CN102021175A - Optimized grass carp alpha interferon encoding gene and application thereof - Google Patents

Abstract

The invention discloses an optimized grass carp interferon-alpha coding gene and its application. The optimized grass carp interferon-alpha encoding gene disclosed in the present invention is the DNA shown in sequence 2 of the sequence listing. The expression ability of the gene is much higher than that of the existing gene, the gene is inserted into the multi-cloning site of pBV220 to obtain a recombinant plasmid, and the recombinant plasmid is introduced into Escherichia coli DH5α to obtain a recombinant bacterium. The recombinant bacteria can produce a large amount of ChIFNα protein through simple induction culture. The gene, recombinant plasmid and recombinant bacterium provided by the invention have great economic value for the production of alpha interferon, and also have great value for the aquaculture industry.

Description

Optimized grass carp interferon-α coding gene and its application

technical field

The invention belongs to the field of genetic engineering and relates to an optimized grass carp alpha interferon coding gene and application thereof.

Background technique

In recent years, my country's high-density and intensive aquaculture methods have led to frequent occurrence of various diseases, especially various viral diseases, which have become a key factor restricting the sustainable development of my country's aquaculture industry. For a long time, people have been using various chemicals to treat fish viral diseases, but such drugs can easily lead to excessive drug residues, coupled with the increasing requirements for food health at home and abroad, making the aquaculture industry difficult , so it is imminent to find new antiviral fish medicines that are safe and residue-free.

Fish is a lower vertebrate, and non-specific immunity plays an important role. As an efficient non-specific immune factor, the interferon system plays an important role in the anti-infection immunity of fish. At present, there have been reports on the gene sequence and amino acid sequence of fish interferon at home and abroad, and the fermentative expression of interferon gene in prokaryotic or eukaryotic expression system has been realized, which provides a basis for the large-scale production and application of fish genetically engineered interferon in accordance with.

Contents of the invention

The purpose of the present invention is to provide an optimized grass carp interferon-alpha coding gene and its application.

The optimized grass carp interferon-alpha encoding gene provided by the present invention is the DNA shown in sequence 2 of the sequence listing.

Recombinant vectors, recombinant bacteria, expression cassettes or transgenic cell lines containing the DNA shown in Sequence 2 of the sequence listing all belong to the protection scope of the present invention.

The recombinant vector can be a recombinant plasmid obtained by inserting the DNA shown in Sequence 2 of the sequence listing into the multiple cloning site of pBV220. The recombinant vector is preferably a recombinant plasmid obtained by inserting the DNA shown in Sequence 2 of the sequence listing between the BamHI and EcoRI sites of pBV220.

The recombinant bacterium can be a recombinant bacterium obtained by introducing the DNA shown in Sequence 2 of the sequence listing into Escherichia coli DH5α. The recombinant bacterium is preferably a recombinant bacterium obtained by introducing the recombinant vector into Escherichia coli DH5α. The recombinant bacterium is most preferably Escherichia coli (Escherichia coli) DH5α/pBV220-GCIFNα, which has been preserved in the General Microbiology Center (CGMCC for short) of the China Committee for the Collection of Microbial Cultures on August 23, 2010, and the address is: Beijing No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing), and the preservation number is CGMCC No.4111.

The present invention also protects a method for preparing interferon-alpha, which is to induce the recombinant bacteria at 40-43°C for 4-8 hours (preferably at 42°C for 6 hours), then crush the bacteria, collect inclusion bodies, and wash the inclusion bodies 1. Dissolving the inclusion body and refolding it to obtain a solution containing alpha interferon.

The method also includes the step of purifying the protein from the solution containing interferon-alpha.

The method of protein purification is as follows: adjust the pH value of the solution containing alpha interferon to 5.5-9.5 with acetic acid, perform cation exchange chromatography with HiLoad 26/10 SP Sepharose High Performance, and wash 5 samples with washing buffer A column volume, then elute 1-10 column bed volumes with washing buffer B, collect the eluate with an ultraviolet absorption value greater than 200 under 280nm light, and the resulting solution is alpha interferon solution; the washing buffer A pH is 5.5-9.5, composed of Tris Cl, NaCl and water, the concentration of Tris Cl is 0.1mol/L, and the concentration of NaCl is 0-150mM; the pH of the washing buffer B is 5.5-9.5, composed of Tris Cl , NaCl and water, the concentration of Tris·Cl is 0.1mol/L, and the concentration of NaCl is 150-2000mM.

The method for the protein purification is as follows: the pH value of the solution containing grass carp interferon alpha is adjusted to 6.5 with acetic acid, and HiLoad 26/10 SP Sepharose High Performance (inner diameter 26mm, bed height 100mm, GE LifeScience) is used to carry out cation exchange chromatography Add the sample to the chromatography column at a rate of 1ml/min, wash 5 column volumes at a rate of 5ml/min with washing buffer A, elute 3 column volumes at a rate of 5ml/min with washing buffer B, and collect Under 280nm illumination, the eluent whose UV absorption value is greater than 200, the solution obtained is alpha interferon solution (0.22um filter membrane can be used to filter and sterilize); the pH of the washing buffer first is 6.5, prepared by Tris Cl, Composed of NaCl and water, the concentration of Tris Cl is 0.1mol/L, and the concentration of NaCl is 50mM; the pH of the washing buffer B is 6.5, consisting of Tris Cl, NaCl and water, and the concentration of Tris Cl is 0.1mol/L , the NaCl concentration was 500 mM.

The alpha interferon solution prepared by the method also belongs to the protection scope of the present invention.

The DNA, the recombinant vector, the recombinant bacteria, the expression cassette or the transgenic cell line can be used to prepare alpha interferon.

The DNA, the recombinant vector, the recombinant bacteria, the expression cassette or the transgenic cell line can be used to prepare virus inhibitors. The virus may be infectious hematopoietic necrosis virus (IHNV) and/or grass carp hemorrhagic disease virus (GCHV). The infectious hematopoietic necrosis virus (IHNV) can specifically be a WRAC strain.

The DNA, the recombinant vector, the recombinant bacteria, the expression cassette or the transgenic cell line can be used to prepare the following (a) and/or (b);

(a) preparations for the treatment and/or prevention of fish viral diseases;

(b) A formulation that increases the survival rate of shrimp.

The fish viral disease can specifically be grass carp hemorrhagic disease. The shrimp can specifically be Penaeus sinensis.

The present invention has discovered an optimized ChIFNα gene, the expression ability of which is much higher than that of existing genes, and inserted the gene into the multi-cloning site of pBV220 to obtain a recombinant plasmid, which was introduced into Escherichia coli DH5α to obtain a recombinant bacteria. The recombinant bacteria can produce a large amount of ChIFNα protein through simple induction culture. The gene, recombinant plasmid and recombinant bacterium provided by the invention have great economic value for the production of alpha interferon, and also have great value for the aquaculture industry.

Description of drawings

Fig. 1 is a sequence alignment of GcIFN-α-1 gene (DNA shown in sequence 2) and GcIFN-α-2 gene (DNA shown in sequence 3).

Fig. 2 is the agarose gel electrophoresis figure of the PCR amplification result of grass carp interferon gene (GcIFN-α-1 and GcIFN-α-2); M is DNA molecular weight standard, and L1 is negative control result (with water as template) , L2 is the result of GcIFN-α-1 gene amplification, L3 is the result of GcIFN-α-2 gene amplification.

Figure 3 is the result of SDS-PAGE detection of grass carp interferon genes (GcIFN-α-1 and GcIFN-α-2) expressed in Escherichia coli; L1 is before induction of control bacteria, L2 is after induction of control bacteria, and L3 is recombinant engineering bacteria Before induction, L4 is induced by recombinant engineered bacteria, and M is low molecular weight protein Marker.

Figure 4 is the SDS-PAGE detection result of the purified grass carp interferon; M is the protein marker, L1 is the GcIFN-α-1 solution, and L2 is the GcIFN-α-2 solution.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. All primer synthesis and sequencing work were completed by Beijing Biomed Technology Development Co., Ltd. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged. % in the following examples, unless otherwise specified, are all percentages by mass.

Polyinosinic acid cytidylic acid (polyinosinic polycytidylic acid, PolyI: C): purchased from Shanghai Hefeng Pharmaceutical Co., Ltd., the article number is: Guoyao Zhunzi H20003599. Grass carp kidney cell line (GIK): China Center for Type Culture Collection, number GDC081. Infectious Hematopoietic Necrosis Virus (IHNV) (ATCC, number VR-1392, strain name WRAC).

Grass Carp Hemorrhage Virus (GCHV): the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences; Characteristics [J]. Acta Hydrobiology, 1990, 14(2): 153-159).

2*YT culture medium is composed of solute and water, the solute and its concentration are as follows: peptone 16g/L, yeast powder 10g/L, sodium chloride 5g/L.

The fermentation medium is composed of solute and water, and the solute and its concentration are as follows: peptone 5 g/l, yeast powder 5 g/l, KH ₂ PO ₄ 2 g/l, K ₂ HPO ₄ 4 g/l, Na ₂ HPO ₄ 12H ₂ O 7 g/L, (NH ₄ ) ₂ SO ₄ 1.2 g/L, NH ₄ Cl 0.2 g/L, MnSO ₄ 5H ₂ O 0.001 g/L, CoCl ₂ 6H ₂ O 0.004 g/L , Na ₂ MoO ₄ 2H ₂ O 0.002 g/L, ZnCl ₂ 0.002 g/L, CuSO ₄ 5H ₂ O 0.001 g/L, H ₃ BO ₄ 0.005 g/L, FeSO ₄ 7H ₂ O 0.02 g /L, CaCl·2H ₂ O 0.02 g/L, MgSO ₄ ·7H ₂ O 0.3 g/L, defoamer 0.2 g/L.

The feed medium is composed of solute and water, and the solute and its concentration are as follows: glycerol 150mL/L, peptone 30g/L, yeast powder 30g/L, MgSO ₄ ·7H ₂ O 5.5mg/L.

PBS buffer has a pH of 8.0 and is composed of solute and water. The solute and its concentration are as follows: NaCl 137mmol/L, KCl 2.7mmol/L, Na ₂ HPO ₄ 4.3mmol/L, KH ₂ PO ₄ 1.4mmol/L.

2×Loading Buffer is composed of solute and water, the solute and its concentration are as follows: 100mM Tris-HCl (pH6.8), 4% SDS, 0.2% bromophenol blue, 20% glycerol.

The denaturing buffer has a pH of 8.5 and is composed of solutes and water. The solutes and their concentrations are as follows: 6mol/L guanidine hydrochloride, 2mmol/L EDTA, 50mmol/L Tris Cl, 10mmol/L DTT.

Refolding buffer pH is 8.0, is made up of solute and water, and solute and its concentration are as follows: 0.5mol/L L-arginine (L-arg), 2mmol/L EDTA, 20% (volume percentage composition) glycerol, 0.9 mmol/L oxidized glutathione (GSSG), 0.1mol/L Tris Cl.

Embodiment 1, the artificial synthesis of the optimized grass carp interferon alpha gene and control gene

1. Artificial synthesis of optimized grass carp interferon-α gene (GcIFN-α-1 gene)

According to the degeneracy of codons and the preference for amino acid codons in Escherichia coli, referring to the published grass carp interferon-α gene sequence DQ357216 in Genebank, the coding gene of grass carp interferon-α containing codons favored by Escherichia coli was designed, as shown in the sequence As shown in Sequence 2 in the list, the optimized gene does not change the amino acid sequence of interferon-α of grass carp and is a gene sequence containing codons preferred by Escherichia coli. The DNA shown in Sequence 2 of the Sequence Listing was named GcIFN-α-1 gene (480 nucleotides in total).

The GcIFN-α-1 gene used as a template in Examples 2, 3 and 4 was synthesized by Shanghai Bioengineering Co., Ltd.

2. Artificial synthesis of control gene (GcIFN-α-2 gene)

The control gene is the grass carp interferon alpha gene that has been published in Genebank, as shown in GENBANK ACCESSION NO.DQ357216 from the 101st to 577th nucleotides at the 5' end, and at the 101st nucleotide from the 5' end of GENBANK ACCESSION NO.DQ357216 The start codon ATG is added before the acid, as shown in sequence 2 of the sequence listing. The DNA shown in Sequence 3 of the Sequence Listing was named GcIFN-α-2 gene (480 nucleotides in total).

The GcIFN-α-2 gene used as a template in Examples 2, 3 and 4 was synthesized by Shanghai Bioengineering Co., Ltd.

3. Sequence Alignment of GcIFN-α-1 Gene and GcIFN-α-2 Gene

The sequence alignment of GcIFN-α-1 gene and GcIFN-α-2 gene is shown in Fig. 1, GcIFN-α-1 gene and GcIFN-α-2 gene all encode the grass carp (Ctenopharyngodon idellus) interference shown in the sequence 1 of sequence list white.

Embodiment 2, the construction of recombinant plasmid and recombinant bacterium and the expression of gene

1. Construction of recombinant plasmid and engineering bacteria containing GcIFNα-1 gene

1. The GcIFNα-1 gene is used as a template, and a primer pair composed of F1 and R1 is used for PCR amplification to obtain a PCR amplification product.

F1: 5'-CGC GAATTC ATGTGCGAATGGCTG-3' (the underlined part is the EcoRI restriction site);

R1: 5'-CGC CTCGAG TTAACGACGGTTAGC-3' (the underlined part is the restriction site of BamHI).

The electropherogram of the PCR amplification product is shown in the swimming lane L2 of FIG. 2 .

2. The PCR amplification product was double-digested with restriction endonucleases BamHI and EcoRI to obtain the digested product.

3. The vector pBV220 (prokaryotic expression vector, purchased from Shanghai Jierui Bioengineering Co., Ltd., GV0302) was double-digested with restriction endonucleases BamHI and EcoRI, and the vector skeleton was recovered.

4. Ligate the digestion product of step 2 with the vector backbone of step 3 to obtain a ligation product.

5. The ligation product was transformed into Escherichia coli DH5α (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., catalog number: CW0808) competent, and a single colony was picked for colony PCR and enzyme digestion identification. The primers used in the colony PCR were the primer pair consisting of F1 and R1, and the colonies with about 480 bp DNA were positive colonies. The enzymes used for enzyme digestion identification are restriction endonucleases BamHI and EcoRI, and colonies with DNA of about 480 bp are positive colonies.

Colony PCR and enzyme digestion identification are positive colonies are recombinant bacteria (engineering bacteria). The host bacteria of the recombinant bacteria is Escherichia coli DH5α, which contains the recombinant plasmid GcIFN-α/pBV220. The recombinant plasmid GcIFN-α/pBV220 is a recombinant plasmid obtained by inserting the GcIFNα-1 gene gene shown in Sequence 2 of the sequence table between the BamHI and EcoRI restriction sites of the pBV220 vector.

An engineering strain was named DH5α/pBV220-GCIFNα, and the recombinant strain was preserved in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: West Beichen Road, Chaoyang District, Beijing) on August 23, 2010 No. 1 Courtyard No. 3), the preservation number is CGMCC No.4111.

The engineered bacteria DH5α/pBV220-GCIFNα were cultured at 30°C, 200 rpm (rotation radius: 13mm) with shaking (to OD600=1), and then induced at 42°C for 6 hours. Take 1 ml of the culture solution before and after induction in EP tubes, centrifuge at 5000g for 10 minutes, discard the supernatant, add 50uL of PBS buffer and 50uL of 2×Loading Buffer, boil for 10 minutes to lyse the bacteria, and then load 10uL of the sample for SDS-PAGE electrophoresis detection. The results of electrophoresis detection are shown in Figure 3 (lane L3 is before induction, and lane L4 is after induction). A protein band appeared at about 19kD in the bacteria collected after induction, which was consistent with the expected size. The protein in this band was recovered for N-terminal sequencing, and the five amino acids at the N-terminus were Cys-Glu-Trp-Leu-Gly, respectively, indicating that the protein was indeed a GcIFN-α protein, and the GcIFNα-1 gene was correctly expressed in E. coli.

The ratio of the interferon product expressed by the engineering bacteria DH5α/pBV220-GCIFNα in the protein of the whole bacteria is greater than 65%, as determined by a thin-layer gel scanner.

2. Construction of recombinant plasmids containing control genes and engineering bacteria

1. The GcIFNα-2 gene is used as a template, and a primer pair composed of F2 and R2 is used for PCR amplification to obtain a PCR amplification product.

F25'CGC GAATTC ATGTGCGAATGGCTC (the underlined part is the EcoRI restriction site);

R25'CGC CTCGAG TTATCGTCTGTTGGC 3' (the underlined part is the restriction site of BamHI).

The electropherogram of the PCR amplification product is shown in the swimming lane L3 of FIG. 2 .

Steps 2 to 4 are the same as Step 1 to 2 to 4.

5. Transform the ligation product into Escherichia coli DH5α competent, and pick a single colony for colony PCR and enzyme digestion identification. The primers used in colony PCR were primer pairs consisting of F2 and R2, and the colonies with about 480 bp DNA were positive colonies. The enzymes used for enzyme digestion identification are restriction endonucleases BamHI and EcoRI, and colonies with DNA of about 480 bp are positive colonies.

Colony PCR and enzyme digestion identification of positive colonies are control recombinant bacteria (control bacteria). The host bacteria of the control bacteria was Escherichia coli DH5α, which contained plasmid pBV220.

The control recombinant bacteria were subjected to shaking culture at 30° C., 200 rpm (rotation radius: 13 mm) (to bacterial liquid OD600 = 1), and then induced at 42° C. for 6 hours. Take 1 ml of the culture solution before and after induction in EP tubes, centrifuge at 5000g for 10 minutes, discard the supernatant, add 50uL of PBS buffer and 50uL of 2×Loading Buffer, boil for 10 minutes to lyse the bacteria, and then load 10uL of the sample for SDS-PAGE electrophoresis detection. The results of electrophoresis detection are shown in Figure 3 (lane L1 is before induction, and lane L2 is after induction). A protein band appeared at about 19kD in the bacteria collected after induction, which was consistent with the expected size. The protein in this band was recovered for N-terminal sequencing, and the five amino acids at the N-terminus were Cys-Glu-Trp-Leu-Gly, respectively, indicating that the protein was indeed a GcIFN-α protein, and the GcIFNα-2 gene was correctly expressed in Escherichia coli.

It was determined by a thin-layer gel scanner that the interferon product expressed by the control bacteria accounted for about 40% of the whole bacterial protein, which was far lower than the ratio of the interferon in the recombinant bacteria described in step 1 to the total bacterial protein.

3. Expression of GcIFNα-1 gene and GcIFNα-2 gene

The engineering bacteria DH5α-GcIFN-α/pBV220 and the control bacteria were fermented respectively to produce grass carp interferon-alpha. The specific steps are as follows:

1. Seed preparation

Inoculate the engineering bacteria (or control bacteria) bacteria solution into 20ml LB liquid medium (containing 100μg/ml ampicillin), the inoculation amount is 1% (volume percentage content), 30°C, 200rmp shaking culture for 8-10h; then heat up Induce culture at 42°C for 4 hours, scratch a plate, pick 5-10 single bacteria and culture them in LB liquid medium (containing 100μg/ml ampicillin) in shake flasks (to OD600≈0.5); 250ul 50% (volume percentage) glycerin aqueous solution is the seed, and it is stored at -20°C for later use.

2. Preparation of fermented seed liquid

Inoculate the seeds prepared in step 1 into 200ml 2*YT culture solution, the inoculation amount is 0.05% (volume percentage content), and cultivate at 30°C and 220rpm for 10-12h, and the obtained bacterial solution is the fermented seed solution.

3. Production of grass carp interferon alpha by fermentation

After the fermentation medium is sterilized at high temperature, add 1mL of 100mg/ml ampicillin to each liter of medium, then inoculate the fermentation seed liquid in step 2, the inoculation amount is 5-10% (volume percentage content), 35 ℃ aeration and stirring culture 4 After 4 hours, cool down to 30°C and continue to cultivate for 2-3 hours, then raise the temperature to 42°C and continue to cultivate for 6 hours to obtain a fermentation broth; during the cultivation process, the sugar (glycerol) in the medium is gradually consumed, When the carbon source is exhausted, the thalline no longer grows, and the dissolved oxygen rises (by about 30%), and feed medium is started to flow, and the flow rate is controlled by dissolved oxygen (set DO=30%, when DO is greater than 30%, flow Add the pump to open the feeding medium, and the DO will gradually decrease. When the DO drops below 30%, the feeding pump will be closed), and feed 26-35ml of the medium about every hour; use 3M NaOH aqueous solution and 10% phosphoric acid aqueous solution to maintain the pH during the cultivation process Value 7.2.

4. Purification of grass carp interferon-alpha

Take 5L of the fermentation broth obtained in step 3, centrifuge at 5000g for 10 minutes to collect the bacteria, wash with PBS buffer, and then resuspend with PBS buffer; ultrasonic (Φ10 probe, ultrasonic 7 seconds interval 5 seconds) for 30 minutes to break the wall of the bacteria , centrifuge at 10000g, 4°C for 10 minutes to collect the precipitate; wash the precipitate (inclusion body) with PBS buffer, then dissolve the inclusion body with denaturing buffer, and collect the supernatant by centrifugation at 10000g, 4°C for 10 minutes, which is the denatured protein solution; Slowly add to the refolding buffer, let stand at 4°C for 48 hours, centrifuge at 10,000g at 4°C for 20 minutes to collect the supernatant, which is the solution containing grass carp interferon-α.

Adjust the pH value of the solution containing grass carp interferon-α to 6.5 with acetic acid, and perform cation exchange chromatography. The cation exchange chromatography column adopts HiLoad 26/10 SP Sepharose High Performance (inner diameter 26mm, bed height 100mm, GE LifeScience); the sample is added to the chromatography column at a speed of 1ml/min, washed with buffer A (0.1mol/L Tris Cl, 50mM NaCl, pH 6.5) was washed with 5 column bed volumes at a rate of 5ml/min, and was eluted with washing buffer B (0.1mol/L Tris Cl, 500mM NaCl, pH 6.5) at a rate of 5ml/min 3 column bed volumes, use a UV detector to detect and elute the target protein at a wavelength of 280nm, and collect the eluate with a UV absorption value greater than 200.

The collected eluate is sterilized by filter membrane (0.22um), and the obtained solution is the grass carp interferon alpha solution. The grass carp interferon-α solution obtained by fermenting engineering bacteria was named GcIFNα-1 solution (batch 2008001), and the grass carp interferon-α solution obtained by fermenting control bacteria was named GcIFNα-2 solution (batch 2008001).

GcIFNα-1 solution and GcIFNα-2 solution were detected by SDS-PAGE electrophoresis. The detection results are shown in Figure 4 (lane M is the low molecular weight protein Marker, lane L1 is the purified product of GcIFNα-1 solution, and lane L2 is the purified product of GcIFNα-2 solution). The results showed that there was a protein band around 19kD in GcIFNα-1 solution and GcIFNα-2 solution, which was consistent with the expected result.

4. Comparison of GcIFN-α protein content in GcIFNα-1 solution and GcIFNα-2 solution

The solution to be tested is the GcIFNα-1 solution (batch 2008001) or GcIFNα-2 solution (batch 2008001) obtained in Step 3.

Take solution A and solution B in the BCA protein quantification kit (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., product number: CW0014) at a volume ratio of 50:1, mix them into a working solution, and dilute the BSA standard to 500 mg /ml, 400mg/ml, 300mg/ml, 200mg/ml, 100mg/ml, 50mg/ml, 25mg/ml, take 20μl of each concentration of standard or solution to be tested, add 200μl of working solution to mix, seal with plastic wrap Incubate at 37°C for 30 min, and read the OD ₅₆₅ absorbance value with a microplate reader after returning to room temperature. Draw a standard curve according to the standard substance concentration and absorbance value, and then calculate the protein concentration in the protein sample to be tested according to the standard curve.

The GcIFNα-1 solution (batch 2008001) had a protein concentration of 3 mg/ml and the GcIFNα-2 solution (batch 2008001) had a protein concentration of 1.9 mg/ml. The results show that the expression ability of the GcIFNα-1 gene provided by the present invention is much higher than that of the existing GcIFNα-2 gene.

Example 3, Expression of GcIFNα-1 Gene and GcIFNα-2 Gene

1. Construction of recombinant plasmid and engineering bacteria containing GcIFNα-1 gene

1. The GcIFNα-1 gene is used as a template, and a primer pair composed of F1 and R1 is used for PCR amplification to obtain a PCR amplification product.

F1 5'CGC GAATTC ATGTGCGAATGGCTG 3' (the underlined part is the EcoRI restriction site);

R1 5'CGC CTCGAG TTAACGACGGTTAGC 3' (the underlined part is the restriction site of BamHI).

2. The PCR amplification product was double-digested with restriction endonucleases BamHI and EcoRI to obtain the digested product.

3. The vector pBV220 (prokaryotic expression vector, purchased from Shanghai Jierui Bioengineering Co., Ltd., GV0302) was double-digested with restriction endonucleases BamHI and EcoRI, and the vector skeleton was recovered.

4. Ligate the digestion product of step 2 with the vector backbone of step 3 to obtain a ligation product.

5. The ligation product was transformed into Escherichia coli DH5α (purchased from Beijing Kangwei Century Biotechnology Co., Ltd., catalog number: CW0808) competent, and a single colony was picked for colony PCR and enzyme digestion identification. The primers used in the colony PCR were the primer pair consisting of F1 and R1, and the colonies with about 480 bp DNA were positive colonies. The enzymes used for enzyme digestion identification are restriction endonucleases BamHI and EcoRI, and colonies with DNA of about 480 bp are positive colonies.

Colony PCR and enzyme digestion identification are positive colonies are recombinant bacteria (engineering bacteria). The host bacteria of the recombinant bacteria is Escherichia coli DH5α, which contains the recombinant plasmid GcIFN-α/pBV220. The recombinant plasmid GcIFN-α/pBV220 is a recombinant plasmid obtained by inserting the GcIFNα-1 gene gene shown in Sequence 2 of the sequence table between the BamHI and EcoRI restriction sites of the pBV220 vector.

2. Construction of recombinant plasmids containing control genes and engineering bacteria

1. The GcIFNα-2 gene is used as a template, and a primer pair composed of F2 and R2 is used for PCR amplification to obtain a PCR amplification product.

F2 5'CGC GAATTC ATGTGCGAATGGCTC (the underlined part is the EcoRI restriction site);

R2 5'CGC CTCGAG TTATCGTCTGTTGGC 3' (the underlined part is the restriction site of BamHI).

Steps 2 to 4 are the same as Step 1 to 2 to 4.

5. Transform the ligation product into Escherichia coli DH5α competent, and pick a single colony for colony PCR and enzyme digestion identification. The primers used in colony PCR were primer pairs consisting of F2 and R2, and the colonies with about 480 bp DNA were positive colonies. The enzymes used for enzyme digestion identification are restriction endonucleases BamHI and EcoRI, and colonies with DNA of about 480 bp are positive colonies.

Colony PCR and enzyme digestion identification are positive colony is the recombinant bacteria (control bacteria). The host bacteria of the control bacteria was Escherichia coli DH5α, which contained plasmid pBV220.

3. Expression of GcIFNα-1 gene and GcIFNα-2 gene

Ferment engineering bacteria and control bacteria respectively to produce grass carp interferon alpha, the specific steps are the same as Step 3 in Example 2.

The grass carp interferon-α solution obtained by fermenting engineering bacteria was named GcIFNα-1 solution (batch 2008002), and the grass carp interferon-α solution obtained by fermenting control bacteria was named GcIFNα-2 solution (batch 2008002).

4. Comparison of GcIFN-α protein content in GcIFNα-1 solution and GcIFNα-2 solution

The solution to be tested is the GcIFNα-1 solution (batch 2008002) or GcIFNα-2 solution (batch 2008002) obtained in Step 3. The detection method is the same as Step 4 of Example 2.

The GcIFNα-1 solution (batch 2008002) had a protein concentration of 2.8 mg/ml and the GcIFNα-2 solution (batch 2008002) had a protein concentration of 1.75 mg/ml. The results show that the expression ability of the GcIFNα-1 gene provided by the present invention is much higher than that of the existing GcIFNα-2 gene.

Example 4, Expression of GcIFNα-1 Gene and GcIFNα-2 Gene

1. Construction of recombinant plasmid and engineering bacteria containing GcIFNα-1 gene

Same as Step 1 of Example 3.

Colony PCR and enzyme digestion identification are positive colonies are recombinant bacteria (engineering bacteria). The host bacteria of the recombinant bacteria is Escherichia coli DH5α, which contains the recombinant plasmid GcIFN-α/pBV220. The recombinant plasmid GcIFN-α/pBV220 is a recombinant plasmid obtained by inserting the GcIFNα-1 gene gene shown in Sequence 2 of the sequence table between the BamHI and EcoRI restriction sites of the pBV220 vector.

2. Construction of recombinant plasmids containing control genes and engineering bacteria

Same as Step 2 of Example 3.

Colony PCR and enzyme digestion identification are positive colony is the recombinant bacteria (control bacteria). The host bacteria of the control bacteria was Escherichia coli DH5α, which contained plasmid pBV220.

3. Expression of GcIFNα-1 gene and GcIFNα-2 gene

Ferment engineering bacteria and control bacteria respectively to produce grass carp interferon alpha, the specific steps are the same as Step 3 in Example 2.

The grass carp interferon-α solution obtained by fermenting engineering bacteria was named GcIFNα-1 solution (batch 2008003), and the grass carp interferon-α solution obtained by fermenting control bacteria was named GcIFNα-2 solution (batch 2008003).

4. Comparison of GcIFN-α protein content in GcIFNα-1 solution and GcIFNα-2 solution

The solution to be tested is the GcIFNα-1 solution (batch 2008003) or GcIFNα-2 solution (batch 2008003) obtained in Step 3. The detection method is the same as Step 4 of Example 2.

The GcIFNα-1 solution (batch 2008003) had a protein concentration of 3.5 mg/ml and the GcIFNα-2 solution (batch 2008003) had a protein concentration of 2.25 mg/ml. The results show that the expression ability of the GcIFNα-1 gene provided by the present invention is much higher than that of the existing GcIFNα-2 gene.

Embodiment 5, the biological activity assay of grass carp interferon alpha

The three batches of GcIFNα-1 solutions prepared in Examples 2 to 4 were divided into two groups: the first group: stored at 4°C-8°C, stored at 6 months, 12 months, 18 months and Samples were taken at 24 months to detect interferon activity; the second group: stored at 25°C, samples were taken at 1 month, 3 months, and 6 months to detect interferon activity; and then the biological activity of alpha interferon was measured. Determination method is with reference to 2005 " Pharmacopoeia of the People's Republic of China " three appendices X C cell activity inhibition method, uses grass carp kidney cell line (GIK) (China Type Culture Collection Center, numbering GDC081)-infectious hematopoietic organ necrosis virus (Infectious Hematopoietic Necrosis Virus) Necrosis Virus, IHNV) system, using CPE (cytopathic effect) inhibition based inhibition micro-assay, the highest dilution per milliliter of interferon test product can still protect half of the cells (50%) from virus attack The reciprocal of the dilution is the interferon unit.

The specific method for detecting interferon activity is as follows:

1. Cell preparation

Take well-grown GIK cells, prepare cell suspension with DMEM containing 10% FBS after digestion; count the cells, adjust the cell concentration to 5× ¹⁰⁵ /mL suspension, and then add it to a 96-well cell culture plate (100 μl/well), cultured at 37° C., 5% CO ₂ for 8-10 hours to make monolayer cells.

2. Add interferon to treat the cells

The GcIFNα-1 solution was pre-diluted with DMEM containing 10% FBS to a final concentration of 0.001 mg/ml as the mother solution, and then the mother solution was diluted 4 times and diluted 6 times to obtain various dilutions.

3. Aspirate the culture solution in each well of the cell culture plate in step 1; add 100 μl/well of cell culture solution to 6 wells (3 positive control wells and 3 negative control wells) of the cell culture plate, and the remaining wells are respectively Add the dilution solution of step 2 (100 μl/well, three replicates for each gradient); incubate at 37° C., 5% CO ₂ for 12-15 h.

4. Virus infection

Take the IHNV virus and dilute it with serum-free DMEM to a virus dilution solution with a final concentration of 1000TCID ₅₀ /ml; add 100 μl virus dilution solution to each well of the positive control well, add 100 μl serum-free DMEM culture solution to each well of the negative control well, and add 100 μl serum-free DMEM culture solution to each well of the remaining wells. Add 100 μl of virus dilution to the wells; incubate for 24 hours.

5. Crystal violet staining

The cell culture medium was discarded, and 100 μl of crystal violet staining solution was added to each well, and left at room temperature for 30 minutes.

6. Decolorization

Discard the dye, and rinse the uncolored dye with tap water or double distilled water, then add 100 μl of decolorization solution to each well, and let stand at room temperature for 10 min.

7. Use a microplate reader to measure and record the OD ₅₇₀ value.

8. Data processing

The content of interferon capable of inhibiting 50% cytopathy was defined as an activity unit, and the interferon titer was calculated by the Reed-Muench method, that is, the dilution factor capable of inhibiting 50% cytopathy. The results are shown in Table 1.

Table 1 Calculation of interferon titer by Reed-Muench method

X: OD ₅₇₀ average value of negative control (no virus control);

Y: Mean _OD570 of the positive control (virus control).

Calculate each value according to the above table, and finally calculate the interferon titer according to the following formula according to G item (assuming that the calculated value of G4 item in the above calculation is greater than 0.5, and the calculated value of G5 item is less than 0.5):

Interferon titer to be detected (U/0.001mg) = pre-dilution multiple × 4 ^{(4+(G4-0.5)/(G4-G5))} .

The test results of interferon titer are shown in Table 2.

Table 2 Interferon titer test results

The results show that: the solution prepared by the method of the present invention is stored at 4°C-8°C for 24 months, and stored at 25°C for 3 months, and the biological activity has no obvious change.

Embodiment 6, the disease resistance test of grass carp alpha interferon (GcIFN-alpha protein)

1. Resistance test of grass carp α-interferon (GcIFN-α protein) to grass carp hemorrhagic disease

Grass carp has an average body length of about 340mm, an average weight of about 750g, and a breeding water temperature of 23-27°C. Divide into 5 groups, each group of 100 tails, carry out the following treatments respectively (the volume of each group injecting grass carp interferon alpha is all the same, and the volume of every group injecting grass carp hemorrhagic disease virus is all identical):

The first group: intraperitoneal injection of GcIFNα-1 solution (batch 2008001) prepared in Example 2, 10000U/tail; 24 hours later, dorsal fin subcutaneous injection of grass carp hemorrhagic disease virus (GCHV), 500TCID ₅₀ /tail; raise 2 weeks, day by day observe;

The second group: subcutaneous injection of grass carp hemorrhagic disease virus (GCHV) in the dorsal fin, 100TCID ₅₀ /tail; immediately intraperitoneal injection of the GcIFNα-1 solution (batch 2008001) prepared in Example 2, 10000U/tail; raise for 2 weeks, observe daily;

The third group: dorsal fin subcutaneous injection of grass carp hemorrhagic disease virus (GCHV), 100TCID ₅₀ /tail; 24 hours later, intraperitoneal injection of the GcIFNα-1 solution (batch 2008001) prepared in Example 2, 10000U/tail; raising for 2 weeks, daily observe;

The fourth group: Grass carp hemorrhagic disease virus (GCHV) was injected subcutaneously into the dorsal fin, 100 TCID ₅₀ /tail; fed for 2 weeks, observed daily;

The fifth group: the dorsal fin was subcutaneously injected with PBS buffer; it was fed for 2 weeks and observed daily.

The results are shown in Table 3, GcIFNα-1 solution injection has obvious antiviral effect, the survival rate of the administration group is 64% higher than that of the control group (the fourth group), and the immune protection rate reaches 72.3%.

Table 3 The results of the resistance test of grass carp hemorrhagic disease by injection of grass carp interferon

fish (tail) Number of deaths (tail) Survival number (tail) Survival rate (%) Protection (%) First group 100 25 75 75 75 Second Group 100 28 72 72 72 The third group 100 30 70 70 70 Fourth group 100 100 0 0 0 fifth group 100 0 100 100 --

2. Grass carp α-interferon (GcIFN-α protein) test on improving the resistance of prawns

Chinese prawns are divided into six groups, with 1000 tails in each group, three groups are experimental groups, and three groups are control groups, which are respectively processed as follows:

Experimental group: the GcIFNα-1 solution (batch 2008001) prepared in Example 2 was administered at a dose of 3 million active units/1000 tails, and the treatment method was soaking for 3 hours;

Control group: without any treatment.

After one month of normal feeding, the natural mortality rate was counted, and the immune protection rate (RPS) was calculated.

The results are shown in Table 4. Compared with the control group, the survival rate of the experimental group was significantly improved, and the immune protection rate reached 34.6%, indicating that the GcIFNα-1 solution can better improve the immune and disease resistance of Chinese prawns.

Table 4 The test results of soaking grass carp interferon on improving the resistance of Chinese prawns

Claims (10)

1. DNA shown in sequence 2 of the sequence listing. 2. A recombinant vector, recombinant bacterium, expression cassette or transgenic cell line containing the DNA shown in Sequence 2 of the sequence listing. 3. The recombinant vector according to claim 2, characterized in that: the recombinant vector is the recombinant plasmid obtained by inserting the DNA shown in sequence 2 of the sequence listing into the multiple cloning site of pBV220; the recombinant vector is preferably the The recombinant plasmid obtained by inserting the DNA shown in Sequence 2 of the sequence listing between the BamHI and EcoRI sites of pBV220. 4. The recombinant bacterium according to claim 2, characterized in that: the recombinant bacterium is the recombinant bacterium obtained by introducing the DNA shown in sequence 2 of the sequence listing into Escherichia coli DH5α; the recombinant bacterium is preferably the recombinant bacterium according to claim 3 The recombinant bacterium obtained by introducing the recombinant vector into Escherichia coli DH5α; the most preferred recombinant bacterium is Escherichia coli (Escherichia coli) DH5α/pBV220-GCIFNα, and its preservation number is CGMCC No.4111. 5. A method for preparing interferon-alpha, comprising: inducing the recombinant bacterium according to claim 2 or 4 at 40-43°C for 4-8 hours, preferably at 42°C for 6 hours, then crushing the bacterium, collecting inclusion bodies, Washing the inclusion body, dissolving the inclusion body and renaturation to obtain a solution containing alpha interferon. 6. The method according to claim 5, characterized in that: the method further comprises the step of purifying the protein from the solution containing interferon-alpha; The method of protein purification is as follows: adjust the pH value of the solution containing alpha interferon to 5.5-9.5 with acetic acid, perform cation exchange chromatography with HiLoad 26/10 SP Sepharose High Performance, and wash 5 samples with washing buffer A column volume, then elute 1-10 column bed volumes with washing buffer B, collect the eluate with an ultraviolet absorption value greater than 200 under 280nm light, and the resulting solution is alpha interferon solution; the washing buffer A pH is 5.5-9.5, composed of Tris Cl, NaCl and water, the concentration of Tris Cl is 0.1mol/L, and the concentration of NaCl is 0-150mM; the pH of the washing buffer B is 5.5-9.5, composed of Tris Cl , NaCl and water, the concentration of Tris·Cl is 0.1mol/L, and the concentration of NaCl is 150-2000mM. 7. The alpha interferon solution prepared by the method according to claim 5 or 6. 8. The application of the DNA according to claim 1 or the recombinant vector, recombinant bacterium, expression cassette or transgenic cell line according to any one of claims 2 to 4 in the preparation of alpha interferon. 9. The application of the recombinant vector, recombinant bacteria, expression cassette or transgenic cell line described in claim 1 or any one of claims 2 to 4 in the preparation of virus inhibitors; the virus is infectious hematopoietic organ necrosis virus and/or grass carp hemorrhagic disease virus; the infectious hematopoietic organ necrosis virus is specifically WRAC strain. 10. the application of the recombinant vector, recombinant bacterium, expression cassette or transgenic cell line described in claim 1 or any one of claims 2 to 4 in the preparation of the following (a) and/or (b); (a) preparations for the treatment and/or prevention of fish viral diseases; (b) preparations for improving the survival rate of shrimp; The fish viral disease is specifically grass carp hemorrhagic disease; the shrimp is specifically Chinese prawn.

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