CN116640787A - Iridovirus DNA vaccine - Google Patents
Iridovirus DNA vaccine Download PDFInfo
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Abstract
The present invention provides iridovirus DNA vaccines. The invention is based on a lysosome related membrane protein LAMP1 gene, and uses a main capsid protein MCP gene of iridovirus particles to modify the LAMP1 gene to obtain a nucleotide sequence as shown in SEQ ID:1 or SEQ ID:2, the two DNA molecules can be transcribed and express related antigens with high efficiency, have immunogenicity, can induce specific humoral immunity and/or cellular immune response, can be applied to the preparation of iridovirus DNA vaccine, and the prepared vaccine has good control effect on iridovirus, thereby realizing effective protection on organisms infected with iridovirus.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to an iridovirus DNA vaccine.
Background
With the increase of aquaculture varieties and the expansion of aquaculture scale, the aquaculture environment is worsened, various infectious diseases are continuously outbreaked and popular, and huge economic loss is caused for aquaculture, so that the aquaculture method has become a main bottleneck for restricting the healthy and sustainable development of aquaculture industry.
Iridovirus (Iridovirus) is a type of regular icosahedral double-stranded DNA virus capable of forming inclusion bodies in the cytoplasm of a host, has a diameter of 125-380 nm and a genome size of 140-303 kb, and is named Iridovirus because its virions are regularly arranged in periodically spaced abnormalities in a virus-infected host or in a purified concentrated viral precipitate, form lattice planes and overlap with each other, and appear blue or purple iridescence when irradiated with oblique light. Iridoviruses are divided into 2 subfamilies, 5 subfamilies, iridoviruses alpha subfamilies, including frog virus, megalopsis cell virus, lymphocyst virus, and iridoviruses beta subfamilies, including iridoviruses and green iridoviruses, which infect invertebrates. Iridovirus is one of the most serious viral pathogens of current seawater and freshwater farmed fish, not only seriously threatens the diversity and ecological safety of the fish, but also causes great economic loss for the aquaculture industry. Therefore, how to prevent and treat iridovirus has become an urgent problem to be solved in the aquaculture industry today.
Vaccines are an effective means of controlling viral spread as a replacement for chemicals and antibiotics. The vaccine mainly comprises three major types of inactivated vaccine, DNA vaccine and attenuated vaccine. DNA vaccines have received considerable attention for their many advantages over traditional antigen vaccines. The DNA vaccine refers to a recombinant DNA plasmid obtained by connecting a fragment of a coding target gene to a plasmid vector, wherein the recombinant DNA plasmid can be expressed in a host body to generate antigen protein and induce the body to generate immune response so as to achieve the aim of protecting the body from pathogenic attack.
Whole virus inactivated vaccines and DNA vaccines have been developed against iridovirus. Cell culture and virus amplification are needed in the preparation process of the whole virus inactivated vaccine, the consumption requirements on cell culture technology, equipment use, culture medium, materials such as serum and the like are increased, and the situation that the immune effect is unstable due to the fact that virus antigen is lost and the antigen structure is destroyed possibly exists in the preparation process of the whole virus inactivated vaccine, so that the immune effect of the whole virus inactivated vaccine is reduced. The existing iridovirus DNA vaccine mainly utilizes the main structural protein of iridovirus particles, namely main capsid protein (Major Capsid Protein, MCP) genes, to be connected with plasmids to obtain recombinant expression plasmids, and the recombinant expression plasmids are introduced into an organism to be capable of efficiently transcribing and expressing related antigens and inducing specific humoral immunity and/or cellular immune response, so that the organism has a certain immune effect on iridovirus, but the control effect of the iridovirus DNA vaccine is poor, and the requirements of aquaculture industry on iridovirus control cannot be met. Therefore, there is an urgent need to develop a vaccine having an excellent control effect against iridovirus.
Disclosure of Invention
The invention provides an iridovirus DNA vaccine, which is based on a lysosome related membrane protein LAMP1 gene, and uses a main capsid protein MCP gene of iridovirus particles to modify the LAMP1 gene to obtain a nucleotide sequence as shown in SEQ ID:1 or SEQ ID:2, the two DNA molecules can be transcribed and express related antigens with high efficiency, have immunogenicity, are applied to the preparation of iridovirus DNA vaccines, and have good control effects on iridovirus.
According to a first aspect of the present invention there is provided a DNA molecule having a nucleotide sequence as set forth in SEQ ID no: 1 or SEQ ID: 2.
The lysosome related membrane protein LAMP1 comprises a signal peptide, a Luminal structural domain, a transmembrane region and an intracellular region, the LAMP1 gene is used as a basis, and the LAMP1 gene is modified by utilizing a main capsid protein MCP gene of iridovirus particles to obtain a nucleotide sequence shown as SEQ ID:1 or SEQ ID:2, wherein the nucleotide sequence is shown as SEQ ID:1 is obtained by ligating the MCP gene behind the transmembrane region and intracellular region of the LAMP1 gene, and has a nucleotide sequence as set forth in seq id no: 2 is obtained by replacing a gene encoding a Luminal structural domain of LAMP1 with an MCP gene, and the two DNA molecules can be transcribed and express related antigens with high efficiency, have immunogenicity, can induce specific humoral immunity and/or cellular immune response, can be applied to preparation of iridovirus DNA vaccines, and have good control effect on iridovirus.
According to a second aspect of the present invention there is provided a recombinant expression vector comprising a DNA molecule as described above.
The nucleotide sequence provided by the invention is shown as SEQ ID:1 or SEQ ID:2, the recombinant expression vector containing the DNA molecule can be directly inoculated into a body, can induce the body to generate specific humoral immunity and/or cellular immune response, has good control effect on iridovirus, improves the immune protection effect of the body on iridovirus, and realizes effective protection of the body.
Preferably, the recombinant expression vector is a plasmid containing the DNA molecule.
Preferably, the plasmid is pcDNA3.1-3HA.
Preferably, the nucleotide sequence of the recombinant expression vector is shown in SEQ ID:3 or SEQ ID: 4.
The nucleotide sequence provided by the invention is shown as SEQ ID:1 or SEQ ID:2 and plasmid pcDNA3.1-3HA, both of which can be transcribed and express related antigens in the body with high efficiency, and have immunogenicity, can induce specific humoral immunity and/or cellular immune response, and can greatly improve the resistance of the body to iridovirus, thereby achieving the purpose of effectively protecting the body infected with iridovirus.
According to a third aspect of the present invention there is provided the use of a recombinant expression vector as described above in the preparation of an iridovirus vaccine.
The invention provides a polypeptide containing SEQ ID:1 or SEQ ID:2, the prepared vaccine can greatly reduce the death rate of the organism infected with the iridovirus after being inoculated and immunized, thereby realizing the effective protection of the organism infected with the iridovirus.
Preferably, the iridovirus is grouper iridovirus.
According to a fourth aspect of the present invention there is provided an iridovirus vaccine comprising a recombinant expression vector as described above.
Preferably, the iridovirus vaccine described above further comprises a pharmaceutically acceptable adjuvant.
Preferably, the iridovirus vaccine is a garrupa iridovirus vaccine.
Drawings
FIG. 1 is a schematic diagram showing the structure of SGIV-MCP, ec-LAMP1, LAMCP and MLAMP amplified by PCR technique in example 1.
S represents a signal peptide, L represents a Luminal domain, and T+C represents a transmembrane region+an intracellular region.
FIG. 2 is a schematic diagram of the construction of the recombinant expression vector provided in example 1.
FIG. 3 is a graph showing experimental results of verification of recombinant expression vectors by agarose gel nucleic acid electrophoresis in example 2.
FIG. 4 is a graph of experimental results of verification of expression of recombinant expression vectors using Western blot in example 3.
Fig. 5 is a graph showing the effect of the groupers vaccinated with different DNA vaccines (recombinant expression vectors) on SGIV provided in example 4.
Detailed Description
The technical features of the technical solution provided in the present invention will be further clearly and completely described in connection with the detailed description below, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The invention relates to a lysosome related membrane protein LAMP1, which comprises a signal peptide, a Luminal structural domain, a transmembrane region and an intracellular region, wherein the LAMP1 gene is used as a basis, and a main capsid protein MCP gene (SEQ ID: 5) of iridovirus particles is utilized to modify the LAMP1 gene to obtain an LAMCP gene and an MLAMP gene, wherein the LAMCP gene is obtained by connecting the MCP gene behind the transmembrane region and the intracellular region of the LAMP1 gene, and the nucleotide sequence of the LAMCP gene is shown as SEQ ID:1 is shown in the specification; the MLAMP gene is obtained by replacing a gene encoding the Luminal domain of LAMP1 with a MCP gene, and the nucleotide sequence of the MLAMP gene is shown as SEQ ID: 2.
The LAMP1 gene, the MCP gene, the LAMCP gene and the MLAMP gene are used as templates, and amplified according to the PCR amplification program shown in the table 1 to obtain SGIV-MCP, ec-LAMP1-MCP (LAMCP) and MLAMP, the structures of which are shown in the figure 1, wherein the nucleotide sequence of Ec-LAMP1-MCP (LAMCP) is shown in SEQ ID:1, the nucleotide sequence of the MLAMP is shown as SEQ ID:2, the nucleotide sequence of SGIV-MCP is shown as SEQ ID:5, the nucleotide sequence of LAMP1 is shown as SEQ ID:6 and the lengths of these several nucleotide sequences are summarized in table 2.
TABLE 1PCR amplification procedure
TABLE 2 PCR products
TABLE 3 homologous recombination primers
| Primer(s) | Sequence (5 '-3') |
| pcDNA3.1-MCP-F | TACGCATCAGCGGAAAAGCTTATGACTTGTACAACGGGTGCTG |
| pcDNA3.1-MCP-R | AGTGGATCCGAGCTCGGTACCCAAGATAGGGAACCCCATGGA |
| pcDNA3.1-LAMP1-F | TACGCATCAGCGGAAAAGCTTATGAAGCAGAGCAACAGTATCCC |
| pcDNA3.1-LAMP1-R | AGTGGATCCGAGCTCGGTACCGATGGTTTGGTAGCCAGCGT |
| pcDNA3.1-LAMCP-F1 | CTTGGTACCGAGCTCGGATCCATGAAGCAGAGCAACAGTATCCC |
| pcDNA3.1-LAMCP-R1 | GTCATGATGGTTTGGTAGCCAGCGT |
| pcDNA3.1-LAMCP-F2 | GGCTACCAAACCATCATGACTTGTACAACGGGTGCTG |
| pcDNA3.1-LAMCP-R2 | AACGGGCCCTCTAGACTCGAGCAAGATAGGGAACCCCATGGA |
| pcDNA3.1-MLAMP-F1 | CTTGGTACCGAGCTCGGATCCATGAAGCAGAGCAACAGTATCCC |
| pcDNA3.1-MLAMP-R1 | CAAGTCATAGCGAAACCCTGGTGCAGG |
| pcDNA3.1-MLAMP-F2 | CAGGGTTTCGCTATGACTTGTACAACGGGTGCTG |
| pcDNA3.1-MLAMP-R2 | ACATCAAGATAGGGAACCCCATGGA |
| pcDNA3.1-MLAMP-F3 | GGGGTTCCCTATCTTGATGTTGATCCCCATCATAGTCGG |
| pcDNA3.1-MLAMP-R3 | AACGGGCCCTCTAGACTCGAGGATGGTTTGGTAGCCAGCGT |
The recombinant expression vectors pcDNA3.1-MCP, pcDNA3.1-LAMP1, pcDNA3.1-LAMCP and pcDNA3.1-MLAMP shown in figure 2 are constructed by taking the plasmids pcDNA3.1-3HA (5541 bp) as vectors and inserting the SGIV-MCP, ec-LAMP1, LAMCP and MLAMP obtained by the amplification into the multiple cloning sites of the pcDNA3.1-3HA through a homologous recombination mode, wherein the homologous recombination steps are as follows:
(1) Based on the ORF sequence and predicted domain of Ec-LAMP1, the ORF sequence of MCP, and the cleavage site of pcDNA3.1-3 XHA vector plasmid, homologous recombination primers of pcDNA3.1-MCP, pcDNA3.1-LAMP, pcDNA3.1-MLAMP, and pcDNA3.1-LAMCP were designed using the Vazyme primer design program, as shown in Table 3.
(2) The specific primer is used for PCR amplification to obtain a fragment of the gene with the homologous recombination arm, the target gene fragment is separated by agarose gel electrophoresis, and the PCR product gel cutting recovery kit is used for purifying the product.
(3) The pcDNA3.1-3 XHA vectors BamH I and Xho I were used with restriction endonucleases; cleavage of HindIII and KpnI cleavage sites gives linearized pcDNA3.1-3 XHA vectors and purification.
(4) Using ClonExpress II One Step Cloning Kit or ClonExpress Ultra One Step Cloning Kit homologous recombinase system, mixing, reacting at 37 deg.C for 30min, cooling to 4 deg.C, wherein the single-segment cloning system is shown in Table 4; the multi-fragment was reacted at 50℃for 15min and then cooled to 4℃with the multi-fragment cloning system shown in Table 5.
(5) The product obtained in the above steps is used as a template, amplified by PCR, and verified by electrophoresis with 1% agarose gel.
(6) The connection product is transformed into competent escherichia coli DH5 alpha for resuscitating culture, and then the competent escherichia coli is coated on Amp-resistant LB solid medium for culture at 37 ℃ for 14-16 h.
(7) Single colonies were picked and grown up using Amp-resistant LB liquid medium. And (3) taking bacterial liquid for PCR positive detection, sequencing the positive bacterial liquid, and comparing sequence results.
TABLE 4 Single-segment cloning System
TABLE 5 Multi-fragment cloning System
Example 2
The purpose of this example was to verify the recombinant expression vectors pcDNA3.1-MCP, pcDNA3.1-LAMP1, pcDNA3.1-LAMCP and pcDNA3.1-MLAMP constructed in example 1 by agarose gel nucleic acid electrophoresis, and the results are shown in FIG. 3. Simultaneously, pcDNA3.1-3HA, pcDNA3.1-MCP, pcDNA3.1-LAMP1, pcDNA3.1-LAMCP and pcDNA3.1-MLAMP are sent to Tsingke biological company and sequenced by using Sanger method, wherein the nucleotide sequence of pcDNA3.1-MCP is shown as SEQ ID:7, the nucleotide sequence of pcDNA3.1-LAMP1 is shown as SEQ ID:8, the nucleotide sequence of pcDNA3.1-LAMCP is shown as SEQ ID:3, the nucleotide sequence of pcDNA3.1-MLAMP is shown as SEQ ID:4, the nucleotide sequence of pcDNA3.1-3HA is shown as SEQ ID:9, and the sizes of pcDNA3.1-3HA, pcDNA3.1-MCP, pcDNA3.1-LAMP1, pcDNA3.1-LAMCP and pcDNA3.1-MLAMP are summarized in Table 6.
TABLE 6 recombinant expression vector sizes
As can be seen from the agarose gel nucleic acid electrophoresis results of FIG. 3 and the test results of Table 6, the recombinant expression vectors pcDNA3.1-MCP, pcDNA3.1-LAMP1, pcDNA3.1-LAMCP and pcDNA3.1-MLAMP provided in example 1 were constructed successfully.
Example 3
The recombinant expression vectors pcDNA3.1-MCP, pcDNA3.1-LAMP1, pcDNA3.1-LAMCP and pcDNA3.1-MLAMP constructed in example 1 were transformed into E.coli by DH 5. Alpha. Competent cells, while pcDNA3.1-3HA was used as a control group, and then E.coli was subjected to expansion culture and endotoxin removal plasmid extraction using Kana-resistant LB medium to obtain five plasmids: pcDNA3.1-3HA, pcDNA3.1-MCP, pcDNA3.1-LAMP1, pcDNA3.1-LAMCP and pcDNA3.1-MLAMP. The DH5 alpha competent cells are transformed into escherichia coli by the following steps:
(1) DH 5. Alpha. Competent cells were thawed on ice.
(2) 10uL of recombinant product was added to 100uL of competent cells, and the mixture was allowed to stand on ice for 30min.
(3) And (5) after heat shock in a water bath at 42 ℃ for 45sec, immediately placing the mixture on ice for cooling for 2-3 min.
(4) 900uL of LB medium (without antibiotics) was added, and the mixture was shaken at 37℃for 1h (rotation speed 200-250 rpm).
(5) The corresponding resistant LB solid medium plates were preheated in a 37℃incubator.
(6) Centrifuge at 5,000rpm for 5min and discard 900uL of supernatant. The bacteria were resuspended in the remaining medium and gently spread on plates containing the correct resistance with sterile spreading bars.
(7) Culturing in an incubator at 37 ℃ for 12-16 h in an inverted mode.
Wherein, the endotoxin removal plasmid extraction operation is carried out according to the specification of Endo-free Plasmid Mini Kit II, and the specific steps are as follows:
(1) The constructed positive bacteria were inoculated into 100mL of LB liquid medium containing Kan resistance, and cultured overnight at 37℃at 180 rpm.
(2) Centrifuging overnight bacterial liquid at 5000 Xg at room temperature for 10min; the supernatant was discarded.
(3) Add 500. Mu.L Solution I/RNase A and blow the resuspended cells with a pipette and transfer to a 2mL EP tube.
(4) Add 500. Mu.L Solution II, gently mix upside down until the bacterial Solution is clear and leave it for 2min at room temperature.
(5) 250. Mu.L of pre-chilled N3 Buffer was added, mixed until a white floc was produced, centrifuged at 12,000Xg for 10min at room temperature and the supernatant transferred to a new 2mL EP tube.
(6) Adding ETR solution with volume of 0.1 times of the supernatant, mixing, and standing on ice for 10min.
(7) The mixture was placed in a water bath at 42℃for 5min.
(8) Centrifuging at room temperature for 3min at 12,000Xg; the supernatant was transferred to a fresh 1.5mL EP tube.
(9) Adding 0.5 times of absolute ethyl alcohol, mixing uniformly and transferring into an adsorption column.
(10) Centrifuge at room temperature for 1min at 10,000Xg, discard the waste liquid.
(11) Add 500. Mu.L HBC Buffer, centrifuge at 10,000Xg for 1min at room temperature, discard the waste.
(12) 700 mu L DNA Wash Buffer and 10,000Xg were added and centrifuged at room temperature for 1min, and the waste liquid was discarded.
(13) Repeating step (12).
(14) Centrifuge at 12,000Xg for 3min.
(15) The column was transferred to a fresh 1.5mL EP tube.
(16) 100. Mu.L of pre-heated elution buffer at 65℃was added, and the mixture was left at room temperature for 2min and centrifuged at 13,000Xg for 1min to elute DNA. Using NanoDrop TM Lite spectrophotometry to determine plasmid concentration, -80 ℃ for preservation.
The above five plasmids were transfected into garrupa spleen cells (GS cells) and cultured, the GS cells were collected 24 hours after transfection, and then the GS cells were lysed using RIPA cell protein lysate to release intracellular proteins, and the five plasmids were examined using Western blot, the results of which are shown in fig. 4, while the sizes of proteins expressed by the above five plasmids (i.e., recombinant expression vectors) were summarized in table 7.
TABLE 7 recombinant expression vector expressed protein size
As can be seen from FIG. 4 and Table 7, the five plasmids were successfully expressed, and the HA protein expressed by pcDNA3.1-3HA was only 3.3kD, which could not be shown in the Western blot results; the MCP protein expressed by pcDNA3.1-MCP is about 53kD; the LAMP1 protein expressed by pcDNA3.1-LAMP1 is about 38kD, and the result of FIG. 4 shows that a plurality of bands exist in the Western blot experiment result, wherein one band is an LAMP1 protein band, and the other band is an LAMP1 protein band subjected to glycosylation modification; the MLAMP protein expressed by pcDNA3.1-MLAMP is about 70kD; the protein expressed by pcDNA3.1-LAMCP is about 90kD. The above results demonstrate that the recombinant expression vectors pcDNA3.1-MCP, pcDNA3.1-LAMP1, pcDNA3.1-LAMCP and pcDNA3.1-MLAMP provided in example 1 can successfully express the corresponding proteins by being introduced into GS cells after successful construction.
EXAMPLE 4 iridovirus DNA vaccine and its immunization Effect
The purpose of this example was to investigate the immune effect of five DNA vaccines (pcDNA3.1-3 HA, pcDNA3.1-MCP, pcDNA3.1-LAMP1, pcDNA3.1-LAMCP and pcDNA3.1-MLAMP) against Epinephelus iridovirus (Singapore grouper iridivirus, SGIV).
Taking 150 healthy groupers, randomly dividing the groupers into 6 groups, taking 5 groups as experimental groups and taking 1 group as control group, inoculating equal amounts of pcDNA3.1-3HA, pcDNA3.1-MCP, pcDNA3.1-LAMP1, pcDNA3.1-LAMCP and pc DNA3.1-MLAMP into the experimental groups, inoculating PBS buffer solution into the control groups, inoculating 2.0 mug/g groupers, injecting SGIV into the groupers of each group after 28 days of immunization, wherein each grouper is injected with 1X 10 parts of the groupers 4 TCID50/mL SGIV virus 100 μl, observing groupers, recording death mantissas of groupers and counting accumulated mortality, and the result is shown in FIG. 5, wherein pcDNA3.1-3HA was inoculatedThe experimental group was designated as pcDNA3.1-HA group, the experimental group inoculated with pcDNA3.1-MCP was designated as pcDNA3.1-MCP group, the experimental group inoculated with pcDNA3.1-LAMP1 was designated as pcDNA3.1-LAMP group, the experimental group inoculated with pcDNA3.1-LAMCP was designated as pcDNA3.1-LAMCP group, the experimental group inoculated with pcDNA3.1-MLAMP was designated as pcDNA3.1-MLAM, and the control group inoculated with PBS buffer was designated as PBS group.
As can be seen from FIG. 5, the first death occurred in the PBS group and pcDNA3.1-HA group, and on day 5, the death occurred in the PBS group and pcDNA3.1-3HA group in 3 and 2 groupers, respectively, and the death did not occur in the other groups; on day 6, the death numbers of the groupers in the PBS group and the pcDNA3.1-HA group continue to increase, the death mantissas of the groupers in the PBS group and the pcDNA3.1-HA group are respectively 6 tails and 5 tails, and the accumulated death mantissas of the groupers are respectively 9 tails and 7 tails; when the time lasts until 9 days, the accumulated death mantissas of the garrupa in the PBS group and the pcDNA3.1-HA group are 23, the survival number of the garrupa is only 2, the garrupa in the pcDNA3.1-LAMP1 group also dies in a large area, and the survival number of the garrupa is only 3; by day 10, the cumulative death mantissa of the groupers of the pcDNA3.1-MCP group was 11 tails, the survival mantissa of the groupers was 14 tails, and the death mantissas of the 2 tails and 1 tail groupers occurred in the pcDNA3.1-MLAMP group on days 9 and 10, respectively, and by day 10, the cumulative death mantissa of the groupers of the pcDNA3.1-MLAMP group was 3 tails, and the death mantissa of the groupers of the pcDNA3.1-LAMCP group occurred only on day 10, and the death mantissa of the groupers of the pcDNA3.1-LAMCP group was only 2 tails on day 10, and continued until day 14.
From the above, it was found that the mortality of each group of groupers was 92%, 88%, 44%, 12% and 8% within 14 days after immunization with PBS, pcDNA3.1-3HA, pcDNA3.1-LAMP, pcDNA3.1-MCP, pcDNA3.1-MLAMP and pcDNA3.1-LAMCP for 28 days and injection of SGIV, respectively.
Table 8 relative immune protection Rate of DNA vaccine against SGIV-infected Epinephelus
In addition, the relative immunoprotection Rate (RPS) of each group of vaccine against SGIV-infected groupers was calculated as shown in table 8, wherein the calculation formula of RPS is as follows: RPS (%) = (1-experimental/control mortality) ×100%. As is clear from Table 8, the relative immunoprotection rates of four DNA vaccines, namely pcDNA3.1-LAMP1, pcDNA3.1-MCP, pcDNA3.1-MLAMP and pcDNA3.1-LAMCP, against SGIV-infected groupers were 4%, 52%, 87% and 91%, respectively.
In summary, the invention is based on LAMP1 gene, and uses the main capsid protein MCP gene of iridovirus particles to modify LAMP1 gene to obtain the nucleotide sequence shown in SEQ ID:1 or SEQ ID:2, wherein the nucleotide sequence is shown as SEQ ID:1 is obtained by ligating the MCP gene behind the transmembrane region and intracellular region of the LAMP1 gene, and has a nucleotide sequence as set forth in seq id no: 2 is obtained by replacing a gene encoding a Luminal structural domain of LAMP1 with an MCP gene, the two DNA molecules are applied to construction of a recombinant expression vector, the recombinant expression vector containing the DNA molecules can be directly inoculated into a body, related antigens can be efficiently transcribed and expressed in the body, immunogenicity is achieved, specific humoral immunity and/or cellular immune response can be induced to the body, and the recombinant expression vector has good control effect on iridovirus, so that effective protection on the body is realized. The nucleotide sequence containing the nucleotide sequence shown in SEQ ID:1 or SEQ ID:2 is applied to the preparation of iridovirus vaccines, the prepared DNA vaccines can greatly improve the resistance of organisms to iridovirus, thereby achieving the purpose of effectively protecting the organisms infected with iridovirus.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention, but these modifications or substitutions are all within the scope of the present invention.
Claims (10)
1. A DNA molecule characterized in that: the nucleotide sequence of the DNA molecule is shown as SEQ ID:1 or SEQ ID: 2.
2. A recombinant expression vector, characterized in that: a DNA molecule according to claim 1.
3. The recombinant expression vector of claim 2, wherein: the recombinant expression vector is a plasmid containing the DNA molecule.
4. The recombinant expression vector of claim 3, wherein: the plasmid is pcDNA3.1-3HA.
5. The recombinant expression vector of claim 4, wherein: the nucleotide sequence of the recombinant expression vector is shown as SEQ ID:3 or SEQ ID: 4.
6. Use of a recombinant expression vector according to any one of claims 2 to 5 in the preparation of an iridovirus vaccine.
7. The use of the recombinant expression vector of claim 6 in the preparation of an iridovirus vaccine, wherein: the iridovirus is garrupa iridovirus.
8. An iridovirus vaccine characterized by: comprising the recombinant expression vector according to any one of claims 2 to 5.
9. The iridovirus vaccine according to claim 8, characterised by: pharmaceutically acceptable adjuvants are also included.
10. The iridovirus vaccine according to claim 8, characterised by: the iridovirus vaccine is an grouper iridovirus vaccine.
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