CN110981945B - Expression preparation and application of porcine circovirus type 2 recombinant Cap protein - Google Patents
Expression preparation and application of porcine circovirus type 2 recombinant Cap protein Download PDFInfo
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Abstract
The invention discloses an expression preparation and application of porcine circovirus type 2 recombinant Cap protein. The sequence of the porcine circovirus type 2 recombinant Cap protein is the sequence 2 in the sequence table. The coding gene is sequence 1 in the sequence table. The preparation method of the porcine circovirus type 2 Cap recombinant protein virion comprises the following steps: 1) the recombinant vector for expressing the porcine circovirus type 2 Cap recombinant protein is used for transforming the competence of escherichia coli BL21(DE3) to obtain recombinant bacteria for expressing porcine circovirus type 2 Cap recombinant protein virions; 2) inducing expression of the recombinant bacteria, collecting and crushing the bacteria, and collecting inclusion bodies; 3) and (4) carrying out dissolution and renaturation on the inclusion body to obtain the assembled porcine circovirus type 2 Cap recombinant protein virion. The invention adopts an escherichia coli expression system to express the porcine circovirus type 2 Cap protein, has large protein expression quantity and simple purification method, greatly reduces the production cost and is beneficial to promoting mass production.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to inclusion body expression, purification, renaturation and application of porcine circovirus type 2 recombinant Cap protein.
Background
Porcine Circovirus (PCV) belongs to the family circoviridae, the genus circovirus, with three serotypes PCV1, PCV2 and PCV 3. The PCV virion is of a regular icosahedral symmetrical structure, has the diameter of about 17nm, is the smallest animal virus known at present, and has no envelope on the surface of the virus. Porcine circovirus disease is an infectious disease caused by porcine circovirus type 2, and is clinically mainly characterized by poor growth and development, emaciation, sow dysreproduction, dyspnea, subcutaneous lymph node high swelling, hemorrhage, necrosis and the like, and is divided into clinical types such as weaned piglet multisystemic failure syndrome (PMWS), porcine dermatitis and nephrotic syndrome (PNDS), Proliferative and Necrotizing Pneumonia (PNP), Reproductive failure (Reproductive failure), Perinatal myocarditis (porcine myocarpis), piglet Congenital tremor (genetic tumors) and the like. The research shows that PCV2 can cause the apoptosis of immune cells in the porcine lymphatic system, cause the immunosuppression, lead the resistance of the organism to be reduced, thereby inducing the mixed infection and the secondary infection of a plurality of bacterial viruses and causing huge economic loss to the breeding industry.
Porcine circovirus is a single negative-strand circular DNA virus, the viral genome containing at least 11 open reading frames, i.e., ORF1-ORF 11. Wherein ORF1 is the largest open reading frame of PCV, encodes the Rep protein (314aa, about 36kDa), a virus replication-related protein responsible for the cross-reactivity of PCV1 and PCV2 antigens, ORF1 is relatively conserved, with about 85% homology between the two serotypes; ORF2 encodes the viral capsid protein Cap protein (233aa, about 27kDa), which is a type-specific antigen with good immunogenicity, no antigenic cross-reactivity between the two serotypes, and only 67% homology between the two serotypes. PCV3 is a new serous type discovered only in recent years, and the amino acid homology of the Cap protein encoded by ORF2 with the Cap protein of PCV2 is about 34%, so relatively few studies on PCV3 are carried out at present.
At present, five types of PCV2 gene subtypes reported in China are PCV2a, PCV2b, PCV2c, PCV2d and PCV2 e. Serological investigation finds that the domestic swinery positive rate is as high as 52.8-100%, the morbidity is as high as 50%, the mortality is generally 5-70% due to different conditions of pig farms, secondary infection and the like, and huge economic loss is caused to the pig industry. Vaccine immunization plays a critical role in the prophylactic control of PCV 2. At present, porcine circovirus vaccines mainly comprise inactivated vaccines, attenuated live vaccines and subunit vaccines, wherein inactivated vaccines are mostly used in China, but PCV2 virus is not easy to propagate on cells, the titer of produced virus is generally low, the immune effect is not ideal enough, and in addition, the inactivated vaccine also has the problems of difficult detection of the inactivated effect, short immune period and the like, and the production cost is increased compared with the inactivated vaccines of other viruses; the safety of subunit vaccines is accepted by the industry, but the level of the subunit vaccines produced in China is different, and the imported subunit vaccines have good immune effect but higher price.
Disclosure of Invention
The invention aims to provide a method for preparing porcine circovirus type 2 recombinant Cap protein by an Escherichia coli expression system, and the protein has good biological activity and immunogenicity.
Based on the purpose, the invention provides a porcine circovirus type 2 recombinant Cap protein, the sequence of which is sequence 2 in a sequence table.
The invention also provides a coding gene of the porcine circovirus type 2 recombinant Cap protein; preferably, the coding gene of the porcine circovirus type 2 recombinant Cap protein is shown as a sequence 2 in a sequence table.
The invention also provides a recombinant vector for expressing the porcine circovirus type 2 recombinant Cap protein, which is obtained by inserting the coding gene of the porcine circovirus type 2 recombinant Cap protein shown in the sequence 1 into an original vector.
The starting vector is preferably pET-30a (+).
The invention also aims to provide a preparation method of the porcine circovirus type 2 Cap recombinant protein virions, which comprises the following steps:
1) transforming the recombinant vector into escherichia coli BL21(DE3) competence to obtain a recombinant bacterium for expressing the porcine circovirus type 2 Cap recombinant protein virion;
2) inducing and expressing the recombinant bacteria obtained in the step 1), collecting and crushing bacteria, and collecting inclusion bodies;
3) dissolving and renaturing the inclusion body obtained in the step 2) to obtain the assembled porcine circovirus type 2 Cap recombinant protein virions.
Specifically, the method comprises the following steps:
(1) synthesizing a target gene: inserting the optimized Cap gene sequence shown in the sequence 1 between Nde I and Xho I enzyme recognition sites of pET-30a (+), obtaining a recombinant vector for expressing the recombined Cap protein, and naming the recombinant vector as pET-CapR;
(2) screening of recombinant strains: transforming the plasmid pET-CapR obtained in the step (1) into Escherichia coli competent BL21(DE 3); obtaining an engineering strain for expressing porcine circovirus type 2 Cap recombinant protein virus particles, and naming the engineering strain as E.coli-PCV 2-FJ-Cap; (3) inducing expression by inclusion bodies: culturing the strain E.coli-PCV2-FJ-Cap obtained in the step (2), and inducing expression protein by IPTG;
(4) and (3) inclusion body purification: centrifugally collecting the recombinant bacteria obtained in the step (3), ultrasonically crushing bacteria, centrifugally removing supernatant, scraping bacterial sludge on the upper layer of sediment, and collecting inclusion bodies;
(5) denaturation and renaturation of inclusion bodies: dissolving the inclusion body by using an inclusion body dissolving solution, slowly adding the dissolved inclusion body into an inclusion body renaturation solution for dilution renaturation, and stirring for 48 hours at 4 ℃; the formula of the renaturation liquid is as follows: 50mM Tris-HCl, 400mM L-arginine, pH adjusted to 8.0;
(6) concentration and purification: slowly adding the replacement solution into the renaturation solution, and concentrating at 4 ℃ to obtain concentrated Cap protein; the formula of the replacement liquid is as follows: 20mM Tris-HCl, 50mM NaCl, pH adjusted to 8.0.
The engineering strain for expressing the porcine circovirus type 2 Cap recombinant protein virions is classified and named as follows: escherichia coli, strain name E.coli-PCV 2-FJ-Cap; has been preserved in China general microbiological culture Collection center (China academy of sciences, institute of microbiology, No. 3, West Lu No.1, Beijing, Chaoyang, North-China) in 16.7.2019, and the preservation number is CGMCC No. 18235.
The invention relates to application of the porcine circovirus type 2 recombinant Cap protein or the coding gene of the porcine circovirus type 2 recombinant Cap protein of claim 2 in preparation of porcine circovirus type 2 recombinant Cap protein virions.
The application of the porcine circovirus type 2 recombinant Cap protein or the coding gene of the porcine circovirus type 2 recombinant Cap protein of claim 2 in the preparation of vaccines for preventing or treating the circovirus type 2 also belongs to the protection scope of the invention.
The invention has the following positive beneficial effects:
the invention adopts an escherichia coli expression system to express the porcine circovirus type 2 recombinant Cap protein, has large protein expression quantity and simple purification method, greatly reduces the production cost and is beneficial to promoting mass production;
the invention utilizes an escherichia coli prokaryotic expression system to express the recombinant Cap protein of PCV2 in the form of inclusion body, solves the technical problem, ensures that the recombinant Cap protein obtained after the inclusion body is processed has higher VLP assembly rate, better biological activity and immunogenicity, greatly reduces the production cost and lays a foundation for the development of subunit vaccine.
The invention optimizes the coding sequence of the porcine circular ring 2 type recombinant Cap protein, improves the protein expression amount, and treats the inclusion body by the protein renaturation liquid and the replacement liquid with specific formulas, so that the finally obtained recombinant Cap protein has better biological activity, higher VLP (VLP) assembly rate and better protein stability;
animal experiments prove that the recombinant Cap protein prepared by the invention can generate high-level antibodies when used for immunizing mice and pigs, and lays a foundation for the establishment of vaccine preparation and detection methods.
Drawings
FIG. 1 is a PCR identification picture of porcine circovirus type 2 recombinant Cap protein CGMCC No.18235 colony;
FIG. 2 is porcine circovirus type 2 recombinant Cap protein CGMCC No.18235SDS-PAGE gel staining picture and VLP assembled electron microscope picture;
FIG. 3 shows the DLS detection result of porcine circovirus type 2 recombinant Cap protein;
FIG. 4 is SDS-PAGE gel staining pictures before and after porcine circovirus type 2 recombinant Cap protein sequence optimization.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
Example 1 preparation of recombinant Cap protein of porcine circovirus type 2
1. Experimental Material
The primer is synthesized from Shanghai Czeri bioengineering GmbH, and Escherichia coli BL21(DE3) is competent for laboratory preservation;
a main reagent DNA plasmid small extraction kit; peptone, yeast extract were purchased from Oxoid; IPTG, L-arginine hydrochloride, Tris-HCL from Amresco; BSA protein standards were purchased from Thermo Fisher Scientific;
2. construction of porcine circovirus type 2 recombinant Cap protein expression plasmid
The sequence of the recombinant Cap gene is from PCV2 epidemic strain separated in the laboratory, the sequence of the recombinant Cap gene is analyzed and compared, the Cap protein sequence is subjected to structure prediction after 41 basic amino acids at the N end are removed, the structure is compared with the analyzed PCV2 Cap protein structure (PDB:3R0R), and CCP4 software package is used for calculating and analyzing the van der Waals force of Contacts between subbunits. Under the premise of keeping the amino acids of main antigen regions such as B folding, C folding, BC loop, C terminal and the like on the surface of virus particles unchanged, carrying out amino acid substitution on main Contacts between 3-axis subbunits and 5-axis subbunits, for example, amino acid residues of DE loop and EF loop participating in hydrophobic interaction are subjected to a plurality of rounds of structural simulation and amino acid substitution, synthesizing a target gene by Nanjing Czeityury organism Limited after codon optimization, constructing the synthesized gene in a pET-30a (+) vector (the vector is stored by the laboratory), inserting two enzyme cutting sites of NdeI and XhoI into 5 'and 3' of a recombinant Cap gene respectively, wherein the sequence of the optimized recombinant Cap gene is shown as a sequence 1 in a sequence table, and the protein sequence expressed by the optimized recombinant Cap gene is shown as a sequence 2 in the sequence table.
3. Inducible expression of porcine circovirus type 2 recombinant Cap protein
The optimized recombinant Cap gene sequence shown in sequence 1 was inserted between Nde I and Xho I enzyme recognition sites of pET-30a (+) to obtain a recombinant vector expressing the recombinant Cap protein, which was designated as pET-CapR.
Respectively transforming the expression plasmid pET-CapR into escherichia coli BL21(DE3) competence, adding 1 mu L of expression plasmid into 100 mu L of competence, after gently shaking and uniformly mixing, carrying out ice bath for 30min, carrying out heat shock in a water bath kettle at 42 ℃ for 90s, continuously carrying out ice bath for 3min, adding 800 mu L of non-resistant LB culture medium, culturing for 1h at 37 ℃ and 200rpm, centrifuging for 5min at 6000rpm, discarding 750 mu L of supernatant, carrying out heavy suspension on the thalli by using the remaining culture medium, uniformly coating the thalli on an LB plate with kanamycin resistance, and culturing at 37 ℃ overnight.
Selecting a single colony for PCR identification, and carrying out PCR amplification on a total reaction system of 25 mu l, wherein the total reaction system contains an upstream primer PCV2-F-1(CATATGAATGGCATCTTCAACACCCG, NdeI restriction site sequence underlined) 1. mu.l, and downstream primer PCV2-R-1 (N-acetyl-L-D-L-PCV)CTCGAGCTTAGGGTTAAGTGGGGG, XhoI cleavage site sequence underlined), 1. mu.l of PCR enzyme MIX 12.5. mu.l, 9.5. mu.l of sterile distilled water, and 1. mu.l of template DNA. Circulating reaction parameters: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45 seconds, annealing at 56 ℃ for 30 seconds, and extension at 72 ℃ for 1 minute for 32 cycles; final extension at 72 ℃ for 10 min.
The PCR products were detected by electrophoresis on a 1% agarose gel (see FIG. 1 for results). As can be seen from FIG. 1, a band of about 600bp was amplified, corresponding to the expected size of the amplified fragment.
The recombinant engineering strain with positive detection is an engineering strain for expressing porcine circovirus type 2 Cap recombinant protein virions, and is classified and named as follows: escherichia coli, strain name E.coli-PCV 2-FJ-Cap; has been preserved in China general microbiological culture Collection center (China academy of sciences, institute of microbiology, No. 3, West Lu No.1, Beijing, Chaoyang, North-China) in 16.7.2019, and the preservation number is CGMCC No. 18235.
Culturing E.coli-PCV2-FJ-Cap positive single colony overnight at the temperature of 37 ℃ at 200rpm to be used as a seed solution, inoculating the seed into LB culture medium with kana resistance according to the proportion of 1:100 the next day, culturing at the temperature of 37 ℃ at 200rpm for 2-3 h, adding IPTG with the final concentration of 1mmol/L when the OD600 of the bacterial solution reaches 0.6-0.8, and continuing to perform induction culture at the temperature of 37 ℃ for 5 h.
4. Collecting and crushing thallus
Centrifuging the thallus for 10min at 5000rpm, collecting the thallus, resuspending the thallus with PBS, concentrating the recombinant Escherichia coli by 10 times, namely centrifuging 50mL of bacterial liquid, then resuspending with 5mL of PBS, ultrasonically crushing the bacteria, and setting parameters of an ultrasonic crusher as follows: the power is 190W, the operation is 5s, the operation is stopped for 10s, the ultrasonic treatment is carried out for 10min, and the thalli are completely broken.
5. Purification of Inclusion bodies
Centrifuging the bacterium solution subjected to the ultrasonic treatment at 12000rpm for 10min at low temperature, discarding the supernatant, scraping the upper layer of gray bacterium mud by using a cell scraper, fully resuspending the inclusion body by using an inclusion body washing solution, centrifuging at 12000rpm for 10min at low temperature, discarding the supernatant, and scraping the bacterium mud again to obtain a primarily purified inclusion body;
the inclusion body washing solution has the following formula: 0.5% Triton X-100, 50mM Tris-HCl, 300mM NaCl, pH adjusted to 8.0.
6. Denaturation and renaturation of Inclusion bodies
The inclusion bodies were weighed, dissolved in a solution at a ratio of 30mg/mL, and stirred thoroughly overnight to completely dissolve the inclusion bodies. 2mL of the inclusion body solution is slowly added into 200mL of renaturation solution, the renaturation is carried out for 48h under the stirring at the temperature of 4 ℃, and the mixture is concentrated to 50mL by a concentration cup.
The inclusion body dissolving solution has the following formula: 8M Urea, 50mM Tris-HCl, 100mM NaCl, 10% glycerol, 10mM EDTA, pH adjusted to 8.0.
The formula of the renaturation liquid is as follows: 50mM Tris-HCl, 400mM L-arginine, pH adjusted to 8.0.
7. Concentration and replacement of renaturation liquid
And slowly adding the replacement solution to the renaturation solution to 1L, stirring at 4 ℃, and concentrating to 50mL to obtain the renaturated Cap protein.
The formula of the replacement liquid is as follows: 20mM Tris-HCl, 50mM NaCl, pH adjusted to 8.0.
8. Protein characterization and electron microscopy
The purity of the recombinant Cap protein was checked by SDS-PAGE and the assembly of VLPs was checked by electron microscopy (see FIG. 2 for results).
9. DSL (dynamic light scattering) detection
The assembly of the formed VLPs was detected by DSL (dynamic light scattering) and the results are shown in fig. 3, which shows that the radius of the particles was 8.87nm, accounting for more than 90% (see fig. 3).
Example 2 preparation of porcine circovirus type 2 recombinant Cap protein subunit vaccine
1. Protein production
pET-CapR was picked and transformed into Escherichia coli BL21(DE3) to obtain recombinant strain single colony E.coli-PCV2-FJ-Cap expressing recombinant Cap protein was prepared according to the method of steps 3-7 in example 1, namely: escherichia coli E.coli-coli-PCV 2-FJ-Cap CGMCC No.18235 is inoculated in 20ml of LB culture medium, the mixture is cultured overnight at 37 ℃ and 200rpm to serve as a seed solution, the seeds are inoculated in 5L of LB culture medium containing kanamycin resistance on the next day according to the proportion of 1:100, the culture is carried out for 2-3 h at 37 ℃ and 200rpm, IPTG with the final concentration of 1mmol/L is added when the OD600 of the bacterial solution reaches 0.6-0.8, and the induction culture is continued for 5h at 37 ℃. Centrifuging the thallus at 5000rpm for 10min, collecting the thallus, re-suspending the thallus by using 500mL PBS, ultrasonically crushing the thallus, and setting parameters of an ultrasonic crusher as follows: power 190W, working 5s, switching off 10s, ultrasound 1 h. Centrifuging at 12000rpm for 10min after ultrasonic treatment, removing the supernatant, scraping off gray bacterial mud, resuspending the inclusion bodies by inclusion body cleaning solution, centrifuging at 12000rpm for 10min, removing the supernatant, scraping off bacterial mud again, and obtaining the preliminarily purified inclusion bodies. The inclusion bodies were dissolved at a ratio of 30mg/mL and thoroughly stirred overnight. 2mL of the inclusion body solution is slowly added into 200mL of renaturation solution, and the renaturation is carried out for 48h under the stirring at the temperature of 4 ℃. And slowly adding the replacement solution to the renaturation solution to 1L, stirring at 4 ℃, and concentrating to 50mL to obtain the renaturated recombinant Cap protein. And finally, detecting the concentration of the recombinant Cap protein by using an SDS-PAGE gel gray scale method.
Meanwhile, the wild-type porcine circovirus type 2 Cap gene (sequence 3 in the sequence table) is inserted between Nde I and Xho I enzyme recognition sites of pET-30a (+) to obtain a recombinant vector for expressing wild Cap protein, and the recombinant vector is named as pET-Cap.
And (2) selecting a single colony of a recombinant bacterium for transforming escherichia coli BL21(DE3) into pET-Cap to obtain competence and expressing a recombinant wild Cap protein, inoculating the single colony into 20ml of LB culture medium, culturing at 37 ℃ overnight at 200rpm to obtain a seed solution, inoculating 5L of LB culture medium containing kanamycin resistance into the seeds on the next day according to the ratio of 1:100, culturing at 37 ℃ for 2-3 hours at 200rpm, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 1mmol/L when the OD600 of the bacterial solution reaches 0.6-0.8, and continuing to perform induction culture at 37 ℃ for 5 hours. Centrifuging the thallus at 5000rpm for 10min, collecting the thallus, re-suspending the thallus by using 500mL PBS, ultrasonically crushing the thallus, and setting parameters of an ultrasonic crusher as follows: power 190W, working 5s, switching off 10s, ultrasound 1 h. Centrifuging at 12000rpm for 10min after ultrasonic treatment, removing the supernatant, scraping off gray bacterial mud, resuspending the inclusion bodies by inclusion body cleaning solution, centrifuging at 12000rpm for 10min, removing the supernatant, scraping off bacterial mud again, and obtaining the preliminarily purified inclusion bodies. The inclusion bodies were dissolved at a ratio of 30mg/mL and thoroughly stirred overnight. 2mL of the inclusion body solution is slowly added into 200mL of renaturation solution, and the renaturation is carried out for 48h under the stirring at the temperature of 4 ℃. And slowly adding the replacement solution to the renaturation solution to 1L, stirring at 4 ℃, and concentrating to 50mL to obtain the renaturated recombinant Cap protein. And finally, detecting the concentration of the recombinant Cap protein by using an SDS-PAGE gel gray scale method.
The result shows that the coding sequence of the porcine circovirus type 2 Cap protein is optimized, the protein expression quantity is improved, the expression quantity of the Cap gene sequence in the inclusion body before and after optimization is compared by an SDS-PAGE method, the result shows that the expression quantity of the sequence-optimized Cap protein inclusion body is improved by about 35 percent (as shown in figure 4) compared with that before and after optimization, the final rate of the sequence-optimized Cap protein is about 160mg/L of culture solution after washing, denaturation and renaturation of the inclusion body, and the final rate of the Cap protein before optimization is about 50mg/L of culture solution.
Preparation of 2-circovirus type 2 recombinant Cap protein subunit vaccine
The recombinant Cap protein subjected to filtration sterilization is mixed with 206 adjuvant of SEPPIC company according to the mass ratio of 1:1 to prepare the vaccine, the final concentration of the Cap protein in the vaccine is 0, 5, 10, 20, 50 and 100 mu g/ml respectively, the emulsification condition is that the adjuvant and the water phase are respectively warm-bathed to 31 ℃, the water phase is slowly added into the adjuvant, the stirring is carried out at 350rpm for 5min, and the cold bath is carried out at 20 ℃ for 1 hour to avoid moving and stirring. And (3) after the mixing is complete, obtaining the vaccine composition, wherein the emulsification quality of the vaccine can reach the quality standard specified by the state.
Example 3: immunogenicity research of porcine circovirus type 2 recombinant Cap protein subunit vaccine
1. Piglet safety test
Screening 35 circular antigen antibody double-negative piglets with the ages of 2-3 weeks for carrying out a safety test of the circular virus type 2 recombinant Cap protein subunit vaccine. Grouping condition: randomly selecting 30 piglets to be evenly divided into 5 groups and 5 piglets per group to serve as a vaccine immunization group, immunizing 4ml of circovirus type 2 recombinant Cap protein subunit vaccine containing 5 mu g/ml recombinant Cap protein by each pig in the immunization group 1, and immunizing 4ml of circovirus type 2 recombinant Cap protein subunit vaccine containing 10 mu g/ml recombinant Cap protein by each pig in the immunization group 2; each pig of the immunization group 3 is immunized with 4ml of a circovirus type 2 recombinant Cap protein subunit vaccine containing 20 mug/ml recombinant Cap protein, each pig of the immunization group 4 is immunized with 4ml of a circovirus type 2 recombinant Cap protein subunit vaccine containing 50 mug/ml recombinant Cap protein, and each pig of the immunization group 5 is immunized with 4ml of a circovirus type 2 Cap protein subunit vaccine containing 100 mug/ml recombinant Cap protein; immunization group 6 Each pig immunized 4ml of circovirus type 2 Cap protein subunit vaccine of wild Cap protein at 100 mug/ml; the remaining 5 heads served as control groups, 4ml of PBS per head and neck muscle injection. Measuring the body temperature every 0-7 d after immunization; continuously observing the appetite and mental state of the swinery for 14 days; and observing and touching whether the local part of the injection part has the tumor or not.
After the circovirus type 2 Cap protein subunit vaccine is used for immunizing all groups of piglets, no obvious body temperature rise phenomenon occurs, and the pigs eat the vaccine normally and have good mental state; the vaccine injection site was free of pain and lumps at 5d post-immunization (table 1). The vaccine is proved to have good safety for the immunized piglets.
Table 1 shows the clinical symptom monitoring results of piglets immunized by porcine circovirus type 2 recombinant Cap protein vaccine
| Group of | Body temperature | Appetite stimulation | Mental state | Injection site | Health condition | Survival |
| Immunization group | ||||||
| 1 | Is normal | Is normal | Good effect | No abnormality | |
100 |
| Immunization group | ||||||
| 2 | Is normal | Is normal | Good effect | No abnormality | |
100 |
| Immunization group | ||||||
| 3 | Is normal | Is normal | Good effect | No abnormality | |
100％ |
| Immunization group 4 | Is normal | Is normal | Good effect | No abnormality | |
100％ |
| Immunization group 5 | Is normal | Is normal | Good effect | No abnormality | |
100％ |
| Immunization group 6 | Is normal | Is normal | Good effect | No abnormality | |
100％ |
| Control group | Is normal | Is normal | Good effect | No abnormality | |
100％ |
2. Mouse immunization test
2.1 mouse immunization procedure
35 5-6 weeks old circular antigen antibody double-negative healthy clean female BALB/c mice are subjected to an antibody level generation test of circular virus type 2 recombinant Cap protein subunit vaccine. Grouping condition: randomly selecting 30 mice to be evenly divided into 6 groups and 5 mice per group as vaccine immunization groups, wherein the immunization groups comprise 0.2ml of circovirus type 2 Cap protein subunit vaccine containing 5 mu g/ml of circovirus recombinant Cap protein under subcutaneous multipoint immunization of the back of each mouse, 0.2ml of circovirus type 2 recombinant Cap protein subunit vaccine containing 10 mu g/ml of circovirus recombinant Cap protein under subcutaneous multipoint immunization of the back of each mouse, 0.2ml of circovirus type 2 recombinant Cap protein subunit vaccine containing 20 mu g/ml of circovirus recombinant Cap protein under subcutaneous multipoint immunization of the back of each mouse, the immunization groups comprise 0.2ml of circovirus type 2 recombinant Cap protein subunit vaccine containing 50 mu g/ml of circovirus recombinant Cap protein under subcutaneous multipoint immunization of the back of each mouse, immunizing a group of mice with 0.2ml of circovirus type 2 recombinant Cap protein subunit vaccine containing 100 microgram/ml of circovirus recombinant Cap protein at multiple points in the back subcutaneous of each mouse; sixthly, performing subcutaneous multipoint immunization on the back of each mouse to obtain 0.2ml of circovirus type 2 Cap protein subunit vaccine containing 100 mu g/ml of circovirus wild Cap protein; the remaining 5 were used as control groups, each with 0.2ml of subcutaneous multi-spot immunization in PBS per back. Blood was collected 3 weeks and 5 weeks after the initial immunization, and serum was separated and measured for PCV2ELISA and neutralizing antibodies.
2.2 mouse antibody detection method
The ELISA antibody detection method is as follows: coating the circular ring Cap protein with pH9.6 carbonate buffer solution, the concentration is 1 μ g/ml, 100 μ l/hole, coating overnight at 4 ℃, adding 300 μ l of 0.5% casein blocking solution to block the plate after washing, and sealing at 37 ℃ for 2 h; diluting the serum to be detected by using a sample diluent in a multiple ratio, adding the diluted sample diluent into a coated plate, setting a negative and positive serum control group at the same time, acting for 1h at 37 ℃, and washing; adding goat anti-mouse secondary antibody (diluted 1: 10000), acting at 37 deg.C for 1h with 100 μ l/well, and washing; adding substrate color development liquid TMB for color development, 100 μ l/hole, and performing reaction at 37 deg.C for 15min, with 2M H2SO4The reaction was terminated. And (4) judging a result: the average value of the negative control OD450nm should be less than or equal to 0.15, otherwise, the negative control OD450nm is invalid; each detection value of the positive control should be between 1.0 and 2.5, otherwise, the positive control is invalid; calculation of the critical value: the cut-off value was 0.18 × the mean value of the positive control OD450nm values. The serum to be detected is determined to be positive if the OD450nm value is greater than or equal to the critical value; determination of OD450nm value in serum to be tested<The critical value is judged to be negative.
2.3 mouse antibody detection results
The results of the mouse ELISA antibodies are shown in table 2 and show: the immunization group produced ELISA antibody at 21 days, and the antibody titer further increased at 35 days, and increased with the increase of the concentration of the recombinant Cap protein, and no ELISA antibody was observed in the negative control group.
Table 2 shows the antibody detection results of porcine circovirus type 2 Cap protein CGMCC No.18235 vaccine immunized mice
3. Immunization test for piglets
3.1 piglet Immunity validation procedure
And (3) carrying out an antibody level generation test of the circular virus type 2 Cap protein subunit vaccine on 35 circular antigen antibody double-negative healthy piglets of 2-3 weeks old. Grouping condition: randomly selecting 30 piglets to be averagely divided into 5 groups and 5 piglets/group as a vaccine immunization group, wherein each piglet of the immunization group I is injected with 2ml of a circovirus type 2 Cap protein subunit vaccine containing 5 mu g/ml of circovirus type 2 Cap protein containing 5 mu g/ml of circovirus type 2 protein subunit vaccine through neck muscles, the immunization group II is injected with 2ml of a circovirus type 2 Cap protein subunit vaccine containing 10 mu g/ml of circovirus type 2 protein through neck muscles, the immunization group III is injected with 2ml of a circovirus type 2 Cap protein subunit vaccine containing 20 mu g/ml of circovirus type 2 protein containing 50 mu g/ml of circovirus type 2 protein subunit vaccine through neck muscles, the immunization group V is injected with 2ml of a circovirus type 2 Cap protein subunit vaccine containing 100 mu g/ml of circovirus type 2 protein through neck muscles, in immunization group VI, 2ml of circovirus type 2 Cap protein subunit vaccine containing 100 mu g/ml of circovirus wild Cap protein is injected into neck muscles of each piglet; and finally, selecting 5 heads as blank control groups, and avoiding immunization. Blood was collected 2 weeks, 3 weeks, and 5 weeks after the first immunization, and serum was separated and measured for PCV2ELISA antibody and neutralizing antibody.
3.2 piglet antibody detection method
3.2.1ELISA antibody detection method
Coating the circular recombinant Cap protein with a carbonate buffer solution with the pH value of 9.6, wherein the concentration is 1 mu g/ml, 100 mu l/hole, coating overnight at the temperature of 4 ℃, washing, adding 300 mu l of 0.5% casein blocking solution to seal the plate, and sealing the plate at the temperature of 37 ℃ for 2 hours; diluting the serum to be detected by using a sample diluent in a multiple ratio, adding the diluted sample diluent into a coated plate, setting a negative and positive serum control group at the same time, acting for 1h at 37 ℃, and washing; adding rabbit anti-pig secondary antibody (diluted 1: 10000), acting at 37 deg.C for 1h in 100 μ l/hole, and washing; adding substrate developing solution TMB for developing, reacting at 37 deg.C for 15min with 100 μ l/well, and treating with 2M H2SO4The reaction was terminated. And (4) judging a result: the average value of the negative control OD450nm should be less than or equal to 0.15, otherwise, the negative control OD450nm is invalid; each detection value of the positive control should be between 1.0 and 2.5, otherwise, the positive control is invalid; calculation of the critical value: the cut-off value was 0.18 × the mean value of the positive control OD450nm values. The serum to be detected is determined to be positive if the OD450nm value is greater than or equal to the critical value; determination of OD450nm value in serum to be tested<The critical value is judged to be negative.
3.2.2 detection of neutralizing antibodies
Inactivating the serum to be detected in 56 deg.C water bath for 30min, filtering, sterilizing, diluting with maintenance solution 2 times to 1:1024 times, and diluting the proliferated PCV2 with maintenance solution to 1 × 106TCID50/ml, then mixed with different gradient diluted serum in equal volume, 37 ℃ water bath for 1 h. The nutrient solution in the 96-well cell plate full of PK15 cells was discarded, and after washing with pure DMEM, the serum virus mixture was added to the wells in 3 replicates per serum dilution set at 100. mu.l/well and incubated at 37 ℃ for 72 h. Simultaneously setting positive and negative serum control and blank controlThen, the neutralizing antibody titer of the serum was measured by IFA method, and the maximum dilution of the serum inhibiting 50% PCV2 infection was used as the neutralizing antibody titer of the serum to be tested.
3.3 piglet antibody test results
3.3.1ELISA antibody detection results
The results of the ELISA antibody detection of the piglets are shown in Table 3, and the results show that the antibodies can be generated 14 days after the immunization of the piglets, the ELISA antibodies are increased along with the increase of the concentration of the Cap protein, and the antibody titer is increased along with the increase of the immune cycle.
Table 3 shows the antibody detection results of porcine circovirus type 2 recombinant Cap protein vaccine immunized piglets
3.3.2 piglet neutralizing antibody detection results
The results of the detection of the neutralizing antibody of the piglets are shown in table 4, and the results show that the neutralizing antibody can be generated 14 days after the piglets are immunized, the neutralizing antibody is increased along with the increase of the concentration of the recombinant Cap protein, and the antibody titer is increased along with the increase of the immune cycle.
Table 4 shows the detection results of neutralizing antibodies of porcine circovirus type 2 recombinant Cap protein vaccines after immunizing piglets
Sequence listing
<110> Zhongmu industries GmbH
<120> expression preparation and application of porcine circovirus type 2 Cap protein
<130> WHOI190107
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 579
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
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gtcagaacgc cctcctggaa tgtggacatg atgagattta atattaatga ttttcttccc 120
ccaggagggg gctcaaaccc cctcactgtg ccctttgaat actacagaat aaggaaggtt 180
aaggttgaat tctggccctg ctccccaatc acccagaatg gtaggggagt gggctccact 240
gctgttattc tagatattaa ctttgtaaca aaggccaatg ccctaatcta tgacccctat 300
gtaaactact cctcccgcca taccataacc cagcccttct cctaccactc ccggtacttt 360
accccgaaac ctgtccttga taggacaatc gattacttcc aacccaataa caaaagaaat 420
caactctggc tgagactaca aactactgga aatgtagacc atgtaggcct cggcactgcg 480
ttcgaaaaca gtatatacga ccaggactac aatatccgta taaccatgta tgtacaattc 540
agagaattta atcttaaaga ccccccactt aaccctaag 579
<210> 2
<211> 193
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Asn Gly Ile Phe Asn Thr Arg Leu Ser Arg Thr Ile Gly Tyr Thr Val
1 5 10 15
Lys Lys Thr Thr Val Arg Thr Pro Ser Trp Asn Val Asp Met Met Arg
20 25 30
Phe Asn Ile Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser Asn Pro Leu
35 40 45
Thr Val Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe
50 55 60
Trp Pro Cys Ser Pro Ile Thr Gln Asn Gly Arg Gly Val Gly Ser Thr
65 70 75 80
Ala Val Ile Leu Asp Ile Asn Phe Val Thr Lys Ala Asn Ala Leu Ile
85 90 95
Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro
100 105 110
Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Arg
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Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu
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Arg Leu Gln Thr Thr Gly Asn Val Asp His Val Gly Leu Gly Thr Ala
145 150 155 160
Phe Glu Asn Ser Ile Tyr Asp Gln Asp Tyr Asn Ile Arg Ile Thr Met
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Lys
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<213> porcine circovirus (porcine circovirus)
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ccctttgaat actacagaat aaggaaggtt aaggttgaat tctggccctg ctccccaatc 180
acccagggtg acaggggagt gggctccact gctgttattc tagatgataa ctttgtaaca 240
aaggccaatg ccctaaccta tgacccctat gtaaactact cctcccgcca taccataacc 300
cagcccttct cctaccactc ccggtacttt accccgaaac ctgtcctgga tcggactatg 360
gattacttcc aacccaataa caaaagaaat caactctggc tgagactaca aactactgga 420
aatgtagacc atgtaggcct cggcactgcg ttcgaaaaca gtatatacga ccaggactac 480
aatatccgta taaccatgta tgtacaattc agagaattta atcttaaaga ccccccactt 540
aaccctaagt ga 552
Claims (10)
1. A porcine circovirus type 2 recombinant Cap protein has a sequence of sequence 2 in a sequence table.
2. The gene encoding the porcine circovirus type 2 recombinant Cap protein of claim 1.
3. The encoding gene of claim 2, wherein the sequence of the encoding gene is shown as sequence 1 in the sequence table.
4. The recombinant vector for expressing the porcine circovirus type 2 recombinant Cap protein of claim 1 is obtained by inserting the coding gene of the porcine circovirus type 2 recombinant Cap protein shown in the sequence 1 into an original vector.
5. The recombinant vector according to claim 4, wherein the starting vector is pET-30a (+).
6. The engineering bacteria for expressing the porcine circovirus type 2 recombinant Cap protein is characterized in that the engineering bacteria are recombinant bacteria for expressing porcine circovirus type 2 Cap recombinant protein virus particles obtained by introducing the recombinant vector of claim 4 or 5 into escherichia coli BL21(DE3) competence and screening.
7. The engineered bacterium expressing porcine circovirus type 2 recombinant Cap protein according to claim 6, wherein the engineered bacterium is Escherichia coli E.coli-PCV2-FJ-Cap, which is deposited at the China general microbiological culture Collection center at 7-16.2019 with the deposit number of CGMCC No. 18235.
8. A preparation method of porcine circovirus type 2 Cap recombinant protein virus particles comprises the following steps:
1) transforming the recombinant vector of claim 4 or 5 into escherichia coli BL21(DE3) competence to obtain a recombinant bacterium for expressing porcine circovirus type 2 Cap recombinant protein virions;
2) inducing and expressing the recombinant bacteria obtained in the step 1), collecting and crushing bacteria, and collecting inclusion bodies;
3) dissolving and renaturing the inclusion body obtained in the step 2) to obtain the assembled porcine circovirus type 2 Cap recombinant protein virions.
9. Use of the porcine circovirus type 2 recombinant Cap protein of claim 1 or the gene encoding the porcine circovirus type 2 recombinant Cap protein of claim 2 in the preparation of porcine circovirus type 2 recombinant Cap protein virions.
10. Use of the porcine circovirus type 2 recombinant Cap protein of claim 1 or the gene encoding the porcine circovirus type 2 recombinant Cap protein of claim 2 in the preparation of a vaccine for the prevention or treatment of porcine circovirus type 2.
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