CN102994551A - Method for expressing silkworm antimicrobial peptide CecropinB2 in silkworm larvae using double-promoter expression vector - Google Patents

Abstract

The invention discloses a method for expressing silkworm antimicrobial peptide CecropinB2 in silkworm larvae by using a dual-promoter expression vector. The steps include: respectively cloning the BmCecB2 gene and the polh gene into a promoter expression vector to construct a recombinant double-promoter expression vector; transforming the recombinant double-promoter expression vector into DH10Bac competent cells, extracting the plasmid, and obtaining the recombinant shuttle vector Bacmid; Bacmid Under the mediation of transfection reagent liposome, silkworm cultured cells were transfected to obtain recombinant virus polyhedrons; after quantitative recombinant virus polyhedrons were inoculated orally in fifth-instar silkworm larvae, they were expressed in silkworm larvae to obtain antibacterial activity The antimicrobial peptide of the present invention has the beneficial effects of feeding silkworms orally, so that the silkworm antimicrobial peptide Cecropin B gene can be expressed in silkworms. Compared with artificially injecting silkworms, it saves time and effort, and improves production efficiency.

Description

Method for expressing silkworm antimicrobial peptide CecropinB2 in silkworm larvae using double-promoter expression vector

technical field

The invention relates to a method for expressing silkworm antimicrobial peptide CecropinB2 in silkworm larvae by using a dual-promoter expression vector. the

Background technique

Antimicrobial peptides (AMPs) are biologically active small molecule peptides induced in vivo, which have broad-spectrum antibacterial activity and strong killing effect on bacteria, especially for some drug-resistant pathogenic bacteria. The killing effect has attracted people's attention. With the continuous development of biotechnology, antimicrobial peptides, as new antibiotics and new drugs, play an important role in agriculture, medicine, food and other fields. So far, thousands of antimicrobial peptides have been isolated from natural organisms. Among them, cecropins antimicrobial peptides are the most clearly studied and most effective antibacterial peptides. Five structures, A, B, C, D, and E, have been found. This kind of antimicrobial peptide also has the characteristics of acid resistance, heat resistance, alkali resistance and resistance to drug resistance, and is widely used in microorganisms, animals and plants. the

In recent years, the Bac-to-Bac baculovirus expression system has been widely used. Based on the principle of F factor vector, this system has changed the traditional construction method of insect recombinant baculovirus and constructed a new baculovirus shuttle vector Bacmid. The system has the following advantages: (1) The recombination rate has been significantly improved, almost up to 100%. (2) The screening of recombinant viruses by blue and white spots has the advantage of screening, and there will be no problems of wild-type and non-recombinant virus contamination, and no longer Traditional tedious plaque screening analysis is required to purify recombinant virus (3) greatly shortening the time required for recombinant virus construction, which can be reduced from 4-6 weeks or longer to only 7-10 days. Therefore, this technology enables rapid and simultaneous production of multiple recombinant viruses. This is the fastest and easiest way to produce recombinant virus. the

With the widespread use of antibiotics in feed and people's higher and higher requirements for food and environmental quality, people's understanding of the side effects of antibiotics is increasing, and antimicrobial peptides have broad-spectrum antibacterial effects, which can promote growth and health of livestock and poultry. It has the function of treating diseases and belongs to a class of environmentally friendly preparations with no toxic side effects, no residues, and no bacterial resistance. Bioengineering technology can be applied to produce anti-bacterial transgenic animal and plant products, and at the same time, antibacterial peptides can be expressed in large quantities through genetic engineering technology, making it a source of a new generation of peptide antibacterial drugs, with broad application prospects. the

When using the silkworm baculovirus as an expression vector, the most widely used method is to replace the polh gene with an exogenous target gene, and use Pph to drive the high-efficiency expression of the target gene. The produced recombinant virus can only inoculate the silkworm by subcutaneous injection because it does not have the protection of the polyhedrin shell, resulting in low efficiency. Therefore, a dual-promoter expression vector can be used to construct an expression vector that can simultaneously express the polh gene and the BmCecB2 gene, and obtain The recombinant virus can be orally inoculated into silkworms and express antimicrobial peptides in silkworms. Compared with artificial subcutaneous injection of silkworms, this method has the advantages of saving time and labor, improving production efficiency, and is conducive to large-scale production. the

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for expressing silkworm antimicrobial peptide CecropinB2 in silkworm larvae by using a dual-promoter expression vector. the

The present invention is achieved through the following technical solutions. the

A method for expressing silkworm antimicrobial peptide CecropinB2 in silkworm larvae using a dual promoter expression vector, the steps comprising:

The BmCecB2 gene and polh gene as shown in the sequence table were cloned into the promoter expression vector to construct the recombinant double promoter expression vector; the recombinant double promoter expression vector was transformed into DH10Bac competent cells, and the plasmid was extracted to obtain the recombinant shuttle vector Bacmid ; Bacmid transfected silkworm cultured cells under the mediation of transfection reagent liposomes to obtain recombinant virus polyhedrons; after quantitative recombinant virus polyhedrons were orally inoculated in fifth-instar silkworm larvae, expressed in silkworm larvae, obtained inhibitory polyhedrons. Bacterially active antimicrobial peptides. the

Further, the above-mentioned dual-promoter expression vector is pFastBacDUAL. the

Further, the above-mentioned BmCecB2 gene and polh gene were respectively inserted into the downstream of the MCS of Pp10 and Pph of the dual-promoter expression vector pFastBacDUAL to construct a recombinant dual-promoter expression vector. the

Further, the above-mentioned recombinant shuttle vector Bacmid obtains Gen resistance by transferring the mini-Tn7 transposon from the double promoter expression vector to the mini-attTn7 target site on the shuttle vector Bacmid, and then by coating , Gen, Tet, IPTG, X-gal LB plate for blue and white screening, the white spot was selected overnight shaking culture, and obtained by alkaline lysis extraction. the

Further, the above-mentioned liposome-mediated transfection reagent refers to recombinant Bacmid and liposome, adding serum-free Bm cell culture medium respectively, mixing the two solutions, standing at room temperature, and taking another well-grown Bm cell , add serum-free Bm cell culture medium and recombinant Bacmid/liposome complex, cultivate for a period of time, suck off the supernatant, add Bm cell culture medium containing 10% serum, continue to cultivate, and observe. the

Further, the concentration of the above quantitative recombinant virus polyhedron is 2×10 ⁷ NPB/ml.

Further, the feed amount of the above-mentioned recombinant virus polyhedron is 7 μl per silkworm. the

Beneficial effects of the present invention:

Using the double-promoter expression vector, the recombinant virus obtained in the silkworm Bac-to-Bac baculovirus expression system can be added to the silkworm orally, so that the silkworm antimicrobial peptide Cecropin B gene can be expressed in the silkworm body, which is comparable to the artificial injection of silkworm. Compared, save time and effort, improve production efficiency. Since the host domain of baculovirus is limited to silkworms, it will not infect other animals. Therefore, after high-efficiency expression of antimicrobial peptides, silkworm bodies can be ground and added to feed without purification. As a feed additive, it is expected to become one of the products that can replace antibiotics. First, it has a good market application prospect. the

Description of drawings

Fig. 1 is the schematic flow sheet of the method for expressing silkworm antimicrobial peptide CecropinB2 in the silkworm larvae of the present invention utilizing double-promoter expression vector;

Fig. 2 is the schematic diagram of recombinant double promoter pFastBac Dual expression vector;

Figure 3 is a schematic diagram of the Tricine-SDS-PAGE detection of BmCecB2 gene and polh gene expression products; where M is the protein molecular standard, 1~2 are the blood of normal silkworm on the sixth day and the seventh day respectively, and 3~4 are oral The blood of the silkworm fed with the recombinant virus on the sixth day and the seventh day, and 5~6 are the blood of the silkworm fed on the wild-type baculovirus on the sixth day and the seventh day, respectively, and the arrow points to the target band;

Figure 4 is a schematic diagram of the detection of antibacterial activity of BmCecB2; 1-2 are the blood of the silkworm fed with the recombinant virus on the sixth day and the seventh day respectively, 3 is the blood of the silkworm fed with the wild-type virus, 4 is the blood of the normal silkworm, and 5 means Amp, 6 means sterile water. the

Detailed ways

The present invention will be described in further detail below according to the drawings and embodiments. the

The overall method flow of the present invention is shown in FIG. 1 . the

Example 1

(1) Construction of a dual-promoter expression vector: Using the synthetic PET-32a-BmCecB2 and wild-type virus strain genomic DNA as templates, respectively, synthesize the BmCecB2 gene and polh gene, and insert them into Pp10 and Pph of the dual-promoter expression vector pFastBacDUAL Downstream of the MCS, design synthetic primers as follows:

CecB-F(5'-CATG CCATGG CGCCGGAACCGCGTTGGAAA-3')

CecB-R(5'-CGG GGTACC TCATTATTTACCGATGGCTTTCG-3')

Polh-F(5'-CGC GGATCC ATGCCGAATTATTCATACA-3'),

Polh-R(5'-CCC AAGCTT TTAATACGCCGGACCAGTGAACAG-3')

The inserted restriction site is BmCecB2 (Nco I/Kpn I), polh (BamH I/Hind III)

(2) Recombinant shuttle vector Bacmid: Transform DH10Bac competent cells with the above-mentioned dual-promoter expression vector, transform the constructed recombinant dual-promoter expression vector into E.coli DH10Bac competent cells, culture with shaking at 37°C for 4 hours, recombine The shuttle vector Bacmid was generated by transpositioning the mini-Tn7 transposon from the dual-promoter expression vector to the mini-attTn7 target site on the shuttle vector Bacmid, and acquired Gen resistance. Blue-white screening by coating on LB plates containing Kan, Gen, Tet, IPTG, X-gal. Vitiligo was selected for shaking culture overnight, the recombinant shuttle vector Bacmid was extracted by alkaline lysis, and PCR verification was carried out with M13 primers. The recombinant dual promoter pFastBac Dual expression vector is shown in Figure 2. the

Note: PCR reaction system (25 μL reaction system): 10×Taq Buffer 2.5 μl, dNTPs 1.5 μl, mixed primer (M ₁₃ F+M ₁₃ R) 1 μl, Taq polymerase 0.2 μl, ddH2O up to 25 μl, template (rBacmid) 1 μl.

PCR amplification conditions: pre-denaturation at 93°C for 3min, denaturation at 94°C for 45s, annealing at 55°C for 45s, extension at 72°C for 4min, and 35 cycles. the

(3) Obtain recombinant virus

Take the recombinant shuttle carrier Bacmid (7~10 μg) and liposome 8 μl, and add serum-free Bm cell culture medium to supplement the volume to 100 μl, mix the two solutions, let stand at room temperature for 20 minutes, and take another well-growing Bm cell , washed 2~3 times with serum-free Bm cell culture medium, then added 800μl serum-free Bm cell culture medium and 200μl of the prepared recombinant Bacmid/liposome complex, cultured at 28°C for 3~5h, then sucked off the upper Add 2ml of Bm cell culture medium containing 10% serum, continue culturing at 28°C for 60 hours, observe under the microscope whether the Bm cells have symptoms of infection, absorb the cell supernatant to obtain recombinant virus particles, reinfect silkworm cells, and obtain recombinant Virus. the

(4) Expression of silkworm antimicrobial peptide Cecropin B2 in silkworm body

Quantify the recombinant virus solution to 2×10 ⁷ NPB/ml and prepare silkworm larvae on the first day of the fifth instar. The first area was fed with the recombinant virus solution orally, and each silkworm was fed with 7 μl, and the wild-type stems were fed with the same concentration. Viruses and feeding water area were used as controls. The infection situation of silkworm was observed every day, and the silkworm blood was collected after the 6th day and the 7th day, and the polyhedron was purified, and the viral DNA was extracted from it, and verified by PCR (primers were CecB-F/CecB-R).

Example 2

Identification of silkworm antimicrobial peptides by Tricine-SDS-PAGE

Sample processing: take the infected silkworm blood, boil it at 100°C for 5min, centrifuge at 11000rpm for 5min, add 2X loading buffer (4% SDS, 12% glycerol, 50mM Tris, 2% mercaptoethanol, 0.01% bromophenol blue to the supernatant) , adjust the pH to 6.8 with 6M Hcl), boil for 5 minutes. the

Tricine-SDS-PAGE: prepare 15% concentration of separating gel (urea), 10% concentration of intermediate gel and 4% stacking gel; sample electrophoresis after sample treatment, fixed solution (50% methanol, 10% acetic acid) for 30min , stained with Coomassie Brilliant Blue for 10 min, and then decolorized with decolorizing solution. the

The detection results of Tricine-SDS-PAGE are shown in Figure 3, wherein the molecular weight of polh protein is 29KD, and the molecular weight of BmCecB2 is 4.9KD. The analysis results showed that both polyhedron and silkworm antibacterial peptide cecropin B were expressed. the

Example 3

Antibacterial Activity Detection of Silkworm Antimicrobial Peptide Cecropin B

The silkworm blood was taken, and the supernatant was obtained by centrifugation after the cells were disrupted by ultrasonic, and the agarose hole diffusion experiment was carried out. Mix 400 μl of Escherichia coli suspension (OD ₆₀₀ =0.3) with 100 mL of LB solid medium at 55°C and spread it on a plate. After it solidifies, punch holes with a sterilized hole puncher (diameter 5 mm), and drop Add 200ul of the expression supernatant to be tested, incubate at 37°C for 16h, use the same volume of sterilized water as a negative control, and Amp as a positive control, and measure the diameter of inhibition on the second day. The activity of antimicrobial peptides was determined according to the size of the inhibition zone.

The results of the activity test are shown in Figure 4. The results show that the silkworm antimicrobial peptide cecropin B gene is expressed in the silkworm, and has obvious antibacterial activity. As a positive control, the blood of normal silkworms and the blood of silkworms fed with wild-type virus were added to exclude the expression of the background level. the

The above-mentioned embodiments are only to illustrate the technical concept and characteristics of the present invention, and its purpose is to allow those familiar with the art to understand and implement the content of the present invention, and cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention. the

Claims (7)

1. a method utilizing double promoter expression vector to express silkworm antimicrobial peptide CecropinB2 in silkworm larvae, is characterized in that, step comprises: The BmCecB2 gene and polh gene as shown in the sequence table were cloned into the promoter expression vector to construct the recombinant double promoter expression vector; the recombinant double promoter expression vector was transformed into DH10Bac competent cells, and the plasmid was extracted to obtain the recombinant shuttle vector Bacmid ; Bacmid transfected silkworm cultured cells under the mediation of transfection reagent liposomes to obtain recombinant virus polyhedrons; after quantitative recombinant virus polyhedrons were orally inoculated in fifth-instar silkworm larvae, expressed in silkworm larvae, obtained inhibitory polyhedrons. Bacterially active antimicrobial peptides. 2. The method for expressing silkworm antimicrobial peptide CecropinB2 in silkworm larvae using a dual-promoter expression vector according to claim 1, wherein the dual-promoter expression vector is pFastBacDUAL. 3. utilize double promoter expression vector according to claim 2 to express the method for silkworm antimicrobial peptide CecropinB2 in silkworm larvae, it is characterized in that, described BmCecB2 gene and polh gene are respectively inserted into double promoter expression vector pFastBacDUAL Pp 10 and A recombinant dual promoter expression vector was constructed downstream of the MCS of Pph. 4. utilize double-promoter expression carrier according to claim 1 to express the method for silkworm antibacterial peptide CecropinB2 in silkworm larvae, it is characterized in that, described recombinant shuttle vector Bacmid is by mini-Tn7 transposon from double-promoter The expression vector was translocated to the mini-attTn7 target site on the shuttle vector Bacmid to obtain Gen resistance, and then screened for blue and white spots by coating on LB plates containing Kan, Gen, Tet, IPTG, and X-gal to select white spots Cultured overnight with shaking, extracted by alkaline lysis. 5. utilize double promoter expression carrier according to claim 1 to express the method for silkworm antimicrobial peptide CecropinB2 in silkworm larvae, it is characterized in that, described in transfection reagent liposome mediation refers to recombinant Bacmid and liposome , add serum-free Bm cell culture medium respectively, mix the two solutions, let stand at room temperature, take another well-grown Bm cell, add serum-free Bm cell culture medium and recombinant Bacmid/liposome complex, and cultivate for a period time, suck off the supernatant, add Bm cell culture medium containing 10% serum, continue culturing, and observe. 6 . The method for expressing silkworm antimicrobial peptide CecropinB2 in silkworm larvae using a dual-promoter expression vector according to claim 1 , characterized in that the quantitative concentration of the recombinant virus polyhedron is 2×10 ⁷ NPB/ml. 7. The method for expressing the silkworm antimicrobial peptide CecropinB2 in silkworm larvae using a dual-promoter expression vector according to claim 1, characterized in that the amount of feed added to the recombinant virus polyhedron is 7 μl per silkworm.

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