Fusion gene of grass carp NPM1a and II type reovirus VP38, recombinant protein, composition and application

Abstract

The invention relates to the technical field of aquaculture fish vaccines, in particular to a fusion gene of grass carp NPM1a and II type reovirus VP38, recombinant protein, a composition and application. The nucleotide sequence of the fusion gene is as shown in SEQ ID NO: 1. The fusion gene is obtained by connecting grass carp NPM1a and II type reovirus VP38 through a linker sequence, a BamH restriction enzyme cutting site is added to the 5'end of the fusion sequence, and an XhoI restriction enzyme cutting site is added to the 3 'end of the fusion sequence; the sequence of the linker is as shown in SEQ ID NO: 2. The subunit vaccine of the fusion protein of the grass carp NPM1a and the II type reovirus VP38 is prepared, and indole-3-lactic acid is used as an immunopotentiator in a combined manner, so that the technical bottlenecks of low protection rate, poor intestinal mucosal barrier repairability and the like of the existing vaccine are overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture fish vaccines, and in particular to a fusion gene, recombinant protein, composition and application of grass carp NPM1a and type II reovirus VP38. Background Art

[0002] Grass carp (Ctenopharyngodon idella) is a core economic species in China's aquaculture and an important source of high-quality aquatic protein. However, viral hemorrhagic disease, caused by type II reovirus (GCRV) infection in grass carp, is highly contagious and pathogenic, causing huge economic losses to grass carp aquaculture every year and seriously hindering the green, healthy and sustainable development of the aquaculture industry. Existing methods for the prevention and control of hemorrhagic disease have three major technical bottlenecks: ① Traditional inactivated vaccines lack specific antigen presentation targets, resulting in insufficient neutralizing antibody titers; ② Live attenuated vaccines have the risk of reversion to virulence; ③ Subunit vaccines mostly use a single viral structural protein (such as VP4, VP35 and VP56), which has a low immune protection rate and cannot activate mucosal immune responses. Therefore, the development of a new vaccine system that takes into account both efficient antigen delivery and precise activation of mucosal immunity has become an urgent need for the prevention and control of grass carp hemorrhagic disease.

[0003] Type II reovirus (GCRV) is the primary pathogen causing hemorrhagic disease in grass carp. Its genome contains 11 double-stranded RNA segments encoding proteins including VP1, VP2, VP3, NS79, VP5, VP4, VP56, VP41, VP46, VP38, and VP35. Researchers have conducted limited research on the efficacy of these proteins as vaccines. Studies have shown that immunization of grass carp with different doses of recombinant VP4 protein, followed by challenge, protects against viral infection, with relative survival rates ranging from 47% to 82%. Oral immunization of rare goby with VP6 protein, delivered by lactic acid bacteria, resulted in a relative survival rate of 42.9%. A VP35 protein subunit vaccine significantly increased the number of peripheral blood white blood cells in grass carp, elevated serum antibody levels, and upregulated the expression of immune-related gene mRNAs. Following challenge, the relative survival rate of grass carp was 60%. A fusion of VP4 and VP35 protein subunits in immunized rare goby with challenge resulted in a survival rate of 67%. The VP56 protein was expressed on the surface of spores using Bacillus subtilis as a vector. Oral immunization of grass carp with a VP3, VP4, and VP38 fusion vaccine resulted in a relative survival rate of 56%. Immunization of grass carp with a VP3, VP4, and VP38 triple-protein fusion vaccine resulted in a relative survival rate of 83%, while immunization with a VP4, VP35, and VP56 triple-protein fusion vaccine resulted in a relative survival rate of 62%. However, these vaccine design strategies only target viral structural proteins and are unable to completely block the interaction between the virus and host receptors. This results in incomplete blockade of the viral invasion pathway and limited activation and protection of the intestinal mucosal barrier.

[0004] Nucleophosmin 1 (NPM1) is a nuclear phosphoprotein widely present in the nucleolus of vertebrates. It activates chromatin transcription in an acetylation-dependent manner and maintains the homeostasis of the nucleus. On the other hand, NPM1 is also a key receptor for a variety of viruses to invade host cells. For example, NPM1 can interact with a variety of viral proteins such as hepatitis B virus (HBV), Epstein-Barr virus (EBV), bovine immunodeficiency virus and human immunodeficiency virus, participate in multiple stages of viral infection and affect viral proliferation. In addition, in grass carp, NPM1 can be transferred from the nucleus to the cytoplasm and promote GCRV replication. However, the role of NPM1 in viral infection has not yet been applied to vaccine development.

[0005] Tryptophan (Trp) is an essential amino acid that can only be ingested through the diet and is the only amino acid containing an indole structure. Tryptophan metabolism can produce a variety of molecules, such as kynurenine (Kyn), 5-hydroxytryptamine, indole-3-lactic acid (ILA), and other indole derivatives, which are involved in regulating various physiological processes such as immune response, reproductive development, and cell biological function. Studies have shown that ILA can act as a ligand for the aryl hydrocarbon receptor (AhR), promoting the expression of interleukin-22 and antimicrobial peptides, thereby protecting the intestinal mucosal barrier and inhibiting infection by pathogenic microorganisms. However, its mechanism of action and practical application in fish immune regulation have not been reported, and there have been no studies combining it with subunit vaccines to achieve precise dual activation of the intestinal mucosal barrier and vaccine immunity.

[0006] In summary, there is currently no vaccine system for grass carp that combines "host-virus dual-target fusion antigen" and "intestinal mucosal precision immune enhancer". Summary of the Invention

[0007] The present invention provides a fusion gene, recombinant protein, composition and application of grass carp NPM1a and type II reovirus VP38, the purpose of which is to fill the current domestic gap in the use of NPM1 in preventing and treating grass carp hemorrhagic disease caused by type II reovirus (GCRV). The difference between the present invention and previous patents is that, based on the above background, the present invention proposes an innovative idea of ​​combining "host-virus dual-target fusion antigen" with "intestinal mucosal precision immune enhancer". By preparing a subunit vaccine of grass carp NPM1a and type II reovirus VP38 fusion protein, and combining it with indole-3-lactic acid as an immune enhancer, the technical bottlenecks of low protection rate of existing vaccines and poor repairability of intestinal mucosal barriers are overcome, providing a solution that is both efficient, safe and economical for the prevention and control of grass carp hemorrhagic disease, and has significant industrial application value.

[0008] In order to achieve the above object, the present invention provides a fusion gene of grass carp NPM1a and type II reovirus VP38, the nucleotide sequence of the fusion gene is shown in SEQ ID NO: 1.

[0009] Preferably, the fusion gene is obtained by connecting grass carp NPM1a and type II reovirus VP38 through a linker sequence, and a BamH restriction site is added to the 5' end of the fusion sequence and an XhoI restriction site is added to the 3' end;

[0010] The linker sequence is shown in SEQ ID NO: 2.

[0011] Under the same technical concept, the present invention also provides a recombinant expression vector containing the fusion gene.

[0012] Under the same technical concept, the present invention also provides a fusion recombinant protein of grass carp NPM1a and type II reovirus VP38, the amino acid sequence of the fusion recombinant protein is shown in SEQ ID NO: 3.

[0013] Under the same technical concept, the present invention also provides a method for preparing a fusion recombinant protein of grass carp NPM1a and type II reovirus VP38, comprising the following steps:

[0014] (1) Inserting a linker sequence between the grass carp NPM1a and type II reovirus VP38 genes to synthesize a fusion gene of grass carp NPM1a and type II reovirus VP38;

[0015] (2) The fusion gene of grass carp NPM1a and type II reovirus VP38 was connected to a recombinant expression vector and transformed into an Escherichia coli expression strain;

[0016] (3) Induce the expression of recombinant protein, precipitate and purify it to obtain the fusion recombinant protein of grass carp NPM1a and type II reovirus VP38.

[0017] Preferably, the linker sequence insertion in step (1) specifically comprises: using the Overlap PCR method, adding a BamH restriction site to the 5' end of the fusion sequence and an XhoI restriction site to the 3' end, and connecting the grass carp NPM1a with the type II reovirus VP38 through the flexible polypeptide encoded by the linker sequence;

[0018] The amino acid sequence of the flexible polypeptide encoded by the linker sequence is shown in SEQ ID NO: 4.

[0019] Preferably, the recombinant expression vector in step (2) is a pET28a vector, and the transformation into the Escherichia coli expression strain specifically includes: using the CaCl2 method to transform the recombinant expression vector into Escherichia coli DH5α competent cells to obtain a recombinant plasmid, sequencing verification, and then transforming the recombinant plasmid into the Escherichia coli BL21 expression vector.

[0020] Preferably, the inducing expression of the recombinant protein in step (3) specifically comprises: inoculating 100-200 μl of Escherichia coli BL21 containing the recombinant plasmid into 200 ml of LB liquid culture medium at a ratio of 1:100-1:200, adding kanamycin to the culture medium to a final concentration of 30-50 μg / ml; then placing the culture flask in a 37°C 180-220 rpm shaker and culturing to an OD of approximately 0.4-0.5; adding IPTG to the culture flask to a final concentration of 0.2-0.4 mM, then placing the culture flask in a 16°C 120-140 rpm shaker and culturing; after 6-8 hours, centrifuging to collect the bacteria, adding 5-10 ml of protein lysis buffer, using an ultrasonic disruptor to disrupt the cells at low temperature, and centrifuging at 10,000-12,000 rpm to collect the supernatant, which is the recombinant protein;

[0021] The precipitation purification specifically includes: adding the supernatant to a Ni-NTA protein chromatography column to purify the fusion recombinant protein of grass carp NPM1a and type II reovirus VP38.

[0022] Based on the same technical concept, the present invention also provides a composition comprising a fusion recombinant protein of grass carp NPM1a and type II reovirus VP38, comprising the fusion recombinant protein of grass carp NPM1a and type II reovirus VP38 and indole-3-lactic acid in a mass ratio of 1:5 to 1:10. This ratio can maximize immune protection efficacy, reduce viral load, and avoid excessive inflammation and metabolic toxicity.

[0023] Under the same technical concept, the present invention also provides the application of a composition of a fusion recombinant protein of grass carp NPM1a and type II reovirus VP38. The composition of the fusion recombinant protein of grass carp NPM1a and type II reovirus VP38 is used to improve the grass carp's ability to resist hemorrhagic disease. The application amount of each component in the composition is 0.5-1.0 mg / ml of the fusion recombinant protein and 2.5-5 mg / ml of ILA. The injected grass carp has a body length of 10-12 cm and a weight of 15-20 g, and the injection frequency is one injection.

[0024] The above solution of the present invention has the following beneficial effects:

[0025] The present invention establishes a double barrier to block the virus invasion path by preparing a subunit vaccine of the fusion recombinant protein of grass carp NPM1a and type II reovirus VP38; at the same time, the AhR / IL-22 pathway is activated by indole-3-lactic acid, which can, on the one hand, improve the antioxidant capacity of immune cells, reduce the damage of oxidative stress to immune cells, provide a good internal environment for immune response, and create a "high-efficiency sensing microenvironment" for the recognition of fusion protein antigens and the production of subunit vaccines; on the other hand, the intestinal mucosal barrier is strengthened through the AhR / IL-22 pathway (upregulating tight junction proteins such as ZO-1 and Occludin), which not only directly blocks the invasion path of reovirus through the intestinal mucosa, but also avoids the functional exhaustion of dendritic cells (DC) by reducing endotoxin leakage, making the fusion protein antigen easier to be captured by DC cells, thereby enhancing the antigen uptake and presentation ability of DC, and achieving the spatial synergistic effect of "virus invasion blocking-antigen presentation enhancement-immune response quality improvement". This multi-faceted synergistic effect has significantly improved the grass carp's ability to resist hemorrhagic disease, with a relative survival rate of up to 85.2%, achieving the synergistic effect of "targeted blocking-mucosal repair" and providing an innovative solution for the efficient prevention and control of grass carp viral hemorrhagic disease in the aquaculture industry.

[0026] Other beneficial effects of the present invention will be described in detail in the subsequent specific implementation section. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the SDS-PAGE test results of rCiNPM1a, rVP38, and rCiNPM1a+rVP38 of Example 1 of the present invention;

[0028] Figure 2 The results are as follows: the detection result of the content of LPS in grass carp plasma after GCRV infection in Example 2 of the present invention;

[0029] Figure 3 The results of the detection of the expression of key protective factors in the intestinal mucosal tissue of grass carp after GCRV infection in Example 2 of the present invention are as follows; wherein A is AhR, B is IL-22, C is ZO-1, and D is Occludin;

[0030] Figure 4 The test results of the key antioxidant enzymes in grass carp after GCRV infection in Example 3 of the present invention are shown below; wherein A is SOD, B is CAT, C is ACP, and D is AKP;

[0031] Figure 5 The results of the detection of the expression of antiviral factors of grass carp after GCRV infection in Example 4 of the present invention are as follows; wherein A is IgM, B is IFN1, C is Mx, D is ISG15, E is Viperin, and F is CMPK2;

[0032] Figure 6 This is the test result of the viral load in grass carp after GCRV infection in Example 4 of the present invention;

[0033] Figure 7 The results are as follows: the survival rate of grass carp after GCRV infection in Example 5 of the present invention. DETAILED DESCRIPTION

[0034] To make the technical problems, technical solutions, and advantages to be solved by the present invention more clear, the following is a detailed description with reference to the accompanying drawings and specific embodiments. It is obvious that the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0035] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0036] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood broadly. For example, they may refer to a locking connection, a detachable connection, or an integral connection; they may refer to a mechanical connection or an electrical connection; they may refer to a direct connection or an indirect connection through an intermediate medium; and they may refer to internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0037] The experimental reagents, sequencing, and experimental animals involved in this invention were provided by the following companies:

[0038] 1. Gene sequencing and primer synthesis were completed by Beijing Qingke Biotechnology Co., Ltd.

[0039] 2. Indole-3-lactic acid reagent was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0040] 3. Escherichia coli DH5α and BL21 (DE3) competent cells, protein lysis buffer, and Ni-NTA chromatography purification kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0041] 4. LPS, SOD, CAT, ACP and AKP kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0042] 5. Healthy grass carp were purchased from Hunan Qiaomai Lake High-Quality Fish Research Institute Co., Ltd.

[0043] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0044] Example 1

[0045] A method for preparing a fusion gene and recombinant protein of grass carp NPM1a and type II reovirus VP38, specifically comprising the following steps:

[0046] Step (1): Synthesize the fusion gene sequence

[0047] Overlap PCR was used to fuse grass carp NPM1a (GenBank Accession No. XM_051909362.1) with the type II reovirus VP38 gene (GenBank Accession No. KC201175.1). A linker sequence was added between the two genes, with a BamH restriction site (GGATCC) added to the 5' end of the fusion sequence and an XhoI restriction site (CTCGAG) added to the 3' end. This method linked grass carp NPM1a to type II reovirus VP38 via the flexible polypeptide encoded by the linker sequence (SEQ ID NO: 4GGGGSGGGGSGGGGSGPGPG).

[0048] The linker sequence is as follows:

[0049] SEQ ID NO: 2

[0050] GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTCCGGGTC CGGGT

[0051] The nucleotide sequence of the recombinant fusion gene is shown below:

[0052] SEQ ID NO: 1

[0053]

[0054] Step (2): Construction of fusion sequence expression vector

[0055] The above sequence was double-digested with BamH and XhoI and ligated into the pET28a vector. The recombinant plasmid was transformed into competent E. coli DH5α cells using the CaCl2 method. After verification by sequencing, the recombinant plasmid was transformed into the E. coli BL21 expression vector. Simultaneously, control vectors expressing the grass carp NPM1a protein and the type II reovirus VP38 protein were constructed using the above method.

[0056] Step (3): Purification of recombinant protein

[0057] 100 μl of Escherichia coli BL21 (DE3) containing the recombinant plasmid was inoculated into 200 ml of LB liquid culture medium at a ratio of 1:200, and kanamycin was added to the culture medium at a final concentration of 50 μg / ml. The culture flask was then placed in a 200 rpm shaker at 37°C and cultured until the OD was about 0.4. IPTG was added to the culture flask at a final concentration of 0.04 mM, and then the culture flask was cultured in a 16°C, 120 rpm shaker. After 6 hours, the bacteria were collected by centrifugation and 5 ml of protein lysis buffer was added. The cells were broken at low temperature using an ultrasonic disruptor, and the supernatant was collected by centrifugation at 10,000 rpm. The supernatant was added to a Ni-NTA protein chromatography column, and the recombinant protein (named rCiNPM1a+rVP38 recombinant protein) was purified according to the operating procedures of the kit, and the size of the recombinant protein was detected by SDS-PAGE (see the test results). Figure 1 At the same time, grass carp NPM1a protein and type II reovirus VP38 protein were expressed alone according to the above method and used as control groups, respectively.

[0058] The amino acid sequence of the recombinant fusion protein is shown below:

[0059] SEQ ID NO: 3

[0060] MDLEQMGPQTFLYGCELKAGKDVSFNPEDDDYDHQLSVRMACVDPATKDELNVVEIEGHDSEGQKVKAVLATLKPSTLPSVCLGGFEITPPVVFRLRSGSGPVHISGQHLVIMGGDQSFDEEEEEEEEEEETVTTAKKRPASLTAKPSKKMKLDEQEEEEEEDDDDDD DDDDEIDDEEDDDEDDDDEEEEEESPVKEKKTASKPQTPAQNGKGPKPSTPAKQNKTPEKGNKGDKKAQTPKTPQSPKTPQTPRVFTVPEIKAKMMASVEKGVSLPKLQPKFENYVKNCFKATDAKVIEELWKWRQSVKGGGGSGGGGSGGGGSGPGPGMAGVSLNI NRNISNSASTIFLEDIPLLSCSVRCEPGKGRELPKFNMSCPAINAMGRCLNPMKFIAEHWVPNSSPSRKPSRQHWRNVLNGLEFSNGRGFDVLSFSPAGMAVLRDILTEDSVKYCFDESNTCSLFTLLYTLCCDAAGVEPMDLDSRQTDASARMVSYQDRAIVLTSNEA GDRIEPWNVELDKEFGNPDLLSRLNISYGVQRYGDSKASTDTLTLADAPERSKPALITVQPLLVAMCIKQSLDGLLALSDLRLRFDQYPGYANALMNAMAMYACLDRDLMRFLLRLEMTHASTVSEVAECWRNSRNSRDATGCHIVPRQGLLIIVSGDVEVRRIFAQML

[0061] Table 1 Primers of Example 1

[0062]

[0063] Example 2

[0064] A grass carp NPM1a and type II reovirus VP38 recombinant protein composition and its application, the composition is a fusion protein combined with indole-3-lactic acid (ILA), specifically comprising the following steps:

[0065] Step (1): Immunization of grass carp with fusion protein combined with indole-3-lactic acid

[0066] Healthy grass carp of the same batch with a body length of about 10-12 cm and a body weight of about 15-20 g were selected and divided into five groups, with 15 fish in each group. The temperature of the culture room was maintained at 28℃±0.1:

[0067] ① Normal group: 100 μl of sterile PBS was injected intraperitoneally.

[0068] ②ILA group: 100 μl of sterile PBS containing 4 mg / ml ILA was injected intraperitoneally.

[0069] ③ILA+rCiNPM1a group: injected with 100 μl of sterile PBS containing 4 mg / ml ILA and 0.5 mg / ml rCiNPM1a recombinant protein.

[0070] ④ILA+rVP38 group: injected with 100 μl of sterile PBS containing 4 mg / ml ILA and 0.5 mg / ml rVP38 recombinant protein.

[0071] ⑤ILA+rCiNPM1a+rVP38 group: Inject 100 μl of sterile PBS containing 4 mg / ml ILA and 0.5 mg / ml rCiNPM1a+rVP38 recombinant protein.

[0072] After 21 days, each group of fish was artificially infected by intraperitoneal injection of 100 μl of GCRV, with a virus concentration of 1.0×107TCID50 / ml.

[0073] Step (2): Detecting changes in intestinal mucosal permeability of grass carp

[0074] Two days after infection, blood from each group of fish was collected using a disposable syringe and prepared into plasma. Subsequently, the LPS content in the plasma was detected using an LPS kit to evaluate the effect of the fusion protein combined with indole-3-lactic acid on the intestinal mucosal permeability of grass carp after infection. The experimental results showed that the LPS content of the normal PBS control group was 43.1±4.8pg / ml, the LPS content of the ILA group was 37.8±4.4pg / ml, the LPS content of the ILA+rCiNPM1a group was 30.8±4.0pg / ml, the LPS content of the ILA+rVP38 group was 28.8±4.3pg / ml, and the LPS content of the ILA+rCiNPM1a+rVP38 group was 20.1±4.4pg / ml, indicating that the LPS content of the ILA+rCiNPM1a+rVP38 group was significantly lower than that of the other groups (Mann-Whitney test, P﹤0.01). Figure 2 shown.

[0075] Step (3): Detect changes in key protective factors of the intestinal mucosal barrier of grass carp

[0076] Two days after infection, the intestinal tissue was quickly frozen in liquid nitrogen, and the total RNA of the intestinal tissue was extracted using a total RNA extraction kit, and then reversed into cDNA using a reverse transcription kit. The expression of AhR, IL-22, ZO-1 and Occludin was detected by qRT-PCR to evaluate the effect of fusion protein combined with indole-3-lactic acid on the expression of key protective factors of the intestinal mucosal barrier of grass carp after infection. The primers are shown in Table 1 below, and the internal reference gene is 18S rRNA. The experimental results showed that the expression of AhR, IL-22, ZO-1 and Occludin in the ILA+rCiNPM1a+rVP38 group was significantly higher than that in the normal PBS control group (Mann-Whitney test, P﹤0.01). Figure 3 shown.

[0077] Among them, GCRV has been reported in the literature: Wei Y, Lv Z, Du Z, Xiao T. The structural characteristics and expression characteristics of C1S in response to GCRV infection in grass carp. Fish Shellfish Immunol. 2025, 161: 110-264.

[0078] Table 2 Primers of Example 2

[0079]

[0080] Example 3

[0081] The effect of the grass carp NPM1a and type II reovirus VP38 recombinant protein composition on the antioxidant capacity of grass carp specifically includes the following steps:

[0082] Step (1): Immunize grass carp with the recombinant protein composition of grass carp NPM1a and type II reovirus VP38

[0083] The immunization method is the same as step (1) in Example 2.

[0084] Step (2): Detection of changes in antioxidant capacity of grass carp

[0085] Two days after infection, liver tissues of fish in each group were collected, quickly frozen in liquid nitrogen, and ground to prepare tissue suspensions. Kits were used to detect the levels of four key antioxidant enzymes, SOD, CAT, ACP, and AKP, to evaluate the effects of the fusion protein combined with indole-3-lactic acid on the antioxidant capacity of grass carp after infection. The experimental results showed that the levels of antioxidant enzymes SOD, CAT, ACP, and AKP in the ILA+rCiNPM1a+rVP38 group were significantly higher than those in the normal PBS control group (Mann-Whitney test, P < 0.01). Figure 4 shown.

[0086] Example 4

[0087] The effect of the grass carp NPM1a and type II reovirus VP38 recombinant protein composition on the antiviral ability of grass carp specifically includes the following steps:

[0088] Step (1): Immunize grass carp with the recombinant protein composition of grass carp NPM1a and type II reovirus VP38

[0089] The immunization method is the same as step (1) in Example 2.

[0090] Step (2): Detection of changes in the expression of grass carp IgM antibodies

[0091] Two days after infection, spleen tissue was quickly frozen in liquid nitrogen. Total RNA was extracted using a total RNA extraction kit and converted to cDNA using a reverse transcription kit. qRT-PCR was used to detect IgM expression and evaluate the effect of the fusion protein combined with indole-3-lactic acid on IgM antibody expression in grass carp challenged with the virus. Primers are listed in Table 2 below. The internal reference gene was 18S rRNA. The experimental results showed that the relative expression of IgM in the normal PBS control group was 1.00±0.02, the relative expression of IgM in the ILA group was 1.19±0.18, the relative expression of IgM in the ILA+rCiNPM1a group was 1.96±0.19, the relative expression of IgM in the ILA+rVP38 group was 1.99±0.31, and the relative expression of IgM in the ILA+rCiNPM1a+rVP38 group was 2.70±0.18, indicating that the expression of IgM in the ILA+rCiNPM1a+rVP38 group was significantly higher than that in the other groups (Mann-Whitney test, P﹤0.01). Figure 5 As shown in A.

[0092] Step (3): Detect changes in the expression of key antiviral factors in grass carp

[0093] After obtaining the cDNA in step (2) above, it was reversed to cDNA using a reverse transcription kit. The expression of IFN1, Mx, ISG15, Viperin and CMPK2 was detected by qRT-PCR to evaluate the effect of the fusion protein combined with indole-3-lactic acid on the expression of key antiviral factors in grass carp after infection. The primers are shown in Table 2 below, and the internal reference gene is 18S rRNA. The experimental results showed that the expression of key antiviral factors such as IFN1, Mx, ISG15, Viperin and CMPK2 in the ILA+rCiNPM1a+rVP38 group was significantly higher than that in the other groups (Mann-Whitney test, P﹤0.01). Figure 5 B to Figure 5 As shown in F.

[0094] Step (4): Detecting changes in viral load in grass carp

[0095] After obtaining the cDNA of step (2) above, the expression of VP38 was detected by qRT-PCR method to evaluate the effect of fusion protein combined with indole-3-lactic acid on the GCRV viral load in grass carp after infection. The primers are shown in Table 2 below, and the internal reference gene is 18SrRNA. The experimental results showed that the relative expression of VP38 in the normal PBS control group was 1.00±0.02, the relative expression of VP38 in the ILA group was 0.89±0.03, the relative expression of VP38 in the ILA+rCiNPM1a group was 0.64±0.05, the relative expression of VP38 in the ILA+rVP38 group was 0.65±0.13, and the relative expression of VP38 in the ILA+rCiNPM1a+rVP38 group was 0.52±0.04, indicating that the viral load of the ILA+rCiNPM1a+rVP38 group was significantly lower than that of the other groups (Mann-Whitney test, P﹤0.01). Figure 6 shown.

[0096] Table 3 Primers of Example 4

[0097]

[0098]

[0099] Example 5

[0100] The effect of the grass carp NPM1a and type II reovirus VP38 recombinant protein composition on the survival rate of grass carp against hemorrhagic disease specifically includes the following steps:

[0101] Step (1): Immunize grass carp with the recombinant protein composition of grass carp NPM1a and type II reovirus VP38

[0102] Healthy grass carp with a body length of about 10-12 cm and a body weight of about 15-20 g from the same batch were selected and divided into five groups, with 30 fish in each group. The temperature of the culture room was maintained at 28°C ± 0.1. The rest of the immunization method was the same as step (1) of Example 2.

[0103] Step (2): Detecting the survival rate of grass carp infected with GCRV

[0104] After challenge, the animals were observed for 15 consecutive days, and mortality rates were calculated. The relative protection rate was calculated to evaluate the effect of the fusion protein combined with indole-3-lactic acid on the grass carp's ability to resist hemorrhagic disease. The results showed that after 15 days, 27 fish died in the normal PBS control group, 22 in the ILA group, 13 in the ILA+rCiNPM1a group, 12 in the ILA+rVP38 group, and 4 in the ILA+rCiNPM1a+rVP38 group. The relative survival rate of each group was calculated using the method of relative survival rate = 1-mortality rate of immune group / mortality rate of control group. The results showed that the relative survival rate of the ILA group was 18.5%, the relative survival rate of the ILA+rCiNPM1a group was 51.9%, the relative survival rate of the ILA+rVP38 group was 55.6%, and the relative survival rate of the ILA+rCiNPM1a+rVP38 group was 85.2%, indicating that the relative survival rate of the ILA+rCiNPM1a+rVP38 group was significantly higher than that of the other groups (Log-rank test, P﹤0.01). Figure 7 shown.

[0105] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A fusion gene of grass carp NPM1a and type II reovirus VP38, characterized in that: The nucleotide sequence of the fusion gene is shown in SEQ ID NO:

1.

2. The fusion gene according to claim 1, wherein The fusion gene is obtained by connecting grass carp NPM1a and type II reovirus VP38 through a linker sequence, and a BamH restriction site is added to the 5' end of the fusion sequence and an XhoI restriction site is added to the 3' end; The linker sequence is shown in SEQ ID NO:

2.

3. A recombinant expression vector containing the fusion gene according to claim 1.

4. A fusion recombinant protein of grass carp NPM1a and type II reovirus VP38, characterized in that: The amino acid sequence of the fusion recombinant protein is shown in SEQ ID NO:

3.

5. A method for preparing a fusion recombinant protein of grass carp NPM1a and type II reovirus VP38, characterized in that: The following steps are involved: (1) Inserting a linker sequence between the grass carp NPM1a and type II reovirus VP38 genes to synthesize a fusion gene of grass carp NPM1a and type II reovirus VP38; (2) The fusion gene of grass carp NPM1a and type II reovirus VP38 was connected to a recombinant expression vector and transformed into an Escherichia coli expression strain; (3) Induce the expression of recombinant protein, precipitate and purify it to obtain the fusion recombinant protein of grass carp NPM1a and type II reovirus VP38.

6. The preparation method according to claim 5, wherein Inserting the linker sequence in step (1) specifically includes: using the Overlap PCR method, adding a BamH restriction site to the 5' end of the fusion sequence and an XhoI restriction site to the 3' end, and connecting the grass carp NPM1a with the type II reovirus VP38 through the flexible polypeptide encoded by the linker sequence; The amino acid sequence of the flexible polypeptide encoded by the linker sequence is shown in SEQ ID NO:

4.

7. The preparation method according to claim 5, wherein The recombinant expression vector in step (2) is a pET28a vector, and the transformation into the Escherichia coli expression strain specifically includes: using the CaCl2 method to transform the recombinant expression vector into Escherichia coli DH5α competent cells to obtain a recombinant plasmid, sequencing verification, and then transforming the recombinant plasmid into the Escherichia coli BL21 expression vector.

8. The preparation method according to claim 5, wherein The inducing expression of the recombinant protein in step (3) specifically includes: inoculating 100-200 μl of Escherichia coli BL21 containing the recombinant plasmid into 200 ml of LB liquid culture medium at a ratio of 1:100-1:200, adding kanamycin to the culture medium to a final concentration of 30-50 μg / ml; then placing the culture flask in a 37°C 180-220 rpm shaker and culturing to an OD of approximately 0.4-0.5; adding IPTG to the culture flask to a final concentration of 0.2-0.4 mM, and then placing the culture flask in a 16°C 120-140 rpm shaker for culturing; after 6-8 hours, centrifuging to collect the bacteria, adding 5-10 ml of protein lysis buffer, using an ultrasonic disruptor to disrupt the cells at low temperature, and centrifuging at 10,000-12,000 rpm to collect the supernatant, which is the recombinant protein; The precipitation purification specifically includes: adding the supernatant to a Ni-NTA protein chromatography column to purify the fusion recombinant protein of grass carp NPM1a and type II reovirus VP38.

9. A composition of a fusion recombinant protein of grass carp NPM1a and type II reovirus VP38, characterized in that: The composition comprises a fusion recombinant protein of grass carp NPM1a and type II reovirus VP38 and indole-3-lactic acid, with a mass ratio of 1:5 to 1:

10.

10. An application of a composition of a fusion recombinant protein of grass carp NPM1a and type II reovirus VP38, characterized in that: The composition of the fusion recombinant protein of grass carp NPM1a and type II reovirus VP38 is used to improve the grass carp's ability to resist hemorrhagic disease. The application amount of each component in the composition is 0.5-1.0 mg / ml of the fusion recombinant protein and 2.5-5 mg / ml of ILA. The injected grass carp has a body length of 10-12 cm and a weight of 15-20 g, and the injection frequency is one injection.

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