CN114748617A - Subunit vaccine of micropterus salmoides iridovirus as well as preparation method and application thereof - Google Patents
Subunit vaccine of micropterus salmoides iridovirus as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a vaccine of iridovirus of micropterus salmoides, a preparation method and application thereof. The vaccine of the micropterus salmoides iridovirus is protein encoded by a nucleotide sequence shown in SEQ ID No. 1. The iridovirus vaccine is obtained by constructing plasmid pET21a-LMBV, transforming Escherichia coli BL21(DE3), carrying out induction expression, breaking cells, recovering and purifying recombinant protein, and obtaining the iridovirus vaccine. The recombinant protein rMCP can be mixed with an adjuvant, and the obtained vaccine mixed solution has the effect of immunoprotection against iridovirus LMBV of micropterus salmoides. The vaccine of the iridovirus of micropterus salmoides has stable immune effect, higher relative protection rate and high-efficiency protectiveness, and can be applied to the culture process of micropterus salmoides to protect fry and adult fish against micropterus salmoides iridovirus infection.
Description
The technical field is as follows:
the invention belongs to the fields of molecular biology and immunology, and particularly relates to an immunoprotection iridovirus recombinant protein subunit vaccine for micropterus salmoides, and a preparation method and application thereof.
The background art comprises the following steps:
the micropterus salmoides are freshwater fishes with high economic value, the culture scale of the micropterus salmoides is enlarged in recent years, the annual output of China already exceeds 50 million tons, and the output value exceeds 100 billion yuan. However, as the culture environment deteriorates day by day, various diseases, particularly viral diseases, frequently outbreak, and great economic loss is caused. Large Mouth Bass (LMBV) is an important pathogenic pathogen of the largemouth bass, which causes the death rate of the largemouth bass to be high. The virus is easy to infect micropterus salmoides and has wide prevalence, so that the survival rate of micropterus salmoides in the seedling stage is low, the iridovirus disease is easy to appear in the adult fish culture at a high temperature stage, and serious economic loss is caused. Therefore, how to prevent and treat iridovirus disease becomes an important problem which needs to be solved urgently in the current micropterus salmoides breeding industry.
It is well known that vaccines, as a replacement for chemicals and antibiotics, are the best choice for the treatment of diseases, in particular viral diseases. Vaccination is an effective means of preventing and treating viruses, and includes inactivated vaccines, DNA nucleic acid vaccines, attenuated vaccines, recombinant protein subunit vaccines, and the like. Aiming at the iridovirus LMBV of the micropterus salmoides, complete virus inactivated vaccines and DNA nucleic acid vaccines have been reported in research. The preparation of the whole virus inactivated vaccine requires cell culture and virus amplification, and has higher requirements on cell culture technology, equipment use, culture medium, materials such as serum and the like; the immune effect is unstable due to the possible loss of virus antigen and the damage of antigen structure in the process of preparing the whole virus inactivated vaccine. Although the DNA nucleic acid vaccine is simple to prepare and can generate a durable immune response, the DNA nucleic acid can induce generation of an autoimmune response and continuously express a foreign antigen, and can cause the body to generate immune tolerance to the antigen. The recombinant protein subunit vaccine absolutely does not contain live toxin components, has no toxin dispersing possibility in the production and use processes, eliminates the possibility of pollution, has strong immune specificity and better repeatability, can be produced in a large scale through fermentation, has lower cost, and is a safe vaccine worthy of being actively popularized and used. With the development of molecular biology technology, the research on the structure and function of virus gene is deep, and the development of genetic engineering recombinant protein subunit vaccine is mature day by day, is the main direction of development at home and abroad, and has been widely applied to the production of various human and animal vaccines. At present, no recombinant subunit vaccine for the largemouth bass iridovirus LMBV is reported. Therefore, the recombinant subunit vaccine product of the virus can be produced and applied in a large scale under the condition of low cost, and the propagation of the virus can be quickly, safely and effectively prevented and controlled, thereby ensuring the healthy development of the largemouth black bass breeding industry.
The invention content is as follows:
the invention aims to provide a subunit vaccine of micropterus salmoides iridovirus, which has stable vaccine and high protection rate.
The subunit vaccine of micropterus salmoides iridovirus is characterized in that the subunit vaccine of micropterus salmoides iridovirus is protein encoded by a nucleotide sequence shown in SEQ ID NO. 1.
Further preferably, the protein coded by the nucleotide sequence shown in SEQ ID NO.1 and adjuvant MontanideTMISA 763A VG is mixed to form a subunit vaccine of the micropterus salmoides iridovirus.
The second purpose of the invention is to provide a preparation method of the subunit vaccine of the micropterus salmoides iridovirus, which is characterized in that the nucleotide sequence shown in SEQ ID NO.1 is transferred into an expression vector, the expression vector is transferred into host bacteria to express the protein coded by the nucleotide sequence shown in SEQ ID NO.1, and then the protein is purified and collected to obtain the subunit vaccine of the micropterus salmoides iridovirus.
Preferably, the expression vector is a plasmid pET-21 a.
Preferably, the host bacterium is Escherichia coli BL21(DE 3).
Further preferably, the subunit vaccine of micropterus salmoides iridovirus is prepared by the following method:
1) Construction of plasmid pET-21 a-LMBV: the plasmid pET-21a is cut by BamHI/XhoI enzyme, and a 5.4kb fragment is recovered; using iris micropterus salmoides iridovirus LMBV genome DNA as a template, using primers LMBV-F and LMBV-R to perform PCR amplification, using BamHI/XhoI to perform enzyme digestion after purifying a product, recovering a 1.4kb fragment, connecting the fragment with the 5.4kb fragment, culturing the Escherichia coli DH5 alpha transformed by a connecting solution on an LB solid culture medium containing ampicillin, and screening a transformant to extract a plasmid, namely, the plasmid pET21 a-LMBV;
the primer LMBV-F is 5'-TAAGGATCCATGTCTTCTGTTACGGGTTC-3', and the primer LMBV-R is 5'-AATCTCGAGTTACAGGATGGGGAAACCC-3';
2) induced expression and purification of vaccine proteins: transforming the plasmid pET-21a-LMBV into escherichia coli BL21(DE3) to obtain a transformant BL21/pET21 a-LMBV; BL21/pET21a-LMBV was cultured overnight in LB liquid medium containing ampicillin; adding the overnight culture solution into a fresh LB liquid culture medium, culturing at 37 ℃ until OD600 is 0.6, adding IPTG with the final concentration of 0.8mM, continuously culturing at 16 ℃ for 6h, then adding PBS buffer solution into the centrifuged bacterial solution, crushing the bacteria by adopting ultrasonic waves for 1-2 h, centrifuging the crushed bacteria solution, and recovering the supernatant; and (3) purifying the supernatant by adopting a protein precipitation and Amylose resin combined purification method to obtain protein rMCP coded by the nucleotide sequence shown in SEQ ID No.1, namely the subunit vaccine of the micropterus salmoides iridovirus.
Further preferably, the step 2) of centrifuging the thallus crushing liquid, recovering the supernatant, and purifying the supernatant by protein precipitation and ammonium sulfate resin combination, comprises the steps of firstly precipitating the hybrid protein by using an ammonium sulfate solution with the mass fraction of 30%, then precipitating the target protein by using an ammonium sulfate solution with the mass fraction of 40%, combining the precipitated target protein with an ammonium sulfate column for 2h, eluting the hybrid protein by using a column passing buffer solution with 20 column volumes, then eluting the target protein by using a 10mM maltose solution, and collecting the protein eluted by the primary and secondary 10mM maltose solutions, so as to obtain the protein encoded by the nucleotide sequence of SEQ ID No.1, namely the subunit vaccine of the largemouth black bass iridovirus.
The third purpose of the invention is to provide the application of the protein coded by the nucleotide sequence shown in SEQ ID NO.1 in the preparation of the vaccine of the micropterus salmoides iridovirus.
The iridovirus vaccine is a vaccine of micropterus salmoides iridovirus.
The immunoprotective recombinant protein rMCP (protein coded by a nucleotide sequence shown in SEQ ID NO. 1) or the recombinant protein rMCP is mixed with an adjuvant to obtain a vaccine mixed solution, which has the effect of immunoprotection on largemouth bass iridovirus LMBV. The vaccine is injected into the body of the largemouth black bass, after 28 days, LMBV virus is injected, the death rate of the largemouth black bass is observed and counted, and the relative protection rate of the vaccine is calculated. The result shows that the relative protection rate of rMCP is 83.3 percent, and rMCP protein and adjuvant Montanide are added TMThe relative protection rate of ISA 763A VG mixed and compounded can reach 100 percent.
Each milliliter of vaccine or vaccine mixed solution contains 100 micrograms of immunoprotective subunit vaccine protein (immunoprotective recombinant protein rMCP); the injection of the subunit vaccine or the vaccine mixed solution in the micropterus salmoides body is 100 mul.
The invention has the following advantages:
1. the BL21/pET-LMBV recombinant strain can be subjected to large-scale fermentation culture, the biomass of the fermented strain can reach the wet weight of 20g/L, and the rMCP recombinant protein with the purity of more than 200mg/L and more than 90% is obtained through cell crushing and purification. The vaccine is simple and convenient to produce, low in cost, safe in product, non-toxic, pollution-free, diffusion-free and convenient to store.
2. The subunit vaccine is stable and has high protection rate. The immune protection efficiency of the recombinant subunit vaccine on micropterus salmoides reaches 83.3 percent.
3. Commercial adjuvant MontanideTMISA 763A VG can improve immune protection effect. Montanide is added when the vaccine is appliedTMThe ISA 763A VG adjuvant can improve the immunoprotection efficiency of micropterus salmoides by 100 percent.
Description of the drawings:
FIG. 1 shows prokaryotic expression (A) and purification (B) of vaccine protein rMCP. The arrow indicates rMCP;
FIG. 2 is a statistical population of largemouth bass survival after challenge;
FIG. 3 is statistics of relative protection rates of iridovirus subunit vaccine proteins for micropterus salmoides.
Detailed Description
The present invention will be further described with reference to the following examples. The examples are intended to illustrate the invention, but not to limit it in any way.
The following methods were used for the conventional experiments in the examples of the present invention:
1. plasmid extraction, DNA (PCR) product purification, DNA fragment recovery from gel, and bacterial genomic DNA extraction all use the corresponding kit of Axygen thought Biotechnology (Hangzhou) Inc.
2. Transformation of plasmids, DNA ligation into E.coli was carried out by the Hanahan method (Sambrook and Russell: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press 2001);
all restriction enzymes and ligases were purchased from "Takara Bio-engineering (Dalian) Ltd".
Example 1
The immune protective subunit vaccine is shown by MCP base sequence in a sequence table SEQ ID No. 1.
The preparation method of the immunoprotective subunit vaccine comprises the following steps:
1) construction of plasmid pET21a-LMBV
The plasmid pET-21a (Novagen, USA) was digested with BamHI/XhoI, and a 5.4kb fragment was recovered. The method is characterized in that the iridovirus LMBV DNA of the micropterus salmoides which is automatically separated and stored is taken as a template, and the primer LMBV-F: 5'-TAAGGATCCATG TCTTCTGTTACGGGTTC-3', primer LMBV-R: 5'-AATCTCGAGTTACAGGATGGGGAAAC CC-3' PCR was performed as follows: the template DNA was pre-denatured at 95 ℃ for 180s, and then subjected to extension reaction at 72 ℃ for 10min after 35 cycles of 94 ℃ for 45s, 55 ℃ for 45s, and 72 ℃ for 90 s. Purifying PCR product, digesting with BamHI/XhoI enzyme, recovering 1.4kb fragment, connecting with the 5.4kb fragment, transforming Escherichia coli DH5 alpha after connection, culturing for 12-24 hours on LB solid culture medium containing ampicillin (100 microgram/ml), screening transformant, culturing, extracting plasmid, namely plasmid pET21 a-LMBV; the LB solid medium comprises the following components: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride and 10g/L of agar.
2) Inducible expression and purification of vaccine protein rMCP
The plasmid pET21a-LMBV of step 1) above was transformed into Escherichia coli BL21(DE3) (purchased from Tiangen Biochemical technology Co., Ltd., Beijing) by a conventional method, cultured on LB solid medium containing ampicillin (100. mu.g/ml) for 12 to 24 hours, and ampicillin-resistant transformants were selected, and one transformant was selected and named BL21/pET21 a-LMBV. BL21/pET21a-LMBV was inoculated in LB liquid medium containing ampicillin (100. mu.g/ml) for overnight culture; adding 1ml of overnight culture solution into 100ml of fresh LB liquid culture medium containing ampicillin (100 mu g/ml), shaking and culturing at the rotation speed of 200rpm at 37 ℃ until OD600 is 0.6, adding isoproyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.8mM, continuing shaking and culturing at the rotation speed of 180rpm for 6h at 16 ℃, then centrifuging (5000g, 4 ℃, 10min), collecting thalli, re-suspending with PBS buffer solution, carrying out ultrasonic disruption for 1-2h, re-centrifuging (5000g, 4 ℃, 10min), and collecting supernatant. And (3) precipitating the mixed protein by using a low-concentration ammonium sulfate (mass fraction of 30%) solution in the supernatant, collecting the supernatant, precipitating the target protein by using an ammonium sulfate solution with the mass fraction of 40% in the supernatant of the thallus crushing liquid, and collecting the precipitate. After binding the precipitated target protein to the Amylose column for 2 hours, the heteroprotein was eluted with 20 column volumes of column buffer, followed by elution of the target protein with 10mM maltose solution. Protein purification was checked by SDS-PAGE and the results are shown in FIG. 1. Collecting the protein eluted by the primary and secondary 10mM maltose solution to obtain the immunoprotective subunit vaccine protein coded by the base sequence in the sequence table SEQ ID No.1, which is named subunit vaccine protein rMCP.
Example 2
Immune application of recombinant subunit vaccine
Step 1) preparation of mixed solution of subunit vaccine protein and adjuvant.
Subunit vaccine protein mixture: the subunit vaccine protein rMCP (200mg/L, purity more than 90%) purified in example 1 is adjusted to a vaccine protein solution containing 100mg of base sequence code in the sequence table SEQ ID No1 (namely, containing 100mg of subunit vaccine protein rMCP/L) in each liter of buffer solution by PBS buffer solution, so as to obtain a subunit vaccine protein mixed solution.
Subunit vaccine protein and adjuvant mixture: the subunit vaccine protein rMCP (200mg/L, purity more than 90%) purified in example 1 and adjuvant MontanideTMISA 763A VG with a volume ratio of 1: 1, and mixing uniformly to ensure that each liter of mixed solution contains 100mg of vaccine protein (subunit vaccine protein rMCP) coded by a base sequence in a sequence table SEQ ID No.1 to obtain subunit vaccine protein and adjuvant mixed solution.
And step 2) immune application of the recombinant subunit vaccine.
180 micropterus salmoides (each weighing about 5g) were randomly divided into 3 groups of 60 pieces. These 3 groups were designated as a (mcp subunit vaccine group), B (mcp +763A adjuvant group), and C (PBS control) group, respectively. And (2) injecting 100 mul of the solution containing the vaccine protein (100mg/L) in the step 1) into the abdominal cavity of each fish in the group A respectively [ namely the subunit vaccine protein mixed solution in the step 1) ], and injecting 100 mul of the mixed solution containing the vaccine protein (100mg/L) and the adjuvant into the abdominal cavity of each fish in the group B respectively [ namely the subunit vaccine protein and adjuvant mixed solution in the step 1) ]. In group C, 100. mu.l PBS buffer was intraperitoneally injected into each fish. A. Group B was the test group and group C was the control group.
And 3) detecting the immunoprotection effect of the recombinant subunit vaccine against iridovirus LMBV of micropterus salmoides.
28 days after the vaccine immunization of the largemouth black bass in the step 2) is carried out, 100 mu l of largemouth black bass iridovirus LMBV (TCID) is respectively injected into the abdominal cavity of each fish of A, B, C three groups50＝1.25×102.8) (ii) a The mortality of the three groups of micropterus salmoides was observed A, B, C within 24 days, and the vaccine protection rate was counted (see fig. 2). The result shows that the subunit vaccine produces good protection effect on LMBV virus challenge after the largemouth black bass is immunized, when the virus challenge is carried out for 24 days, the cumulative mortality rate of the group C (PBS control group) is 60%, the cumulative mortality rate of the group A (rMCP, subunit vaccine group) is 10%, and the group B (rMCP +763A, subunit vaccine and adjuvant group) does not die. By the formula RPS ═ 1- (mortality in the immune group/mortality in the control group)]The relative protection rate of the subunit vaccine protein rMCP is 83.3 percent and the relative protection rate of the rMCP after being matched with the adjuvant reaches 100 percent (shown in a figure 3) by calculating multiplied by 100 percent.
Sequence listing
<110> south China university of agriculture
Lingnan modern agriculture science and technology Guangdong province laboratory
<120> a subunit vaccine of largemouth bass iridovirus, and a preparation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1392
<212> DNA
<213> Iris virus of Large bass (Large moth bass virus)
<400> 1
atgtcttctg ttacgggttc tggcatcact agcgggttca ttgatctcgc cacttatgac 60
agccttgata aagcgctgta cggtggaaaa gatgcaacta cttatttcgt caaagagcat 120
tatcccgtgg gttgctttac caatctgcct acggctgcca caaaaacttc tggtacgcct 180
gctttcgggc agcacttttc cgtcggagtg cccagctcgg gcgactatgt gctcaactct 240
tggctggtcc tcaagacccc ccagattaag ctgctggcgg ccaaccagtt taacaatgac 300
ggtaccatca gatggaccaa aaatctcatg cacaacgtcg tggagcacgc cgcactctcg 360
ttcaacgaga ttcaggccca gcagttcaac actgctttcc tggacgcctg gaacgagtac 420
accatgcccg aggccaagcg catcggctac tacaacatga ttggcaacac gagcgatctc 480
gtcaatcccg cccccgccac cggtcaagca ggagctaggg tcctgcccgc caaaaacctg 540
gtccttcctc tcccattctt tttcggcaga gacagcgggc tggccctgcc tacagtcacc 600
ctgccataca acgaaattag aatcaccatc agcctgagat ccattcagga tctcctgatt 660
cttcagcaca agacgaccgg agaagtcaac cccatcgtgg ccacagatct ggaaggaggt 720
ctcccagaca cggtagaggc ccacgtctac atgactgtgg gtctggtgac tgccgccgag 780
cgtcaggcta tgagcagctc agtcagcgac atggtggtgg agcagatgca gatggctccg 840
gtccacatgg tcaaccccaa gaacgccacc gtcttccacg cagacctgcg cttttcccac 900
gccgtcaaag cgctcatgtt tatggtgcaa aacgtcactc acaagtctgt gggttccaac 960
tacacttgcg tcactcctgt tgttggagcg ggtaacaccg tcctggagcc tgccctggcc 1020
gtcgatccgg tcaacagcgc cagtctggtg tacgaaaaca ctaccaggct tccagagatg 1080
agcgtagagt actattccct ggtgcagccc tggtactacg cacccgccat tcccatcagc 1140
actggtcacc acctctactc gtacgccctg agcctcaacg atcctcaccc ttcagggtct 1200
accaatttcg gtcgcctgac caacgcaagc atcaacgtgt ctctgtctgc cgaggccgga 1260
actgccgccg gaggaggagg ggcagacaac tctggctaca aaaaccctca gaaatacgcc 1320
ctggtggtca tggccatcaa ccacaacatt atccgcatca tgaacggttc catgggtttc 1380
cccatcctgt aa 1392
Claims (10)
1. The subunit vaccine of the micropterus salmoides iridovirus is characterized in that the subunit vaccine of the micropterus salmoides iridovirus is protein encoded by a nucleotide sequence shown in SEQ ID NO. 1.
2. The subunit vaccine of claim 1, wherein the protein encoded by the nucleotide sequence of SEQ ID No.1 is mixed with an adjuvant to form the subunit vaccine of iridovirus of micropterus salmoides.
3. The subunit vaccine of claim 2 wherein the adjuvant is MontanideTMISA 763A VG adjuvant.
4. The subunit vaccine of claim 3, wherein the adjuvant and vaccine protein rMCP are mixed in a volume ratio of 1: 1.
5. a preparation method of a subunit vaccine of a micropterus salmoides iridovirus is characterized in that a nucleotide sequence shown in SEQ ID NO.1 is transferred into an expression vector, the expression vector is transferred into host bacteria to express protein coded by the nucleotide sequence shown in SEQ ID NO.1, and then the protein is purified and collected to obtain the subunit vaccine of the micropterus salmoides iridovirus.
6. The method of claim 5, wherein the expression vector is plasmid pET-32 a.
7. The process according to claim 5, wherein the host bacterium is Escherichia coli BL21(DE 3).
8. The method of claim 5, wherein the said sub-unit vaccine of iridovirus of micropterus salmoides is prepared by the following method:
1) construction of plasmid pET-21 a-LMBV: the plasmid pET-21a is cut by BamH I and Xho I enzyme, and a fragment of about 5.4kb is recovered; using iridovirus LMBV genome DNA of largemouth bass as a template, using primers LMBV-F and LMBV-R to perform PCR amplification, using BamHI/XhoI to perform enzyme digestion after product purification, recovering a fragment of about 1.4kb, connecting the fragment with the fragment of 5.4kb, transforming escherichia coli DH5 alpha by using a connecting solution, culturing on an LB solid culture medium containing ampicillin, and screening transformants to extract plasmids, namely plasmid pET21 a-LMBV;
the primer LMBV-F is 5'-TAAGGATCCATGTCTTCTGTTACGGGTTC-3', and the primer LMBV-R is 5'-AATCTCGAGTTACAGGATGGGGAAACCC-3';
2) induced expression and purification of vaccine proteins: transforming the plasmid pET-21a-LMBV into escherichia coli BL21(DE3) to obtain transformant BL21/pET21 a-LMBV; BL21/pET21a-LMBV is cultured in LB liquid medium containing ampicillin overnight; adding the culture solution after overnight into a fresh LB liquid culture medium, culturing at 37 ℃ until OD600 is 0.6, adding IPTG with the final concentration of 0.8mM, continuously culturing at 16 ℃ for 6h, then adding PBS buffer solution into the centrifuged bacterial solution, crushing the bacteria by adopting ultrasonic waves for 1-2 h, centrifuging the crushed bacterial solution, and recovering the supernatant; and (3) purifying the supernatant by adopting protein precipitation and an Amylose resin combined purification method to obtain protein rMCP coded by the nucleotide sequence shown in SEQ ID No.1, namely the subunit vaccine of the micropterus salmoides iridovirus.
9. The preparation method of claim 8, wherein the step 2) of centrifuging the cell disruption solution and recovering the supernatant is carried out, and the protein precipitation and the Amylose resin purification method of the supernatant are carried out by firstly precipitating the impure proteins in the cell disruption solution by using an ammonium sulfate solution with the mass fraction of 30%, then precipitating the target proteins in the supernatant of the cell disruption solution by using an ammonium sulfate solution with the mass fraction of 40%, binding the precipitated target proteins with an Amylose column for 2h, eluting the impure proteins by using a column buffer solution with 20 column volumes, then eluting the target proteins by using a 10mM maltose solution, and collecting the proteins eluted by the primary and secondary 10mM maltose solutions to obtain the protein encoded by the nucleotide sequence of SEQ ID No.1, namely the subunit vaccine of the black bass iridovirus.
The application of the protein coded by the nucleotide sequence shown in SEQ ID NO.1 in preparing the vaccine of the micropterus salmoides rainbow virus.
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