CN116375814A - Megalopsis iridovirus MCP-2 recombinant protein and application thereof - Google Patents
Megalopsis iridovirus MCP-2 recombinant protein and application thereof Download PDFInfo
- Publication number
- CN116375814A CN116375814A CN202211434003.7A CN202211434003A CN116375814A CN 116375814 A CN116375814 A CN 116375814A CN 202211434003 A CN202211434003 A CN 202211434003A CN 116375814 A CN116375814 A CN 116375814A
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- Prior art keywords
- mcp
- protein
- recombinant
- pet32a
- strain
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Links
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Abstract
According to the invention, the core antigen epitope of the virus antigen can be obtained by cutting off the MCP-2 protein fragment expressing the MCP protein, so that the immunoprotection performance is improved. The MCP-2 protein is further prepared into subunit vaccine injection preparation, so that stress response symptoms after injection are reduced, the health-care rate of injection immunity is improved, and a high-strength toxicity attack test shows that the injection preparation has the highest relative immune protection rate. The invention has wide application prospect in developing high-efficiency large-mouth black bass iridovirus vaccine and preventing and controlling the same.
Description
Technical field:
the invention belongs to the technical field of biology, and particularly relates to a megalopsis iridovirus MCP-2 recombinant protein and application thereof.
The background technology is as follows:
the largehead jewfish (Micropterus salmoides) is native in fresh water rivers and lakes in North America, also called California jewfish and jewfish, belongs to the order of Perciformes, the subgrade of Perciformes, the family of Cehtrachidae (Cehtrachidae) and the genus of jewfish (Micropterus), belongs to wide-temperature fishes, has the characteristics of high growth speed, short cultivation period, fresh, tender, rich meat, high price and the like, and is deeply favored by fishermen. The culture of the largemouth black bass has better economic benefit in China, and rapidly develops into main economic culture fish seeds in China in more than ten years. The largehead jewfish is susceptible to various virus diseases in the cultivation process, wherein the damage of the largehead jewfish iridovirus disease (Largemouthbass virus disease) caused by the largehead jewfish iridovirus (Largemouth bass virus, LMBV) is the most serious.
To date, vaccination has remained lacking an effective therapeutic approach to the diseases associated with LMBV, the most effective method of preventing infectious diseases in aquaculture, and thus LMBV-related vaccine development remains a major issue in addressing this viral disease. Up to now, there is no commercialized iridovirus vaccine for micropterus salmoides in domestic and foreign markets, and the development of vaccine for LMBV is also in an exploration stage, mainly comprising nucleic acid vaccine and subunit vaccine. Researchers find that the DNA vaccine of LMBV has better immune effect, and the immune protection rate can reach 63% (Yi et al 2020); although DNA vaccines can provide better immunoprotection, there is a potential risk to the body. In the aspect of subunit vaccine, ma Dongmei and the like (2016) find that the subunit vaccine of the whole length of the MCP gene for encoding the micropterus salmoides iridovirus can achieve 67.7% protection rate; the subunit vaccine constructed by Jia and the like (2020) is used for preventing and treating infection caused by the iridoid virus of the largehead jewfish by a soaking immunization mode, and the immune protection rate can reach more than 80 percent.
Currently, fish vaccines are vaccinated mainly by three means: intraperitoneal or intramuscular injection immunization, bathing or spraying immunization and oral immunization fed with vaccine-containing feed. The injection immunization mainly comprises the steps of injecting the antigen into the fish body, wherein the injection part is generally provided with an abdomen, a muscle, a ventral fin base part and the like, the injection immunization can accurately control the dosage of the antigen, so that the organism generates higher serum antibody level, has lasting immunization effect, and can generate higher immune protection (the protection rate is generally about 40-90 percent) for the organism. However, the injection immunization is complicated in operation, has high requirements on the professionality of the breeder and the inoculation equipment, consumes labor, financial resources and time, is not suitable for the immunization of fish fries or small fish species, is easy to cause stress reaction of fish bodies, and is easy to cause bacterial infection due to accompanying physical damage, so that the survival rate is reduced, the immunization effect is poor, and in the injection immunization, only the vaccine cannot have a good immune protection effect, so that an adjuvant is usually added to improve the immunization effect of the vaccine. Traditional adjuvants (such as mineral oil) are widely applied to fish bacterial vaccines, but the adjuvants are not easy to inject, can cause stronger tissue inflammatory reaction and generate obvious side effects, and in the prior research, carbon nano tube particles, beta-glucan, plant polysaccharide, cytokines and Chinese herbal medicine components are also applied to fish vaccines as adjuvants. In the preparation process of the LMBV vaccine, how to reduce the stress response of fish bodies, reduce death caused by stress and improve the immunity is also a technical problem which needs to be solved at present.
In addition, screening of dominant epitopes of viruses is also a common method for improving vaccine effect, and the dominant epitopes are generally screened to improve vaccine immune protection effect by searching core antigens with strong immunogenicity. Some epitopes on an antigen can induce activation of lymphocytes to generate humoral and cellular immunity, but lymphocytes can only recognize antigen components of some small molecules, and one antigen usually contains a large number of epitopes, so that the discovery of epitopes with strong immunogenicity is a key to developing more effective subunit vaccines. MCP, the major capsid protein, is a viable viral vaccine antigen widely accepted worldwide. Although the LMBVCP gene sequence of the micropterus salmoides is conserved among different strains, early experiments show that the dominant epitope distribution information of the LMBVCP of the micropterus salmoides is difficult to accurately obtain through sequence analysis. At present, reports of vaccine product development and application by utilizing dominant epitope of LMBV MCP of micropterus salmoides are not seen.
The invention comprises the following steps:
in order to solve the technical problems, on the basis of the early MCP subunit vaccine research, other possibilities of the largehead jewfish iridescent subunit vaccine are further researched so as to enrich the largehead jewfish iridescent subunit vaccine and explore a more effective immunization scheme. The invention aims to provide an MCP-2 recombinant protein containing MCP main epitope and application thereof, wherein the recombinant protein has good immunogenicity and has positive significance for preparing subunit vaccine.
In order to solve the technical problems, the invention adopts the following technical scheme:
the recombinant protein MCP-2 based on the largemouth black bass iridovirus antigen is characterized in that the amino acid sequence of the recombinant protein MCP-2 is shown as SEQ ID No: shown at 5.
A gene encoding said recombinant protein MCP-2, characterized in that: the nucleotide sequence of the gene is shown as SEQ ID No: 2.
An expression vector, which is pET32a-MCP-2, constructed on the basis of the pET32a expression vector, comprising the amino acid sequence of SEQ ID No:2, and a nucleotide sequence shown in the following formula.
A genetically engineered strain for preparing the recombinant protein MCP-2 is a recombinant strain obtained by converting a recombinant plasmid pET32a-MCP-2 into E.coli BL21 (DE 3).
A method for preparing said recombinant protein MCP-2, comprising the steps of:
step one: constructing a recombinant escherichia coli E.coli BL21/pET32a-MCP-2 strain; the synthetic sequence is SEQ ID No:2, cloning the sequence between corresponding enzyme cutting sites on a pET-32a expression vector to obtain a recombinant plasmid pET32a-MCP-2, and converting the recombinant plasmid into E.coli BL21 (DE 3) to obtain a recombinant Escherichia coli E.coli BL21/pET32a-MCP-2 strain;
step two: inducible expression of recombinant E.coli BL21/pET32a-MCP-2 strain: selecting a small amount of bacterial liquid from a seed ring of a production strain E.coli BL21/pET32a-MCP-2, streaking and inoculating the bacterial liquid to an LB solid culture medium plate, standing and culturing the bacterial liquid at 37 ℃ for 16-18 hours, selecting single bacterial colony and inoculating the bacterial colony to an LB liquid culture medium, and culturing the bacterial colony at 160-180r/min for 12-16 hours at 37 ℃ to serve as first-stage seeds; inoculating 1% by volume of the first seed into LB liquid medium, culturing at 37deg.C at 160-180r/min for 14-16 hr as second seed, inoculating 1% by volume of the second seed into LB medium, adding ampicillin to final concentration of 100 μg/ml, fermenting at 37deg.C for 5-7 hr with aeration and culturing with dissolved oxygen of 30-40% until OD of the bacterial liquid is reached 600 When the value is 1.1-1.3, adding IPTG to the final concentration of 0.001mol/L, and performing induction culture at 37 ℃ for 6 hours, and stopping fermentation;
step three: treating bacterial liquid and performing ultrasonic crushing;
step four: purification of protein: by Ni 2+ And carrying out affinity chromatography separation on the chelate affinity chromatography column to obtain the protein, and dialyzing to obtain the purified protein.
The injection preparation of the MCP-2 subunit vaccine of the largemouth black bass iridovirus is characterized by comprising the following components: MCP-2 recombinant protein 100 μg/mL, astragalus polysaccharide 5mg/mL, taurine 2mg/mL, sodium selenite 50 μg/mL, zinc sulfate 20 μg/mL, inosine 2mg/mL, sodium ascorbate 500 μg/mL, span-8050mg/mL; the amino acid sequence of the recombinant protein MCP-2 is shown as SEQ ID No: shown at 5.
The nucleotide sequence of the coding recombinant protein MCP-2 is shown as SEQ ID No: 2.
The preparation method of the recombinant protein MCP-2 comprises the following steps:
step one: constructing a recombinant escherichia coli E.coli BL21/pET32a-MCP-2 strain; the synthetic sequence is SEQ ID No:2, cloning the sequence between corresponding enzyme cutting sites on a pET-32a expression vector to obtain a recombinant plasmid pET32a-MCP-2, and converting the recombinant plasmid into E.coli BL21 (DE 3) to obtain a recombinant Escherichia coli E.coli BL21/pET32a-MCP-2 strain;
step two: inducible expression of recombinant E.coli BL21/pET32a-MCP-2 strain: selecting a small amount of bacterial liquid from a seed ring of a production strain E.coli BL21/pET32a-MCP-2, streaking and inoculating the bacterial liquid to an LB solid culture medium plate, standing and culturing the bacterial liquid at 37 ℃ for 16-18 hours, selecting single bacterial colony and inoculating the bacterial colony to an LB liquid culture medium, and culturing the bacterial colony at 160-180r/min for 12-16 hours at 37 ℃ to serve as first-stage seeds; inoculating 1% by volume of the first seed into LB liquid medium, culturing at 37deg.C at 160-180r/min for 14-16 hr as second seed, inoculating 1% by volume of the second seed into LB medium, adding ampicillin to final concentration of 100 μg/ml, fermenting at 37deg.C for 5-7 hr with aeration and culturing with dissolved oxygen of 30-40% until OD of the bacterial liquid is reached 600 When the value is 1.1-1.3, adding IPTG to the final concentration of 0.001mol/L, and performing induction culture at 37 ℃ for 6 hours, and stopping fermentation;
step three: treating bacterial liquid and performing ultrasonic crushing;
step four: protein purification using Ni 2+ And carrying out affinity chromatography separation on the chelate affinity chromatography column to obtain the protein, and dialyzing to obtain the purified protein.
The invention also claims the application of the recombinant protein of the megaphone iridovirus MCP-2 in the preparation of iridovirus subunit vaccine, and the recombinant protein of the MCP-2 can be used for injection immunization of the megaphone iridovirus, and can stimulate the organism to produce stronger corresponding antibodies compared with the MCP protein.
Based on the technical scheme, the invention has the following advantages and beneficial effects:
firstly, on the basis of the prior research of companies, on the basis of effective immune protein MCP protein, the invention can obtain the core epitope of virus antigen by cutting off the MCP-2 protein fragment expressing the MCP protein, thereby improving the immune protection performance. Animal experiments show that compared with MCP recombinant protein injection immunization, the MCP-2 recombinant protein can generate a neutralizing antibody level with higher titer, and the highest dose immunization group immune related index of the MCP-2 recombinant protein is obviously higher than that of the MCP group on the 7 th day after immunization. Based on the test results, the invention has better immunogenicity compared with the MCP-2 protein fragment after intercepting the MCP-2 protein fragment expressing the MCP protein, can stimulate the organism to generate higher neutralizing antibody level, and has positive significance for preventing LMBV.
Secondly, the invention optimizes and adjusts the formulation of the injection preparation aiming at the problems of fish death and the like caused by stress reaction, wound infection and the like in aquatic injection immunity, and simultaneously adds taurine, sodium selenite, zinc sulfate, inosine and the like to the injection preparation, thereby playing a synergistic effect among the components.
In conclusion, the invention can obtain the core epitope of the virus antigen and improve the immune protection performance by cutting off the MCP-2 protein fragment of the MCP protein. The MCP-2 protein is further prepared into subunit vaccine injection preparation, so that stress response symptoms after injection are reduced, the health-care rate of injection immunity is improved, and a high-strength toxicity attack test shows that the injection preparation has the highest relative immune protection rate. The invention has wide application prospect in developing high-efficiency large-mouth black bass iridovirus vaccine and preventing and controlling large-mouth black bass iridovirus.
Description of the drawings:
FIG. 1 is the identification of truncated recombinant plasmids. Wherein M: maker DL 5000;1-4: double cleavage products of MCP-1, MCP-2, MCP-3 and MCP-4; the marker is the target gene.
FIG. 2 shows SDS-PAGE identification of the expression of LMBV-MCP-2 recombinant protein. Wherein M: maker;1: e.coli BL21/pET32a-MCP-2 cultures after induction.
FIG. 3 shows WB identification of LMBV-MCP-2 recombinant protein. Wherein M: maker;1: uninduced E.coli BL21/pET32a-MCP-2 cultures; 2: e.coli BL21/pET32a-MCP-2 cultures after induction.
FIG. 4 shows the results of serum antibody titer determination after immunization.
Specific examples:
example 1:
construction and identification of recombinant E.coli BL21/pET32a-MCP-2 strain.
1. Materials and methods
1.1 materials
1.1.1 plasmids and strains
The pET-32a-MCP plasmid and the E.coli BL-21/pET-32a-MCP prokaryotic expression strain are preserved by aquatic animal disease laboratory of the university of northwest agriculture and forestry science and technology animal sciences.
1.1.2 reagents
Sodium chloride, absolute ethyl alcohol, glycerol, and the like are purchased from national pharmaceutical group chemical reagent limited company; the positive serum immunized by the LMBVCP protein of the largehead jewfish is preserved by an aquatic disease laboratory of the university of North agriculture and forestry science and technology animal sciences; TMB enzyme chromogenic kit, BCA protein concentration determination kit, PBST purchased from Beijing Soy Bao technology Co., ltd; dialysis bags, murine 6 Xhistidine (His) tag monoclonal antibodies, horseradish peroxidase (HorseradishPeroxidase, HRP) -labeled goat anti-mouse IgG monoclonal antibodies, his tag protein purification kits were purchased from Shanghai, inc.; primers were synthesized by Shanghai Biotechnology Co. Protein markers were purchased from Tiangen Biochemical technologies (Beijing); tryptone and yeast extract powder were purchased from sigma company in the united states; IPTG and ampicillin were purchased from shanghai microphone Biochemical technologies limited.
1.1.3 test instruments
Electronic balance ALC-1100.2, beijing Sidoris instruments systems Co., ltd; ultra clean bench YT-CJ-2ND, beijing Asia Thai Instrument technology Co., ltd; t100 type PCR instrument, bio-Rad company, USA; H1650-W type desk-top high-speed microcentrifuge, hunan instrument laboratory instrument development Co., ltd; KQ-500DE type numerical control ultrasonic cleaner, kunshan ultrasonic instruments Inc.; 1-15K high-speed refrigerated centrifuge, sigma Co., USA; ultrasonic cell disruption apparatus, ningbo Xinzhi biotechnology Co., ltd; DYCZ-24DN vertical electrophoresis apparatus, six instrument factories in Beijing; thermo Multiskan MK3 enzyme-labeled instrument, U.S. thermoelectric Sieimer Feier technology Co.
1.2 test methods and results
1.2.1 Gene validation and expression plasmid construction of each truncated form of MCP
Based on the LMBV FS001 strain MCP protein gene sequence (Accession No.: OM 319463), the gene sequence was truncated into 4 segments and designated MCP-1 (SEQ ID No: 1), MCP-2 (SEQ ID No: 2), MCP-3 (SEQ ID No: 3) and MCP-4 (SEQ ID No: 4), with 30bp overlapping bases between the two segments, and sent to the Bio-company for synthesis of an expression vector ligated to pET-32 a.
And respectively carrying out double enzyme digestion identification and sequencing identification on the constructed segmented gene recombinant plasmids pET-32a-MCP-1, pET-32a-MCP-2, pET-32a-MCP-3 and pET-32a-MCP-4. After enzyme digestion, the result of electrophoresis detection (shown in figure 1) is combined with the sequencing result of plasmids, which shows that each plasmid is constructed correctly, wherein the corresponding band size of the MCP gene is 1392bp, and the corresponding band sizes of the segmented genes MCP-1, MCP-2, MCP-3 and MCP-4 are 375, 378 and 369bp respectively.
1.2.2 Construction of prokaryotic expression strains of various truncated genes of MCP
The segmented gene recombinant plasmids are named as pET-32a-MCP-1, pET-32a-MCP-2, pET-32a-MCP-3 and pET-32a-MCP-4, so that corresponding prokaryotic expression strains E.coli BL-21/pET-32a-MCP-1, E.coli BL-21/pET-32a-MCP-2, E.coli BL-21/pET-32a-MCP-3 and E.coli BL-21/pET-32a-MCP-4 are obtained.
1.2.3 Prokaryotic expression of MCP and its truncations
The four truncated prokaryotic expression strains and the expression strain containing the untruncated MCP gene constructed in the previous step are respectively inoculated into 100mL of LB liquid medium containing ampicillin (100 mu g/mL), and placed in a shaking table at 37 ℃ and 180rpm for shaking culture until the bacteria grow to a logarithmic phase (OD) 600 =1.1 to 1.5), isopropyl- β -D-thiogalactoside (IPTG) was added to a final concentration of 0.001mol/L in the medium, shaking culture was continued for 6 to 8 hours, and induction was completed.
1.2.4 Isolation and purification of MCP and its truncations
Collecting the bacterial liquid induced to express in the last step, centrifuging for 10min at the temperature of 12000g and 4 ℃, and discarding the supernatant. An equal volume of phosphate buffer (Phosphate buffered saline, PBS) was added to the pellet for resuspension, followed by sonication with a sonicator (power 300w, sonication 2s, interval 3 s) until the bacterial solution was clear and transparent (reaction was performed on ice). Then by containing His tag Ni 2+ Purifying the obtained protein liquid by using a chelate affinity chromatographic column, freeze-drying the purified protein solution to prepare freeze-dried powder, and storing at a low temperature of-20 ℃ to be dissolved in sterile water when in use.
1.2.5 Evaluation of immunogenicity of MCP truncations
Immunogenicity was assessed using an enzyme-linked adsorption assay (Enzyme linked immunosorbent assay, ELISA) as follows: firstly, diluting the micropterus salmoides positive serum by using ELISA coating liquid at a ratio of 1:1000, adding 100 mu L of diluted serum into each hole of an ELISA plate as antigen, and coating for 24 hours at the temperature of 4 ℃. After removing the liquid from the wells, 200. Mu.L of 1% bovine serum albumin was added to each well and the wells were blocked at 37℃for 1 hour. Rinsing with phosphate Tween buffer (Phosphate buffered saline with Tween-20, PBST) 3 times for 3min. Subsequently, 100. Mu.L of the protein solution from which the expression products of the different recombinant plasmids were isolated and purified was added to the wells, and incubated at 37℃for 1 hour. PBST was rinsed 3 times, followed by incubation at 37℃for 1h, respectively, with murine 6 XHis-tag monoclonal antibody as primary antibody and HRP-labeled goat anti-mouse IgG monoclonal antibody as secondary antibody. After PBST is rinsed for 3 times, TMB color development liquid is added, and the mixture is placed in a dark place at 37 ℃ for 3-5min for color development reaction. Then 50. Mu.L of a color-development stop solution was added to each well, and the absorbance (OD) of each microplate at a wavelength of 450nm was measured by a microplate reader in 20 minutes 450 ) The results are shown in Table 1.
TABLE 1 immunogenicity evaluation of MCP truncations
Under the condition that the target protein content is the same, table 1 shows that the OD value of MCP-2 is obviously higher than that of MCP and other sections of MCP, and proves that the immunogenicity of MCP-2 is obviously higher than that of MCP and other sections of MCP, and the result provides a basis for finally screening and obtaining dominant antigen epitopes of main capsid proteins of the megaphone iridovirus.
1.2.6 Identification of MCP dominant antigen epitope protein MCP-2
The truncated protein MCP-2 with the best result in the last step is selected for SDS-PAGE detection and identification, and the specific flow is as follows: carrying out denaturation treatment on a sample by using a 5 XSDS-PAGE loading buffer solution, respectively preparing a lower layer separating gel and an upper layer concentrating gel, immersing the gel in an electrophoresis buffer solution after the gel is solidified, adding the denatured sample into a gel hole, respectively carrying out electrophoresis for 20 and 80 minutes under 80V and 120V voltages, and respectively carrying out WesternBlot analysis on electrophoresis results by using a Coomassie brilliant blue method for dyeing, decolorizing and observing and using a murine His tag monoclonal antibody as a primary antibody and using an HRP-marked goat anti-mouse IgG monoclonal antibody as a secondary antibody.
SDS-PAGE analysis was performed on the protein product expressed by the recombinant plasmid containing the dominant MCP-2 epitope. In the analysis results, the size of the expressed target protein containing MCP-2 was found to be about 35kDa (FIG. 2), which is the same as the expected result; in the Western Blot analysis, it was seen that there was a distinct, design-compliant recognition band at 35kDa (FIG. 3). These results indicate that recombinant plasmid pET-32a-MCP-2 can effectively express MCP-2 truncated protein in colibacillus.
Example 2
Immune effect of dominant antigen epitope MCP-2 subunit vaccine of main capsid protein of largemouth black bass iridovirus.
2 materials and methods
2.1 materials
2.1.1 test animals and viruses
Healthy largehead jewfish (1.0.+ -. 0.5g,4-5 cm) was purchased from a farm in the city of Buddha, guangdong. The water temperature for raising is 25+/-1 ℃, and the dissolved oxygen is kept above 6 mg/L. Feeding twice at 8 a day and 5 a afternoon and timely removing residual bait and feces at the bottom of the fish tank, and changing water for half volume of the fish tank every 2 d. After 14d of temporary rearing, the test was performed. The used iridovirus of the largehead black bass is preserved in aquatic animal disease laboratory of northwest university of agriculture and forestry science and technology.
2.1.2 reagents
MCP truncations (specifically MCP-2) were prepared in the laboratory for aquatic animal diseases at the university of North Western agriculture and forestry, animal sciences, as described in example 1 above. The murine His-tag monoclonal antibody, HRP-labeled goat anti-mouse IgG monoclonal antibody were purchased from Biotechnology (Shanghai) Inc. The other reagents were all analytically pure.
2.1.3 test instruments
ALC-1100.2 electronic balance, beijing Sidoris instruments systems Co., ltd; HH-4 digital display constant temperature water bath, shanghai Ten mechanical equipments limited company; H1650-W type desk-top high-speed microcentrifuge, hunan instrument laboratory instrument development Co., ltd; 1-15K high-speed refrigerated centrifuge, sigma Co., USA; ultrasonic cell disruption apparatus, ningbo Xinzhi biotechnology Co., ltd; DYCZ-24DN vertical electrophoresis apparatus, six instrument factories in Beijing; thermo MultiskanMK3 enzyme-labeled instrument, U.S. thermoelectric Sieimer Feier technology Co; t100 type PCR apparatus, burley, USA.
2.2 test methods
2.2.1 immunization
And (3) performing injection immunization on the healthy largemouth bass after temporary culture for 14 days by adopting a dorsal fin basal intramuscular injection mode, wherein the injection volume of each fish is 10 mu L. The prepared MCP and MCP-2 are respectively dissolved in sterile PBS at corresponding concentrations to serve as vaccines, and diluted into different concentration gradients to evaluate the effect of the vaccines at different immune doses. The treatment dose was 10.0 μg/tail for the MCP group and 1.0, 5.0 and 10.0 μg/tail for the MCP-2 group (see Table 2 for specific groupings), each vaccine treatment group contained 60 fish and each group contained three replicates.
TABLE 2 immune grouping Condition
2.2.2 serum immune antibody titer determination
Blood was collected from micropterus salmoides 7d, 14d, 21d, 28d after immunization, 3 tail fish per sampling for antibody titer determination. The collected blood sample was allowed to stand at room temperature for 2 hours, and then left at 4℃overnight to allow it to spontaneously coagulate. And finally, centrifuging for 10min by using a low-temperature refrigerated centrifuge at 5000g, collecting upper serum, and storing at-20 ℃ for later antibody titer determination. When the antibody titer is measured, purified MCP is taken as an antigen, the serum of the micropterus salmoides is taken as the serum to be measured, the murine-His tag monoclonal antibody is taken as a primary antibody, the HRP goat anti-mouse IgG monoclonal antibody is taken as a secondary antibody, the dilution ratio is 1:1000, and the antibody titer in the serum is measured by adopting an ELISA method. After color development, the absorbance at a wavelength of 450nm was measured by an enzyme-labeled instrument.
The results of the serum immune antibody titer measurement of the micropterus salmoides after injection immunization are shown in fig. 4, the antibody titer levels of the MCP and MCP-2 vaccine treatment groups are gradually increased along with the extension of the immunization time at the beginning of 14d after immunization, the antibody titer levels are remarkably improved compared with the Control group at each detection time point, and the highest antibody titer level is reached at 28d after immunization. The antibody titer level of each dose of MCP-2 vaccine treatment group after immunization is obviously higher than that of the Control group, and the antibody titer of the highest dose of vaccine treatment group (10 mug/tail) is obviously higher than that of other dose of vaccine treatment groups, which can reach about 3 times of the Control group. Furthermore, 28d after immunization, the antibody titer of the MCP-2 vaccine treated group with 5 μg/tail dose was already higher than that of the MCP vaccine treated group (10 μg/tail).
2.2.3 toxicity test
After 28d immunization, 37 micropterus salmoides are randomly taken from each group, and placed in a new fish tank under the same feeding condition for toxicity test. Injecting 3.50X10 of each fish by intraperitoneal injection 6 TCID 50 /mL LMBV virus solution (50. Mu.L). Continuously observing for 14d, checking and recording the morbidity at regular time, and finally calculating the death rate and the relative immune protection rate:
relative immune protection rate = (1-vaccine treatment group mortality/Control group mortality) ×100%.
After the immune 28d largehead jewfish is subjected to virus attack by using LMBV virus, the morbidity and mortality of the largehead jewfish are recorded every day, and after the virus attack is performed for 14d, each group of largehead jewfish reaches a relatively stable state and the quantity change is small. The results of statistics on mortality and relative immunoprotection rates of the groups of micropterus salmoides within 14d are shown in Table 3, which shows that the mortality rate of the Control group is 100%, the mortality rate of all vaccine treatment groups is lower than that of the Control group, and the relative immunoprotection rate is higher than that of the Control group. After 14d challenge, the relative immune protection rate of the MCP-2 group (specifically the highest dose vaccine treatment group) was highest, reaching 73.0%, whereas the relative immune protection rate of the MCP vaccine treatment group was only 45.9%.
TABLE 3 toxicity counteracting mortality after immunization and relative immune protection Rate
Based on the test results, the MCP-2 recombinant protein has relatively better immunogenicity compared with the MCP recombinant protein, can stimulate organisms to generate higher antibody levels in early immune stage, realizes immune protection in early infection stage, has positive significance for epidemic prevention of fish shoals in early disease stage, can reach appropriate antibody levels in a shorter time, and has positive significance for preventing mass death of fish fries in early infection stage.
Example 3.
Preparation of a dominant antigen epitope MCP-2 subunit vaccine injection preparation of the main capsid protein of the largehead jewfish iridovirus.
The invention further researches how to enhance the immune effect of MCP-2 subunit vaccine injection preparation and reduce weever death caused by stress reaction in the injection immune process. Wherein the MCP-2 recombinant protein is protein obtained by separation and purification (the purity is more than or equal to 60 percent); astragalus polysaccharides (analytically pure) were purchased from Wuhan Hua Xiangke Biotechnology Co., ltd., CAS:89250-26-0; taurine, sodium selenite, zinc sulfate, inosine, sodium ascorbate and span-80 are all purchased from reagent companies, and meet the related requirements of pharmacopoeia injection.
The MCP-2 subunit vaccine injection preparation provided by the invention comprises the following components: MCP-2 recombinant protein 100 μg/mL, astragalus polysaccharide 5mg/mL, taurine 2mg/mL, sodium selenite 50 μg/mL, zinc sulfate 20 μg/mL, inosine 2mg/mL, sodium ascorbate 500 μg/mL, span-8050mg/mL.
The preparation method of the MCP-2 subunit vaccine injection preparation comprises the following steps:
firstly, weighing MCP-2 recombinant protein, astragalus polysaccharide, taurine, sodium selenite, zinc sulfate, inosine, sodium ascorbate and span-80 according to parts by weight;
secondly, dissolving the weighed astragalus polysaccharide, taurine, sodium selenite, zinc sulfate, inosine and sodium ascorbate in normal saline, stirring and dissolving, then adding span-80 and MCP-2 recombinant protein, adopting normal saline to carry out constant volume, and rapidly and uniformly stirring to obtain mixed emulsion;
thirdly, filtering and sterilizing the mixed emulsion, and sub-packaging to obtain the MCP-2 subunit vaccine injection preparation.
Example 4.
During the course of the study of the injection according to the invention, numerous experiments and studies were carried out on the selection of the components and the determination of the amounts used in order to obtain the optimal injection formulation, in particular the following experiments were also carried out during the course of the study according to the invention, which are shown here as a comparison:
table 4 composition ratio test of injection preparation
The subunit vaccine injectable preparation of the present invention and the injectable preparations of controls 1 to 5 were prepared by the method described in example 3, respectively, in example 3 and in controls 1 to 5.
Example 5.
Animal experiments with MCP-2 subunit vaccine injection formulations.
5.1 safety and health after immunization:
taking 100-120g of healthy largemouth black bass 350 tails, temporarily raising for 14 days, and dividing the largemouth black bass into an immune group and a blank control group, wherein each group has 50 tails. Injecting and immunizing by adopting a dorsal fin basal intramuscular injection mode after anesthesia, injecting 0.1mL of corresponding vaccine into each immune group, replacing vaccine by normal saline in a blank control group, transferring into a normal culture water body for reviving and feeding after injection, continuously observing for 28 days, and recording health and activity conditions of each group. The specific results are shown in Table 5 below.
TABLE 5 health conditions 28 days after injection immunization
Based on the results of table 5, it is seen that within 28 days after injection, weever is killed to some extent due to stress reaction, wound, and the like of injection. The blank control group dies 10 animals, the feed intake is obviously reduced in the feeding process, obvious stress symptoms are shown, the injection wounds of the dead weever animals all show a certain infection phenomenon, the health-care rate is 80%, the example 3 and the control groups 1-3 show higher health-care rates, particularly, the example 3 of the optimal formula obtained by the invention only dies 1 animal, the health-care rate is up to 98%, the stress response symptoms of the weever animals in the group are lighter, the feed intake is maintained at the normal level before immunization, and the fish flexibility and other aspects are not different from those before immunization. In addition, based on comparison of control groups 2 and 3 and example 3, the components such as taurine, sodium selenite, zinc sulfate, inosine and the like are added simultaneously, and compared with the components, the anti-stress capability of weever is improved to a large extent, the stress response symptom after injection is reduced, and the health-care rate of injection immunity is improved.
5.2 toxicity test
After 28d immunization, 37 micropterus salmoides are randomly taken from each group, and placed in a new fish tank under the same feeding condition for toxicity test. Injecting 3.50X10 of each fish by intraperitoneal injection 6 TCID 50 /mL LMBV virus solution (50. Mu.L). Continuously observing for 14d, checking and recording the morbidity at regular time, and finally calculating the death rate and the relative immune protection rate:
relative immunoprotection = (1-vaccine treatment group mortality/placebo group mortality) ×100%.
After the immune 28d largehead jewfish is subjected to virus attack by using LMBV virus, the morbidity and mortality of the largehead jewfish are recorded every day, and after the virus attack is performed for 14d, each group of largehead jewfish reaches a relatively stable state and the quantity change is small. The results of statistics on mortality and relative immunoprotection rates for each group of micropterus salmoides within 14d are shown in Table 6.
TABLE 6 toxicity counteracting mortality and relative immunoprotection rates after immunization of different injectable formulations
| Healthy and alive mantissa | Mortality (%) | Relative immune protection Rate (%) | |
| Example 3 | 33 | 10.8％ | 89.2％ |
| |
0 | 100.0％ | 0.0 |
| Control | |||
| 1 | 29 | 21.6％ | 78.4 |
| Control | |||
| 2 | 30 | 18.9％ | 81.1 |
| Control | |||
| 3 | 30 | 18.9％ | 81.1 |
| Control | |||
| 4 | 28 | 24.3％ | 75.7％ |
| Control 5 | 31 | 16.2％ | 83.8％ |
Based on the results in table 6, the mortality rate of the placebo group was 100%, and the mortality rate of all vaccine-treated groups was lower than that of the placebo group, while the relative immunoprotection rate was higher than that of the placebo group. After 14d of challenge, the relative immune protection rate of example 3 was highest, reaching 89.2%, and increased by approximately 13.5% relative to 75.7% for control group 4; compared with the control group 1-3, the injection preparation has a certain synergistic effect among the components, and can reduce the immune effect under the condition of lacking astragalus polysaccharide, zinc sulfate, sodium selenite and other components, and compared with the control group 5, the specific proportion limitation of the injection preparation also improves the immune effect to a greater extent.
The above description is only of the preferred embodiments of the present invention and should not be taken as limiting the scope of the invention, and therefore, equivalent changes according to the claims of the present invention still fall within the scope of the present invention.
Claims (8)
1. The recombinant protein MCP-2 based on the largemouth black bass iridovirus antigen is characterized in that the amino acid sequence of the recombinant protein MCP-2 is shown as SEQ ID No: shown at 5.
2. The recombinant protein MCP-2 encoding gene of claim 1, wherein: the nucleotide sequence of the gene is shown as SEQ ID No: 2.
3. The expression vector of recombinant protein MCP-2 of claim 1, said expression vector being pET32a-MCP-2, characterized in that it is an expression vector constructed on the basis of a pET-32a expression vector, comprising the sequence of SEQ ID No:2, and a nucleotide sequence shown in the following formula.
4. A genetically engineered strain for preparing the recombinant protein MCP-2 according to claim 1, wherein the recombinant strain is obtained by transforming a recombinant plasmid pET32a-MCP-2 into E.coli BL21 (DE 3).
5. A method for preparing the recombinant protein MCP-2 of claim 1, comprising the steps of:
step one: constructing a recombinant escherichia coli E.coliBL21/pET32a-MCP-2 strain; the synthetic sequence is SEQ ID No:2, cloning the sequence between corresponding enzyme cutting sites on a pET-32a expression vector to obtain a recombinant plasmid pET32a-MCP-2, and transforming the recombinant plasmid into E.coliBL21 (DE 3) to obtain a recombinant Escherichia coli E.coliBL21/pET32a-MCP-2 strain;
step two: inducible expression of recombinant E.coli strain E.coli BL21/pET32 a-MCP-2: selecting a small amount of bacterial liquid from a seed ring of a production strain E.coliBL21/pET32a-MCP-2, streaking and inoculating the bacterial liquid to an LB solid culture medium plate, standing and culturing for 16-18 hours at 37 ℃, inoculating a single colony to an LB liquid culture medium, and culturing for 12-16 hours at 160-180r/min at 37 ℃ to serve as first-stage seeds; inoculating 1% by volume of the first seed into LB liquid medium, culturing at 37deg.C at 160-180r/min for 14-16 hr as second seed, inoculating 1% by volume of the second seed into LB medium, adding ampicillin to final concentration of 100 μg/ml, fermenting at 37deg.C for 5-7 hr with aeration and culturing with dissolved oxygen of 30-40% until OD of the bacterial liquid is reached 600 When the value is 1.1-1.3, adding IPTG to the final concentration of 0.001mol/L, and performing induction culture at 37 ℃ for 6 hours, and stopping fermentation;
step three: treating bacterial liquid and performing ultrasonic crushing;
step four: purification of protein: by Ni 2+ And carrying out affinity chromatography separation on the chelate affinity chromatography column to obtain the protein, and dialyzing to obtain the purified protein.
6. The injection preparation of the MCP-2 subunit vaccine of the largemouth black bass iridovirus is characterized by comprising the following components: MCP-2 recombinant protein 100 μg/mL, astragalus polysaccharide 5mg/mL, taurine 2mg/mL, sodium selenite 50 μg/mL, zinc sulfate 20 μg/mL, inosine 2mg/mL, sodium ascorbate 500 μg/mL, span-8050mg/mL; the amino acid sequence of the recombinant protein MCP-2 is shown as SEQ ID No: shown at 5.
7. The injection preparation of the largemouth black bass iridovirus MCP-2 subunit vaccine according to claim 7, wherein the nucleotide sequence encoding the recombinant protein MCP-2 is as set forth in SEQ id no: 2.
8. The injection preparation of the largemouth black bass iridovirus MCP-2 subunit vaccine according to claim 7, wherein the preparation method of the recombinant protein MCP-2 includes the steps of:
step one: constructing a recombinant escherichia coli E.coliBL21/pET32a-MCP-2 strain; the synthetic sequence is SEQ ID No:2, cloning the sequence between corresponding enzyme cutting sites on a pET-32a expression vector to obtain a recombinant plasmid pET32a-MCP-2, and converting the recombinant plasmid into E.coliBL21 (DE 3) to obtain a recombinant Escherichia coli E.coliBL21/pET32a-MCP-2 strain;
step two: inducible expression of recombinant E.coli strain E.coli BL21/pET32 a-MCP-2: selecting a small amount of bacterial liquid from a seed ring of a production strain E.coliBL21/pET32a-MCP-2, streaking and inoculating the bacterial liquid to an LB solid culture medium plate, standing and culturing for 16-18 hours at 37 ℃, inoculating a single colony to an LB liquid culture medium, and culturing for 12-16 hours at 160-180r/min at 37 ℃ to serve as first-stage seeds; inoculating 1% by volume of the first seed into LB liquid medium, culturing at 37deg.C at 160-180r/min for 14-16 hr as second seed, inoculating 1% by volume of the second seed into LB medium, adding ampicillin to final concentration of 100 μg/ml, fermenting at 37deg.C for 5-7 hr with aeration and culturing with dissolved oxygen of 30-40% until OD of the bacterial liquid is reached 600 When the value is 1.1-1.3, adding IPTG to the final concentration of 0.001mol/L, and performing induction culture at 37 ℃ for 6 hours, and stopping fermentation;
step three: treating bacterial liquid and performing ultrasonic crushing;
step four: purification of protein: by Ni 2+ And carrying out affinity chromatography separation on the chelate affinity chromatography column to obtain the protein, and dialyzing to obtain the purified protein.
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| CN118324901B (en) * | 2024-06-13 | 2024-08-27 | 深圳万可森生物科技有限公司 | Single-chain antibody protein for resisting largemouth black bass iridovirus, screening method and application thereof |
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