Fusion protein, micropterus salmoides virus subunit vaccine and application of micropterus salmoides virus subunit vaccine

Abstract

The invention discloses a fusion protein, a micropterus salmoides virus subunit vaccine and application of the micropterus salmoides virus subunit vaccine, and belongs to the technical field of bioengineering. The fusion protein is obtained by fusing an sfGFP tag with any one of the following proteins: A1) Micropterus salmoides iridovirus MCPL protein; a2) largemouth bass rhabdovirus GM protein; wherein the amino acid sequence of the sfGFP tag is as shown in SEQ ID NO: 1. After the sfGFP tag subjected to surface ion optimization is added into the Micropterus salmoides iridovirus MCPL protein or Micropterus salmoides rhabdovirus GM protein, the soluble expression quantity of the MCPL protein and the soluble expression quantity of the GM protein can be remarkably increased, and a new technical scheme is provided for efficiently preventing Micropterus salmoides iridovirus and / or rhabdovirus at low cost; therefore, the method has a relatively good application prospect in fish soaking vaccines.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a fusion protein, a largemouth bass virus subunit vaccine and applications thereof. Background Art

[0002] Largemouth bass (Micropterus salmoides), also known as California bass. This fish has strong temperature tolerance and rapid growth ability, can survive at lower temperatures, and has tender meat and is easy to catch. However, with the expansion of aquaculture scale, the increase in aquaculture density and the deterioration of water quality, the California bass aquaculture industry is facing increasingly serious health problems. Among many diseases, viral diseases are particularly serious. Once an outbreak occurs, the mortality rate is extremely high and difficult to control. For example, largemouth bass iridovirus disease caused by Largemouth bass ranavirus (LMBV) and California bass rhabdovirus disease caused by Micropterus salmoides rhabdovirus (MSRV) have become important threats to the global California bass aquaculture industry.

[0003] In fishery production, vaccination is an important measure to improve the health of fish and prevent the spread of diseases. At present, subunit vaccines prepared by genetic engineering technology are an emerging prevention method, which has the advantages of good immune effect, high safety, wide application range and low production cost. Therefore, it has a good application prospect in the prevention of largemouth bass virus.

[0004] Due to the particularity of different vaccines, fish species and breeding conditions, in order to achieve the best immune effect, it is critical to choose the right immunization method. At present, the common immunization methods in commercial breeding mainly include injection immunization, immersion immunization and oral immunization. Among them, for fry and small fish, the immersion method is usually used for vaccination. This is because immersion immunization is easy to operate, suitable for large-scale immunization, and does not cause stress response in fish. However, during the immersion immunization process, the dosage of the vaccine needs to be increased to achieve the best immune effect. Therefore, it is of great significance to provide a method to increase the yield of the target protein in the subunit vaccine of largemouth bass virus and reduce the cost of industrial production for largemouth bass breeding. Summary of the invention

[0005] The purpose of the present invention is to provide a fusion protein, a largemouth bass virus subunit vaccine and applications thereof. In the present invention, by fusing the sfGFP tag after surface ion optimization with the largemouth bass iridovirus MCPL protein and the largemouth bass rhabdovirus GM protein, the soluble expression of the sfGFP-MCPL fusion protein and the sfGFP-GM fusion protein is significantly increased. In addition, by optimizing the fermentation process, the soluble expression of the sfGFP-MCPL fusion protein and the sfGFP-GM fusion protein is further increased, providing a new technical solution for the efficient and low-cost prevention of largemouth bass iridovirus and / or rhabdovirus.

[0006] In a first aspect, the present invention provides a fusion protein, which is obtained by fusing an sfGFP tag with any of the following proteins: A1) MCPL protein of largemouth bass iridovirus; A2) GM protein of largemouth bass rhabdovirus; wherein the amino acid sequence of the sfGFP tag is shown in SEQ ID NO: 1.

[0007] In the present invention, the inventors have found that adding a surface ion-optimized sfGFP tag to the MCPL protein of largemouth bass iridovirus or the GM protein of largemouth bass rhabdovirus can significantly increase the soluble expression level of MCPL protein and GM protein.

[0008] In some embodiments, the fusion protein is obtained by connecting the sfGFP tag to the N-terminus of the largemouth bass iridovirus MCPL protein or the largemouth bass rhabdovirus GM protein through a linker; wherein the amino acid sequence of the largemouth bass iridovirus MCPL protein is shown in SEQ ID NO: 2; the amino acid sequence of the largemouth bass rhabdovirus GM protein is shown in SEQ ID NO: 3; and the amino acid sequence of the linker is shown in SEQ ID NO: 4.

[0009] In some embodiments, the fusion protein is a protein as shown in any of the following: B1) a protein having an amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 6; B2) a protein having the same function obtained by changing one or more amino acid residues in the amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 6, wherein the change is selected from at least one of substitution, deletion, and addition; B3) a protein having more than 80% sequence identity with the amino acid sequence defined in B1) or B2) and having the same function.

[0010] The above-mentioned fusion protein provided by the present invention can be a natural, recombinant or synthetic active polypeptide, and the active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (such as Escherichia coli) or a eukaryotic host (such as yeast, higher plants) using recombinant technology.

[0011] In a second aspect, the present invention provides a nucleic acid molecule encoding any of the above-mentioned fusion proteins, and the nucleic acid molecule is any of the following: C1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 11 or SEQ ID NO: 12; C2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in C1) under stringent conditions and encodes any of the above-mentioned fusion proteins; C3) a nucleic acid molecule that has a sequence identity of more than 90% with the nucleic acid molecule defined in C1) or C2) and encodes any of the above-mentioned fusion proteins.

[0012] The nucleic acid molecules provided by the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0013] As used herein, the term "hybridize under stringent conditions" means that two nucleic acid molecule fragments hybridize to each other under standard hybridization conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (1989) (Cold Spring Lane Laboratories, New York, USA) in the section "Expression of cloned genes in Escherichia coli". Such conditions are, for example, hybridization in 6.0×SSC at 45° C., followed by a washing step in 2×SSC at 50° C. In order to select stringency, the salt concentration in the washing step can be selected, for example, between 2.0×SSC at 50° C. for low stringency and 2.0×SSC at 50° C. for high stringency. In addition, the temperature in the washing step can be varied between room temperature of about 22° C. for low stringency and 65° C. for high stringency.

[0014] As used herein, the term "sequence identity" can be evaluated by the naked eye or by computer software (such as the software program described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology). When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. A "sequence identity" in which a polynucleotide sequence or an amino acid sequence has a certain percentage (e.g., 90%, 95%, 98% or 99%) with another sequence means that when the sequences are aligned, the bases or amino acids in the two sequences being compared are the same.

[0015] In a third aspect, the present invention provides a recombinant vector comprising the above nucleic acid molecule.

[0016] The recombinant vectors in the present invention include cloning vectors and expression vectors. The cloning vectors are used to replicate related sequences, and the expression vectors are used to express related genes. The vector used in constructing the expression vector can be a pET23a vector.

[0017] In a fourth aspect, the present invention provides a recombinant cell comprising the above nucleic acid molecule or the above recombinant vector.

[0018] In some embodiments, the method for preparing a recombinant cell comprises the step of transforming the above-mentioned recombinant vector into an expression host cell.

[0019] In a fifth aspect, the present invention provides a method for preparing a fusion protein, comprising the following steps: Fermenting and culturing the recombinant cells, inducing expression, and obtaining a culture; Any of the above fusion proteins is isolated from the culture.

[0020] In some embodiments, the culture medium in the fermentation culture is selected from any one of fermentation culture medium B or fermentation culture F; the separation specifically comprises: using a membrane washing buffer to separate the fusion protein from the culture; wherein the fermentation culture medium B comprises: 15-25g / L tryptone, 5-15g / L yeast extract, 5-15g / L NaCl, 3-7g / L K2HPO4, 3-7g / L glucose; the fermentation culture F comprises: 20-30g / L tryptone, 15-20g / L yeast extract, 6-10g / L K2HPO4, 3-7g / L KH2PO4, 3-7g / L (NH4)2SO4, 1-5g / L MgSO4, 0.1-0.5g / L MnSO4, 0.03-0.07g / L CaCl2, 14-18g / L glucose; the membrane washing buffer comprises: 30-70mM NaCl, 30-70mM Tris-HCl, 3-7mM EDTA, 0.5-1.5mM SDS, pH8.0.

[0021] In the present invention, the inventors further studied and found that by optimizing the fermentation process, the soluble expression levels of sfGFP-MCPL fusion protein and sfGFP-GM fusion protein can be further increased.

[0022] In a sixth aspect, the present invention provides a largemouth bass virus subunit vaccine, comprising any one of the above-mentioned fusion proteins or a fusion protein prepared by any one of the above-mentioned preparation methods.

[0023] The largemouth bass virus subunit vaccine provided by the invention can be used as an immersion vaccine for the prevention and treatment of fish viruses.

[0024] In the seventh aspect, the present invention provides the use of any of the above-mentioned fusion proteins, the above-mentioned nucleic acid molecules, the above-mentioned recombinant vectors, the fusion proteins prepared by any of the above-mentioned preparation methods, or the above-mentioned largemouth bass virus subunit vaccines in the preparation of products for preventing largemouth bass iridovirus and / or rhabdovirus.

[0025] The beneficial effect of the present invention is that, different from the prior art, after adding the sfGFP tag after surface ion optimization to the largemouth bass iridovirus MCPL protein or the largemouth bass rhabdovirus GM protein, the present invention can significantly increase the soluble expression level of the MCPL protein and the GM protein, and provides a new technical solution for the efficient and low-cost prevention of largemouth bass iridovirus and / or rhabdovirus; therefore, it has a good application prospect in fishery immersion vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The SDS-PAGE images of the MCPL fusion proteins with different tags in Example 1 of the present invention, wherein A is the SDS-PAGE image of the Trxa-MCPL fusion protein, B is the SDS-PAGE image of the XXA-MCPL fusion protein, C is the SDS-PAGE image of the sfGFP-MCPL fusion protein, and D is the SDS-PAGE image of the sfGFP-Trxa-MCPL fusion protein. Lane M: protein marker; Lane W: total protein of whole cell lysis; Lane S: supernatant of whole cell lysis; Lane P: precipitated protein; Figure 2 The SDS-PAGE images of GM fusion proteins with different tags in Example 1 of the present invention, wherein A is the SDS-PAGE image of Trxa-GM fusion protein, B is the SDS-PAGE image of sfGFP-GM fusion protein, C is the SDS-PAGE image of sfGFP-Trxa-GM fusion protein, and D is the SDS-PAGE image of XXA-GM fusion protein. Lane M: protein marker; Lane W: total protein of whole cell lysis; Lane S: supernatant of whole cell lysis; Lane P: precipitated protein; Figure 3 It is an SDS-PAGE diagram of the sfGFP-MCPL fusion protein expressed by the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria in different fermentation media in Example 2 of the present invention, wherein lanes AF represent fermentation media AF in sequence; Figure 4 It is an SDS-PAGE diagram of the sfGFP-GM fusion protein expressed by the BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria in different fermentation media in Example 2 of the present invention, wherein lanes AF represent fermentation media AF in sequence; Figure 5A This is an SDS-PAGE image of the sfGFP-MCPL fusion protein expressed by the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria in Example 3 of the present invention after three batch fermentation tests in a 50L high-density fermenter, wherein lanes 1-3 are the expression levels of the sfGFP-MCPL fusion protein after batch fermentation, and lanes 4-6 are BSA standards with concentrations of 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively; Figure 5B This is an SDS-PAGE image of the sfGFP-GM fusion protein expressed by the BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria in Example 3 of the present invention after three batches of fermentation tests in a 50L high-density fermenter, wherein lanes 1-3 are the expression levels of the sfGFP-GM fusion protein after batch fermentation, and lanes 4-6 are BSA standards with concentrations of 0.0625 mg / mL, 0.125 mg / mL, and 0.25 mg / mL, respectively; Fig. 6A This is an SDS-PAGE image of the sfGFP-MCPL fusion protein in the supernatant after washing the membrane of the fermented BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria using a buffer in Example 4 of the present invention, wherein lanes 1-4 are: whole cells, whole cell lysis supernatant, precipitated protein, and membrane washing supernatant, respectively; Figure 6B The SDS-PAGE diagram of the sfGFP-GM fusion protein in the supernatant after washing the membrane of the fermented BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria in Example 4 of the present invention using different buffers, wherein lanes 1-3 are: whole cells, whole cell lysis supernatant, and membrane washing supernatant, respectively; Fig. 7A This is a diagram showing the effect of using sfGFP-MCPL fusion protein to protect largemouth bass against iridovirus immunity in Example 5 of the present invention; Figure 7B This is a diagram showing the immune protection effect of sfGFP-GM fusion protein against largemouth bass rhabdovirus in Example 5 of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F. Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0029] Example 1 Expression and solubility analysis of fusion protein 1.1 Expression and solubility analysis of MCPL fusion protein 1.1.1 Vector construction The sfGFP tag (amino acid sequence as shown in SEQ ID NO: 1) and the N-terminus of the MCPL protein of the largemouth bass iridovirus (amino acid sequence as shown in SEQ ID NO: 2) were operably linked using a linker (amino acid sequence as shown in SEQ ID NO: 4) to obtain a sfGFP-MCPL fusion protein. The amino acid sequence of the sfGFP-MCPL fusion protein is shown in SEQ ID NO: 5, and the nucleotide sequence of the gene encoding the sfGFP-MCPL fusion protein is shown in SEQ ID NO: 11.

[0030] Among them, the amino acid sequences of the sfGFP tag, largemouth bass iridovirus MCPL protein and linker are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 4, respectively; the nucleotide sequences of the genes encoding the sfGFP tag, largemouth bass iridovirus MCPL protein and linker are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 10, respectively.

[0031] The above-mentioned nucleotide sequence encoding the sfGFP-MCPL fusion protein was fully synthesized, and the synthesized gene fragment replaced the sequence between BamH I and Xho I in the pET23a vector to obtain the pET23a-sfGFP-MCPL recombinant plasmid, which was sent for sequencing after colony PCR verification to obtain the pET23a-sfGFP-MCPL recombinant plasmid with the correct sequence.

[0032] Similarly, the Trxa tag, XXA tag and sfGFP-Trxa tag were operably linked to the N-terminus of the MCPL protein of the largemouth bass iris virus to obtain Trxa-MCPL fusion protein, XXA-MCPL fusion protein and sfGFP-Trxa-MCPL fusion protein; the amino acid sequences of the Trxa-MCPL fusion protein, XXA-MCPL fusion protein and sfGFP-Trxa-MCPL fusion protein are shown in SEQ ID NOs: 13-15, respectively, and the nucleotide sequences encoding the Trxa-MCPL fusion protein, XXA-MCPL fusion protein and sfGFP-Trxa-MCPL fusion protein are shown in SEQ ID NOs: 19-21, respectively.

[0033] The above-mentioned nucleotide sequences encoding Trxa-MCPL fusion protein, XXA-MCPL fusion protein and sfGFP-Trxa-MCPL fusion protein were fully synthesized, and the synthesized gene fragments replaced the sequence between BamH I and Xho I in the pET23a vector to obtain pET23a-Trxa-MCPL recombinant plasmid, pET23a-XXA-MCPL recombinant plasmid and pET23a-sfGFP-Trxa-MCPL recombinant plasmid, respectively. After verification by colony PCR, they were sent for sequencing to obtain pET23a-Trxa-MCPL recombinant plasmid, pET23a-XXA-MCPL recombinant plasmid and pET23a-sfGFP-Trxa-MCPL recombinant plasmid with correct sequences, respectively.

[0034] 1.1.2 Expression and solubility analysis of MCPL fusion protein The pET23a-sfGFP-MCPL recombinant plasmid, pET23a-Trxa-MCPL recombinant plasmid, pET23a-XXA-MCPL recombinant plasmid and pET23a-sfGFP-Trxa-MCPL recombinant plasmid constructed in 1.1.1 were transformed into Escherichia coli BL21 (DE3), and after screening and identification, BL21 (DE3)-pET23a-sfGFP-MCPL recombinant bacteria, BL21 (DE3)-pET23a-Trxa-MCPL recombinant bacteria, BL21 (DE3)-pET23a-XXA-MCPL and BL21 (DE3)-pET23a-sfGFP-Trxa-MCPL recombinant bacteria were obtained, respectively. The above recombinant bacteria were cultured, and then IPTG was added at a final concentration of 1mM for induction for 12h, and the bacteria were collected by centrifugation and stained with 20mM After the cells were resuspended in PB solution, they were broken by ultra-high pressure homogenizer and centrifuged to obtain the supernatant. The precipitate was resuspended and samples were taken for SDS-PAGE detection of target proteins. Image Lab was used for grayscale scanning to compare the solubilization efficiency of each label protein. The SDS-PAGE results are shown in Figure 2. Figure 1 shown.

[0035] from Figure 1 It can be seen that the fusion protein is expressed in the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria, BL21(DE3)-pET23a-Trxa-MCPL recombinant bacteria, BL21(DE3)-pET23a-XXA-MCPL recombinant bacteria and BL21(DE3)-pET23a-sfGFP-Trxa-MCPL recombinant bacteria. Among them, in the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria, the sfGFP-MCPL fusion protein is mainly present in the whole cell lysate supernatant, and the content in the precipitate is relatively small.

[0036] The solubility comparison results of each MCPL fusion protein are shown in Table 1.

[0037] Table 1 Comparison results of MCPL fusion protein solubility

[0038] As can be seen from Table 1, compared with other tag proteins, the sfGFP tag in the present invention can significantly promote the soluble expression of MCPL fusion protein.

[0039] 1.2 Expression and solubility analysis of GM fusion protein 1.2.1 Vector construction The sfGFP tag (amino acid sequence as shown in SEQ ID NO: 1) and the N-terminus of the largemouth bass rhabdovirus GM protein (amino acid sequence as shown in SEQ ID NO: 3) were operably connected using a linker (amino acid sequence as shown in SEQ ID NO: 4) to obtain a sfGFP-GM fusion protein. The amino acid sequence of the sfGFP-GM fusion protein is shown in SEQ ID NO: 6, and the nucleotide sequence of the gene encoding the sfGFP-GM fusion protein is shown in SEQ ID NO: 12.

[0040] Among them, the amino acid sequence of the largemouth bass rhabdovirus GM protein is shown in SEQ ID NO: 3, and the nucleotide sequence of the gene encoding the largemouth bass rhabdovirus GM protein is shown in SEQ ID NO: 9.

[0041] The above-mentioned nucleotide sequence encoding the sfGFP-GM fusion protein was fully synthesized, and the synthesized gene fragment replaced the sequence between BamH I and Xho I in the pET23a vector to obtain the pET23a-sfGFP-GM recombinant plasmid, which was sent for sequencing after colony PCR verification to obtain the pET23a-sfGFP-GM recombinant plasmid with the correct sequence.

[0042] Similarly, the Trxa tag, XXA tag and sfGFP-Trxa tag were operably linked to the N-terminus of the GM protein of the largemouth bass rhabdovirus, respectively, to obtain the Trxa-GM fusion protein, XXA-GM fusion protein and sfGFP-Trxa-GM fusion protein; the amino acid sequences of the Trxa-GM fusion protein, XXA-GM fusion protein and sfGFP-Trxa-GM fusion protein are shown in SEQ ID NOs: 16-18, respectively, and the nucleotide sequences encoding the Trxa-GM fusion protein, XXA-GM fusion protein and sfGFP-Trxa-GM fusion protein are shown in SEQ ID NOs: 22-24, respectively.

[0043] The above-mentioned nucleotide sequences encoding Trxa-GM fusion protein, XXA-GM fusion protein and sfGFP-Trxa-GM fusion protein were fully synthesized, and the synthesized gene fragments replaced the sequence between BamH I and Xho I in the pET23a vector to obtain pET23a-Trxa-GM recombinant plasmid, pET23a-XXA-GM recombinant plasmid and pET23a-sfGFP-Trxa-GM recombinant plasmid, respectively. After verification by colony PCR, they were sent for sequencing to obtain pET23a-Trxa-GM recombinant plasmid, pET23a-XXA-GM recombinant plasmid and pET23a-sfGFP-Trxa-GM recombinant plasmid with correct sequences, respectively.

[0044] 1.2.2 Expression and solubility analysis of GM fusion protein The pET23a-sfGFP-GM recombinant plasmid, pET23a-Trxa-GM recombinant plasmid, pET23a-XXA-GM recombinant plasmid and pET23a-sfGFP-Trxa-GM recombinant plasmid constructed in 1.2.1 were transformed into Escherichia coli BL21 (DE3), and after screening and identification, BL21 (DE3)-pET23a-sfGFP-GM recombinant bacteria, BL21 (DE3)-pET23a-Trxa-GM recombinant bacteria, BL21 (DE3)-pET23a-XXA-GM and BL21 (DE3)-pET23a-sfGFP-Trxa-GM recombinant bacteria were obtained, respectively. The above recombinant bacteria were cultured, and then IPTG was added at a final concentration of 1mM for induction for 12h, and the bacteria were collected by centrifugation and stained with 20mM After the cells were resuspended in PB solution, they were broken by ultra-high pressure homogenizer and centrifuged to obtain the supernatant. The precipitate was resuspended and samples were taken for SDS-PAGE detection of target proteins. Image Lab was used for grayscale scanning to compare the solubilization efficiency of each label protein. The SDS-PAGE results are shown in Figure 2. Figure 2 shown.

[0045] from Figure 2 It can be seen that the fusion protein is expressed in BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria, BL21(DE3)-pET23a-Trxa-GM recombinant bacteria, BL21(DE3)-pET23a-XXA-GM recombinant bacteria and BL21(DE3)-pET23a-sfGFP-Trxa-GM recombinant bacteria. Among them, in BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria, the sfGFP-GM fusion protein is mainly present in the whole cell lysis supernatant, and the content in the precipitate is relatively small.

[0046] The solubility comparison results of each GM fusion protein are shown in Table 2.

[0047] Table 2 Comparison of GM fusion protein solubility

[0048] It can be seen from Table 2 that, compared with other tag proteins, both the sfGFP tag and the XXA tag in the present invention can significantly promote the soluble expression of GM fusion protein.

[0049] Example 2 Optimization of high-density fermentation of fusion protein This example optimizes the types of fermentation medium used in the high-density fermentation process of the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria and the BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria constructed in Example 1, wherein the types of fermentation medium are shown in Table 3 below: Table 3 Types of fermentation media

[0050] Specifically, the recombinant bacterial liquid was inoculated into fermenters containing different fermentation mediums, and fermented at a temperature of 37°C and a rotation speed of 200 rpm. 600 When the OD value was 30-40, IPTG with a final concentration of 1 mM was added for induction, and 10 mL of samples were taken 10 h after induction to measure the OD value. 600 The cells were centrifuged at 7000 rpm for 5 min to compare the wet weight of the cells, and the cells were diluted and resuspended in water at a ratio of 1:10. Finally, SDS-PAGE was used to detect the protein expression. The results were as follows: Figure 3 and 4 shown.

[0051] from Figure 3 It can be seen that the target sfGFP-MCPL fusion protein is well expressed in fermentation medium A, B, E, and F by the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria, while the target sfGFP-MCPL fusion protein is almost not expressed in fermentation medium C and D. Combined with the comparison of growth, wet weight of bacteria, and expression, the optimal medium for high-density fermentation of the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria in the present invention is fermentation medium F.

[0052] from Figure 4 It can be seen that the target sfGFP-GM fusion protein is well expressed in the fermentation medium A and B by the BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria, while the target sfGFP-GM fusion protein is almost not expressed in the fermentation medium C, D, E and F. Combined with the comparison of growth, wet weight of bacteria and expression, the optimal medium for high-density fermentation of the BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria in the present invention is fermentation medium B.

[0053] Example 3 Fusion protein 50L high-density fermentation batch test The fermentation medium F was placed in a 50L high-density fermenter, and then the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria were inoculated and fermented in three batches. The fermentation culture was carried out at a temperature of 30-37°C and a rotation speed of 200-300 rpm. 600 When the OD value was 30-35, IPTG with a final concentration of 1 mM was added for induction, and 10 mL of samples were taken 8 h after induction to measure the OD value. 600 The cells were centrifuged at 7000 rpm for 5 min to compare the wet weight of the cells, and the cells were diluted and resuspended in water at a ratio of 1:10. Finally, SDS-PAGE was used to detect the protein expression in different batches of experiments. The results are shown in Figure 2. Figure 5A shown.

[0054] from Figure 5A It can be seen that in the three batches of experiments, the target sfGFP-MCPL fusion protein was successfully expressed.

[0055] OD between each batch of experiments 600 The values, wet weight of bacteria and proportion of target protein are shown in Table 4 below: Table 4 OD between batches 600 Value, wet weight of bacteria and target protein percentage results

[0056] As can be seen from Table 4, in the three batch fermentation experiments, the OD 600 The values, wet weight of bacteria and proportion of target protein were relatively stable with little difference. The results showed that the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria had good stability in 50L high-density fermentation.

[0057] Similarly, fermentation medium B was placed in a 50L high-density fermenter, and then inoculated with BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria for three batches of fermentation. Fermentation was carried out at a temperature of 30-37°C and a rotation speed of 200-300 rpm. 600 When the OD value was 30-35, IPTG with a final concentration of 0.5 mM was added for induction, and 10 mL of samples were taken 10 h after induction to measure the OD value. 600 The cells were centrifuged at 7000 rpm for 5 min to compare the wet weight of the cells, and the cells were diluted and resuspended in water at a ratio of 1:10. Finally, SDS-PAGE was used to detect the protein expression in different batches of experiments. The results are shown in Figure 2. Figure 5B shown.

[0058] from Figure 5B It can be seen that in the three batches of experiments, the target sfGFP-GM fusion protein was successfully expressed.

[0059] OD between each batch of experiments 600 The values, wet weight of bacteria and proportion of target protein are shown in Table 5 below: Table 5 OD between batches 600 Value, wet weight of bacteria and target protein percentage results

[0060] As can be seen from Table 5, in the three batch fermentation experiments, the OD 600 The values, wet weight of bacteria and proportion of target protein were relatively stable with little difference. The results showed that the BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria had good stability in 50L high-density fermentation.

[0061] Example 4 Demembranation test of fusion protein First, prepare a membrane washing buffer comprising 50 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1 mM SDS, pH 8.0.

[0062] Then, the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria and BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria obtained by fermentation in Example 3 were placed on ice to fully cool, and then centrifuged to collect the bacteria, the culture solution after centrifugation was discarded, and 10 ml of membrane washing buffer was added per gram of bacteria to resuspend them, and then the resuspended bacteria were placed in a silent mixer and incubated overnight at 4°C. After incubation, they were centrifuged at 4000 rpm for 10 min, and the supernatant was collected to observe whether the supernatant turned green; in the actual operation process, the recombinant bacteria can be washed multiple times, and then the supernatants can be combined to increase the concentration of the target protein; finally, SDS-PAGE was used to detect the target protein in the supernatant, and the results were as follows. Fig. 6A and 6B shown.

[0063] from Fig. 6AIt can be seen that the content of the target sfGFP-MCPL fusion protein in the supernatant is significantly increased by using the membrane washing buffer of the present invention to wash the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria. The results show that in the present invention, the target sfGFP-MCPL fusion protein with higher purity can be obtained by directly using the membrane washing buffer to wash the BL21(DE3)-pET23a-sfGFP-MCPL recombinant bacteria without the need for bacterial disruption.

[0064] from Figure 6B It can be seen that the content of the target sfGFP-GM fusion protein in the supernatant is significantly increased by using the membrane washing buffer of the present invention to wash the BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria. The results show that in the present invention, the target sfGFP-GM fusion protein with higher purity can be obtained by directly using the membrane washing buffer to wash the BL21(DE3)-pET23a-sfGFP-GM recombinant bacteria without the need for bacterial disruption.

[0065] Example 5 Test on the immune protection effect of fusion protein on largemouth bass iridovirus and rhabdovirus This example studies the immune protection effect of the fusion protein on largemouth bass iridovirus and rhabdovirus, specifically, comprising the following steps: 1) 320 largemouth black bass of similar length and weight were selected and randomly divided into 8 groups, each with 40 fish, of which 2 groups were treated with PB buffer as control, and the remaining 6 immune groups were respectively treated with 10 mg / L, 30 mg / L, and 50 mg / L sfGFP-MCPL fusion protein (purified in Example 4) and sfGFP-GM fusion protein (purified in Example 4) samples for 6 h; 2) On the 28th day after immunization, the largemouth bass in the sfGFP-MCPL fusion protein immunization group and the PB buffer control group were challenged with 100 μL of 5.1×10 4 TCID 50 At the same time, the largemouth bass in the sfGFP-GM fusion protein immunization group and the PB buffer control group were challenged with LMBV virus liquid by intraperitoneal injection of 100 μL of 5.1×10 4 TCID 50 MSRV virus fluid; 3) Record the mortality of largemouth bass within 14 days and calculate the relative protection rate. The results are as follows: Fig. 7A and 7BThe relative survival rate (RPS) was calculated by the following formula: relative survival rate (RPS) = 1-[mortality percentage of the immune group / mortality percentage of the control group] × 100%.

[0066] from Fig. 7A It can be seen that the sfGFP-MCPL fusion protein has a good immune protection effect on largemouth bass iridovirus, and as the concentration of sfGFP-MCPL fusion protein increases, the protection efficiency increases; Figure 7B It can be seen that the sfGFP-GM fusion protein has a good immune protection effect on largemouth bass rhabdovirus, and as the concentration of the sfGFP-GM fusion protein increases, the protection efficiency increases; the above results show that the sfGFP-MCPL fusion protein and sfGFP-GM fusion protein provided in the present invention have good immune protection effects on largemouth bass iridovirus and rhabdovirus, respectively, and have good application prospects in fish immersion vaccines.

[0067] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0068] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A fusion protein, characterized in that The fusion protein is obtained by fusing the sfGFP tag with any of the following proteins: A1) MCPL protein of largemouth bass iridovirus; A2) Largemouth bass rhabdovirus GM protein; Wherein, the amino acid sequence of the sfGFP tag is shown in SEQ ID NO:

1.

2. The fusion protein according to claim 1, characterized in that The fusion protein is obtained by connecting the sfGFP tag to the N-terminus of the largemouth bass iridovirus MCPL protein or the largemouth bass rhabdovirus GM protein through a linker; Among them, the amino acid sequence of the MCPL protein of the largemouth bass iridovirus is shown in SEQ ID NO: 2; the amino acid sequence of the GM protein of the largemouth bass rhabdovirus is shown in SEQ ID NO: 3; and the amino acid sequence of the Linker is shown in SEQ ID NO:

4.

3. The fusion protein according to claim 1, characterized in that The fusion protein is any of the following proteins: B1) a protein having an amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 6; B2) a protein having the same function obtained by changing one or more amino acid residues of the amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 6, wherein the change is selected from at least one of substitution, deletion and addition; B3) A protein having a sequence identity of more than 80% with the amino acid sequence defined in B1) or B2) and having the same function.

4. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 3, characterized in that: The nucleic acid molecule is any of the following: C1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 11 or SEQ ID NO: 12; C2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in C1) and encodes the fusion protein according to any one of claims 1 to 3; C3) A nucleic acid molecule having a sequence identity of more than 90% with the nucleic acid molecule defined in C1) or C2) and encoding the fusion protein according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that: Comprising the nucleic acid molecule of claim 4.

6. A recombinant cell, characterized in that Comprising the nucleic acid molecule according to claim 4 or the recombinant vector according to claim 5.

7. A method for preparing a fusion protein, characterized in that: The steps include: Fermenting and culturing the recombinant cell according to claim 6, and inducing expression to obtain a culture; The fusion protein of any one of claims 1 to 3 is isolated from the culture.

8. The preparation method according to claim 7, characterized in that: The culture medium in the fermentation culture is selected from any one of fermentation medium B or fermentation medium F; The separation specifically includes: using a membrane washing buffer to separate the fusion protein from the culture; Wherein, the fermentation medium B comprises: 15-25 g / L tryptone, 5-15 g / L yeast extract powder, 5-15 g / L NaCl, 3-7 g / L K2HPO4, and 3-7 g / L glucose; The fermentation culture F comprises: 20-30 g / L tryptone, 15-20 g / L yeast extract, 6-10 g / L K2HPO4, 3-7 g / L KH2PO4, 3-7 g / L (NH4)2SO4, 1-5 g / L MgSO4, 0.1-0.5 g / L MnSO4, 0.03-0.07 g / L CaCl2, and 14-18 g / L glucose; The membrane washing buffer comprises: 30-70 mM NaCl, 30-70 mM Tris-HCl, 3-7 mM EDTA, 0.5-1.5 mM SDS, pH 8.

0.

9. A largemouth bass virus subunit vaccine, characterized in that: The invention comprises the fusion protein according to any one of claims 1 to 3 or the fusion protein prepared by the preparation method according to any one of claims 7 to 8.

10. Use of the fusion protein according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the fusion protein prepared by the preparation method according to any one of claims 7 to 8, or the largemouth bass virus subunit vaccine according to claim 9 in the preparation of products for preventing largemouth bass iridovirus and / or rhabdovirus.

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