvae was recorded twice a day.

The results are shown in Figure 8. The results showed that on the 5th day, the death rate of E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+AcNPV group was 58%; the death rate of AcNPV positive control group was 84%; E.coliOrigamiB(DE3)pLysS (pET-32a(+)) The mortality rate of the broken supernatant+AcNPV control group was 92%. On the 7th day, the mortality rate of E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+AcNPV group was 75%; the mortality rate of AcNPV positive control group was 100%; E.coli OrigamiB(DE3)pLysS(pET-pen) 32a(+)) The mortality rate of the broken supernatant+AcNPV control group was 100%. Antimicrobial peptide Pen24 can effectively inhibit AcNPV infection of third-instar silver-leafed larvae, and has biological activity against AcNPV virus infection.

Example 12 Genetic engineering recombinant prawn peptide Pen24

Biological Activity Against Cotton Bollworm Nucleopolyhedrosis Virus (HaNPV) Infection

The licking method was used to detect the effect of genetic engineering recombinant prawn peptide Pen24 on the resistance of cotton bollworm larvae infected by nuclear polyhedrosis virus (HaNPV).

1. Sample Preparation

(1) Preparation of cotton bollworm nuclear polyhedrosis virus (HaNPV) samples:

Cotton bollworm nuclear polyhedrosis virus (HaNPV) crude extract was purified by sucrose gradient, counted under a microscope, and the HaNPV content was 1.5×10 ⁹ PIB/mL.

(2) E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant sample preparation:

The strain E.coli OrigamiB(DE3)pLysS(pET-pen) was fermented and the cells were collected. 100 grams of wet bacteria were resuspended in 500 mL PBS Buffer, ultrasonically disrupted, centrifuged, and the supernatant was taken.

(3) E.coli OrigamiB(DE3)pLysS(pET-32a(+)) broken supernatant sample preparation:

The strain E.coli OrigamiB(DE3)pLysS(pET-32a(+)) was fermented and the cells were collected. 100 grams of wet bacteria were resuspended in 500 mL PBS Buffer, ultrasonically disrupted, centrifuged, and the supernatant was taken.

2. Determination of biological activity of Pen24 against cotton bollworm nuclear polyhedrosis virus (HaNPV) infection

1.5×10 ⁴ PIB/mL HaNPV was selected as the virus infection dose. Cotton bollworms were raised at 26°C with artificial feed. The third-instar cotton bollworm larvae were divided into 4 groups, and the following 4 samples were added to feed the infection:

①E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+HaNPV group:

E.coli OrigamiB (DE3) pLysS (pET-pen) crushed supernatant sample was mixed with HaNPV suspension, and after 1 hour of action at 26 ° C, 300 μ L was applied to the surface of 1 cm ³ artificial feed to feed cotton bollworm larvae.

②E.coli OrigamiB(DE3)pLysS(pET-32a(+)) broken supernatant+HaNPV group:

E.coli OrigamiB(DE3)pLysS(pET-32a(+)) crushed supernatant sample was mixed with HaNPV suspension, and after acting for 1 hour at 26°C, 300μL was applied to the surface of 1cm ³ artificial feed to feed cotton bollworm larvae.

③HaNPV positive control group:

Apply 300 μL of HaNPV suspension on the surface of 1 cm ³ artificial feed to feed the larvae of cotton bollworm.

④PBS Buffer control group:

Apply 300 μL of PBS Buffer suspension on the surface of 1 cm ³ artificial feed to feed the larvae of cotton bollworm.

Cotton bollworm larvae were reared at 20-25°C. After being infected with HaNPV virus for three days, they were fed with fresh artificial feed every day, and the death of cotton bollworm larvae was recorded twice a day.

The results are shown in Figure 9. The results showed that on the 4th day, the mortality rate of E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+HaNPV group was 33%; the mortality rate of HaNPV positive control group was 75%; E.coliOrigamiB(DE3)pLysS (pET-32a(+)) The mortality rate of the broken supernatant+HaNPV control group was 75%. On the 5th day, the mortality rate of E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+HaNPV group was 67%; the mortality rate of HaNPV positive control group was 92%; E.coli OrigamiB(DE3)pLysS(pET-pen) -32a (+)) broken supernatant + HaNPV control group had a mortality rate of 92%. On the 6th day, the mortality rate of E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+HaNPV group was 83%; the mortality rate of HaNPV positive control group was 100%; E.coli OrigamiB(DE3)pLysS(pET-pen) 32a(+)) The mortality rate of the broken supernatant + HaNPV control group was 100%. The antimicrobial peptide Pen24 can effectively inhibit HaNPV infection to the third instar cotton bollworm larvae, and has biological activity against HaNPV virus infection.

Example 13 Genetic engineering recombinant prawn peptide Pen24

Biological Activity Against Beet Spodoptera Nucleopolyhedrosis Virus (SeMNPV) Infection

The licking method was used to detect the effect of genetic engineering recombinant prawn peptide Pen24 on beet armyworm nucleopolyhedrosis virus (SeMNPV) infection of beet armyworm larvae.

1. Sample Preparation

(1) Preparation of beet armyworm nucleopolyhedrosis virus (SeMNPV) samples:

The crude extract of beet armyworm nuclear polyhedrosis virus (SeMNPV) was purified by sucrose gradient, counted under a microscope, and the content of SeMNPV was 7.8×10 ⁹ PIB/mL.

(2) E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant sample preparation:

The strain E.coli OrigamiB(DE3)pLysS(pET-pen) was fermented and the cells were collected. 100 grams of wet bacteria were resuspended in 500 mL PBS Buffer, ultrasonically disrupted, centrifuged, and the supernatant was taken.

(3) E.coli OrigamiB(DE3)pLysS(pET-32a(+)) broken supernatant sample preparation:

The strain E.coli OrigamiB(DE3)pLysS(pET-32a(+)) was fermented and the cells were collected. 100 grams of wet bacteria were resuspended in 500 mL PBS Buffer, ultrasonically disrupted, centrifuged, and the supernatant was taken.

2. Determination of biological activity of Pen24 against beet armyworm nucleopolyhedrosis virus (SeMNPV) infection

7.8×10 ⁴ PIB/mL SeMNPV was selected as the virus infection dose. The beet armyworm was raised at 26°C and fed with artificial diet. The third-instar beet armyworm larvae were divided into 4 groups, and the following 4 samples were added to feed the infection:

① E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant + SeMNPV group:

E.coli OrigamiB(DE3)pLysS(pET-pen) crushed supernatant sample was mixed with SeMNPV suspension, and after acting for 1 hour at 26°C, 300μL was applied to the surface of ^1cm3 artificial feed to feed beet armyworm larvae.

②E.coli OrigamiB(DE3)pLysS(pET-32a(+)) crushed supernatant+SeMNPV group:

E.coli OrigamiB(DE3)pLysS(pET-32a(+)) crushed supernatant sample was mixed with SeMNPV suspension, and after acting for 1 hour at 26°C, 300μL was applied to the surface of ^1cm3 artificial feed to feed beet armyworm larvae.

③SeMNPV positive control group:

300 μL of SeMNPV suspension was applied to the surface of 1 cm ³ artificial feed to feed beet armyworm larvae.

④PBS Buffer control group:

Apply 300 μL of PBS Buffer suspension on the surface of 1 cm ³ artificial feed to feed beet armyworm larvae.

The beet armyworm larvae were reared at 20-25°C. After being infected with SeMNPV virus for three days, they were fed with fresh artificial feed every day, and the death of beet armyworm larvae was recorded twice a day.

The results are shown in Figure 10. The results showed that on the 4th day, the mortality rate of E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+SeMNPV group was 58%; the mortality rate of SeMNPV positive control group was 75%;

E.coli OrigamiB(DE3)pLysS(pET-32a(+)) broken supernatant + SeMNPV control group had a mortality rate of 75%. On the 5th day, the mortality rate of E.coli OrigamiB(DE3)pLysS(pET-pen) broken supernatant+SeMNPV group was 58%; the mortality rate of SeMNPV positive control group was 100%; E.coli OrigamiB(DE3)pLysS(pET-pen) -32a (+)) crushed supernatant + SeMNPV control group had a mortality rate of 100%. On the 7th day, the mortality rate of E.coliOrigamiB(DE3)pLysS(pET-pen) broken supernatant+SeMNPV group was 75%;

The mortality rate of the SeMNPV positive control group was 100%; the mortality rate of the E.coli OrigamiB(DE3)pLysS(pET-32a(+)) broken supernatant+SeMNPV control group was 100%. The antimicrobial peptide Pen24 can effectively inhibit the infection of SeMNPV to third-instar beet armyworm larvae, and has biological activity against SeMNPV virus infection.

Example 14 Genetic engineering recombinant prawn peptide Pen24

Biological activity against shrimp white spot syndrome virus infection

1. Sample Preparation and Handling

(1) Preparation of shrimp white spot syndrome virus (WSSV) samples

Put Procambarus clarkii frozen at -20°C and infected with white spot syndrome virus (WSSV) on ice, take out the head and chest (gills, liver, heart and other organs), cut into small pieces with surgical scissors, and put them in glass In a homogenizer, homogenize in an ice bath. The homogenate was centrifuged at 12000r/min at 4°C for 20min, and the supernatant was sterilized by filtration with a 0.45μm filter membrane, and then packed into 1.5ml EP tubes and stored at -20°C for later use.

Using the above-mentioned WSSV virus suspension, inject Procambarus clarkii with 150 μl/head WSSV suspension, infect at 22-24° C., and all die within 6.5 days as a result. Procambarus clarkii was injected with 100 μl/head WSSV suspension, infected at 22-24°C, and all died within 9 days as a result. We chose 0.1mL WSSV virus suspension as the dose of WSSV virus infection in the experiment of genetically engineered recombinant prawn peptide Pen24 against prawn white spot syndrome virus infection.

(2) Treatment of four test samples

①Purified samples of genetically engineered recombinant prawn peptide Pen24 test group samples: Mix the quantitative 1 mg/mL purified sample of recombinant prawn peptide Pen24 with WSSV virus suspension in equal volumes, and react at 26°C for 1 hour.

②E.coli OrigamiB(DE3)pLysS(pET-32a(+)) crushing supernatant sample preparation and processing: the strain E.coli OrigamiB(DE3)pLysS(pET-32a(+)) was fermented and the cells were collected. 100 grams of wet bacteria were resuspended in 500 mL PBS Buffer, ultrasonically disrupted, centrifuged, and the supernatant was taken. The crushed supernatant was mixed with equal volumes of WSSV virus suspension, and reacted at 26°C for 1 hour.

③WSSV positive control group sample: WSSV virus suspension was diluted 1 times with PBS, and treated at 26°C for 1 hour.

④Healthy control group sample: PBS Buffer.

2. Determination of biological activity of Pen24 against shrimp white spot syndrome virus (WSSV) infection

Procambarus clarkii was reared at room temperature at 22-24°C, and the prawn bait (No. 0) was purchased from Zhanjiang Yuehua Aquatic Feed Co., Ltd. Divide the Procambarus clarkii into 4 groups, and inject the Procambarus clarkii with the above-mentioned 4 kinds of test samples respectively. Dose 0.15mL/tail, 25 tails in each group, two repetitions. Breed under the condition of natural temperature (15-22 ℃), change the water every day and feed artificial shrimp feed once, and record the death situation of crayfish twice a day.

The results are shown in Table 3 and accompanying drawing 11. The results of feeding at room temperature (20-25°C) showed that Procambarus clarkii was cultured at 22-24°C. On the 4th day, there was no death in the test group of the genetically engineered recombinant prawn peptide Pen24; the mortality rate in the WSSV positive control group was 15%. ; E.coli OrigamiB(DE3)pLysS(pET-32a(+)) crushed supernatant group had a mortality rate of 20%. On the 9th day, the mortality rate of the WSSV positive control group and the E.coli OrigamiB(DE3)pLysS(pET-32a(+)) broken supernatant group were both 100%, and the mortality rate of the genetically engineered recombinant prawn peptide Pen24 purified sample test group was 85%, with a 15% survival rate. It shows that the genetically engineered recombinant prawn peptide Pen24 can effectively resist WSSV infection in Procambarus clarkii, and has biological activity against WSSV virus infection.

Table 3 Determination of recombinant prawn peptide Pen24 against WSSV infection test group Mortality in 4d(%) Mortality in 9d(%) LT ₅₀ (d) Recombinant prawn peptide Pen24 sample test group E.coli OrigamiB(DE3)pLysS(pET-32a(+)) crushed supernatant test group WSSV positive control group Healthy control group 020150 851001000 7.555.50

Sequence Listing 1

SEQUENCE LISTING

<110> Wuhan University

<120>A Genetic Engineering Strain Expressing Recombinant Prawn Peptide Pen24 and Its Application

<130> Nucleotide sequence of a recombinant prawn peptide Pen24

<160>2

<170>PatentIn version 3.1

<210>1

<211>657

<212>DNA

<213> recombinant DNA

<220>

<221> CDS

<222>(1)..(657)

<223>

<400>1

atg agc gat aaa att att cac ctg act gac gac agt ttt gac acg gat 48

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

gta ctc aaa gcg gac ggg gcg atc ctc gtc gat ttc tgg gca gag tgg 96

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

tgc ggt ccg tgc aaa atg atc gcc ccg att ctg gat gaa atc gct gac 144

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

gaa tat cag ggc aaa ctg acc gtt gca aaa ctg aac atc gat caa aac 192

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

cct ggc act gcg ccg aaa tat ggc atc cgt ggt atc ccg act ctg ctg 240

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

ctg ttc aaa aac ggt gaa gtg gcg gca acc aaa gtg ggt gca ctg tct 288

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

aaa ggt cag ttg aaa gag ttc ctc gac gct aac ctg gcc ggt tct ggt 336

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

tct ggc cat atg cac cat cat cat cat cat tct tct ggt ctg gtg cca 384

Ser Gly His Met His His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

cgc ggt tct ggt atg aaa gaa acc gct gct gct aaa ttc gaa cgc cag 432

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

cac atg gac agc cca gat ctg ggt acc gac gac gac gac aag gcc atg 480

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

gct gat atc gga tcc gaa ttc tac agg ggc ggt tac aca ggc ccg ata 528

Ala Asp Ile Gly Ser Glu Phe Tyr Arg Gly Gly Tyr Thr Gly Pro Ile

165 170 175

ccc agg cca cca ccc att gga aga cca ccg ttc aga cct gtt tgc aat 576

Pro Arg Pro Pro Pro Ile Gly Arg Pro Pro Phe Arg Pro Val Cys Asn

180 185 190

gca tgc tac aga ctt tcc gtc tca gat gct cgc aat tgc tgc atc aag 624

Ala Cys Tyr Arg Leu Ser Val Ser Asp Ala Arg Asn Cys Cys Ile Lys

195 200 205

ttc gga agc tgt tgt cac tta gta aaa gga taa 657

Phe Gly Ser Cys Cys His Leu Val Lys Gly

210 215

<210>2

<211>218

<212>PRT

<213> recombinant DNA

<400>2

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Glu Phe Tyr Arg Gly Gly Tyr Thr Gly Pro Ile

165 170 175

Pro Arg Pro Pro Pro Ile Gly Arg Pro Pro Phe Arg Pro Val Cys Asn

180 185 190

Ala Cys Tyr Arg Leu Ser Val Ser Asp Ala Arg Asn Cys Cys Ile Lys

195 200 205

Phe Gly Ser Cys Cys His Leu Val Lys Gly

210 215

Sequence Listing 2

SEQUENCE LISTING

<110> Wuhan University

<120>A Genetic Engineering Strain Expressing Recombinant Prawn Peptide Pen24 and Its Application

<130> Amino acid sequence of a recombinant prawn peptide Pen24

<160>1

<170>PatentIn version 3.1

<210>1

<211>218

<212>PRT

<213> Recombinant protein

<400>1

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Glu Phe Tyr Arg Gly Gly Tyr Thr Gly Pro Ile

165 170 175

Pro Arg Pro Pro Pro Ile Gly Arg Pro Pro Phe Arg Pro Val Cys Asn

180 185 190

Ala Cys Tyr Arg Leu Ser Val Ser Asp Ala Arg Asn Cys Cys Ile Lys

195 200 205

Phe Gly Ser Cys Cys His Leu Val Lys Gly

210 215

Claims (16)

1, the reorganization prawn peptide Pen24 of a kind of gene engineering expression, separation and purifying, it has the sequence with at least 70% homology of aminoacid sequence shown in the sequence table 2.

2, the reorganization prawn peptide Pen24 of a kind of gene engineering expression, separation and purifying, it has the sequence with at least 80% homology of aminoacid sequence shown in the sequence table 2.

3, the reorganization prawn peptide Pen24 of a kind of gene engineering expression, separation and purifying, it has the sequence with at least 90% homology of aminoacid sequence shown in the sequence table 2.

4, protein according to claim 1 and 2 is characterized in that: described proteinic molecular weight is 24 kilodaltons.

5, the reorganization prawn peptide Pen24 of a kind of gene engineering expression, separation and purifying, it has the sequence with at least 50% homology of nucleotide sequence shown in the sequence table 1.

6, the reorganization prawn peptide Pen24 of a kind of gene engineering expression, separation and purifying, it has the sequence with at least 80% homology of nucleotide sequence shown in the sequence table 1.

7, a kind of dna segment is characterized in that comprising as claim 5 or 6 described nucleotide sequences.

8. an engineering strain is characterized in that comprising as claim 5 or 6 described nucleotide sequences.

9, a kind of express recombinant prawn peptide Pen24 engineering strain, it is characterized in that: described bacterial strain is engineering strain E.coli OrigamiB (DE3) pLysS (pET-pen), CCTCC No:M206003.

10, the construction process of the engineering strain of a kind of express recombinant prawn peptide Pen24, it comprises the following steps:

Synthesizing of the extraction of A, the total RNA of Penaeus vannamei Boone and cDNA article one chain: gather the Penaeus vannamei hemolymph, obtain hemocyte, extract total RNA, and undertaken by the Universal Riboclone cDNASynthesis System of Promega company test kit operational manual, cDNA article one chain is synthesized in reverse transcription;

B, pcr amplification Penaeidin-2 gene: with reverse transcription synthetic cDNA article one chain is template, upstream primer P1:5 ' GAATTCTACAGGGGCGGTTACACA 3 '; Downstream primer P2:3 ' GTGAATCATTTTCCTATTTTCGAA 5 ' utilizes the amplification of PCR instrument, obtains the 168bp goal gene;

The structure of C, recombinant plasmid pGEM-T/Pen and evaluation: reclaim the PCR product, the PCR product is connected on the pGEM-T carrier, Transformed E .coli JM109 competent cell, coat and carry out blue hickie screening on the LB flat board that contains X-gal, IPTG and Amp resistance, use the evaluation of PCR and digestion with restriction enzyme, dna sequencing Analysis and Identification recombinant plasmid pGEM-T/Pen;

D, the structure of recombinant expression plasmid pET-pen and evaluation: use EcoR I, Hind III is double digestion expression vector pET-32a (+) and recon pGEM-T/Pen plasmid DNA respectively, with the directed expression vector pET-32a (+) that inserts of Penaeidin-2 gene, obtain recombinant expression plasmid pET-pen, Transformed E .coli Origami B (DE3) pLysS competent cell obtains intestinal bacteria recombination engineering strain E.coli OrigamiB (DE3) pLysS (pET-pen), identifies with PCR and digestion with restriction enzyme, dna sequencing Analysis and Identification recombinant expression plasmid pET-pen.

11, the application of reorganization prawn peptide Pen24 in bacterial-infection resisting.

12, the application of reorganization prawn peptide Pen24 in anti-fungal infection.

13, the application of reorganization prawn peptide Pen24 in anti-virus infection.

14, reorganization prawn peptide Pen24 is as the application of sanitas in makeup.

15, reorganization prawn peptide Pen24 as sanitas in Application in Food.

16, reorganization prawn peptide Pen24 is as the application of sanitas in animal-feed.

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