CN106589077B

CN106589077B - Porcine circovirus type 2 antigen purification and concentration method

Info

Publication number: CN106589077B
Application number: CN201710062983.5A
Authority: CN
Inventors: 乔绪稳; 于晓明; 郑其升; 李鹏成; 陈瑾; 侯立婷; 张元鹏; 侯继波
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Agricultural Science and Technology Transfer Center Co.,Ltd.
Priority date: 2017-01-26
Filing date: 2017-01-26
Publication date: 2020-07-10
Anticipated expiration: 2037-01-26
Also published as: CN106589077A

Abstract

The invention provides a porcine circovirus type 2 antigen purification and concentration method, and relates to the technical field of biomedicine. A method for purifying and concentrating porcine circovirus type 2 antigen comprises the following steps: (1) adding a linker protein with an amino acid sequence shown as SEQ ID NO. 1 into porcine circovirus type 2 antigen, mixing uniformly, and incubating; (2) adding a purification carrier, uniformly mixing, and incubating; the purification carrier is a lactococcus lactis framework; (3) centrifuging and taking the precipitate. The purification and concentration method can remove a large amount of foreign proteins, and has the advantages of high antigen recovery efficiency, high concentration multiple, low requirement on equipment, simple operation and low cost.

Description

Porcine circovirus type 2 antigen purification and concentration method

Technical Field

The invention relates to the technical field of biomedicine, in particular to a porcine circovirus type 2 antigen purification and concentration method.

Background

Porcine circovirus type 2 (PCV 2) is the primary etiological agent causing Postweaning Multisystemic Wasting Syndrome (PMWS). Since the PMWS was first reported in 1991 Canada, the PMWS has spread all over the world, is one of important infectious diseases which are recognized globally and endanger the pig industry, and is very popular in the swinery in China. Immunization with a vaccine is key to solving the problem of PCV2 epidemic.

At present, in the process of developing and producing PCV2 vaccines in China, some unsolved important problems still exist: the PCV2 has poor in vitro proliferation capability and low antigen titer, and the antigen titer is improved by optimizing culture conditions without significant and limited effect; secondly, most of PCV2 vaccines sold in the market at present are whole virus inactivated vaccines, wherein cell protein, bovine serum albumin and the like account for more than 95%, and the vaccines inoculated in a large area have certain side reactions, which can cause the problems of waste of host immune resources, poor immune protection efficacy of the vaccines and the like.

At present, no mature purification method exists in the production of the circovirus vaccine at home, and the membrane filtration clarification technology is matched with the physical purification technology such as the hollow fiber concentration technology and the like to purify and concentrate in the production of individual enterprises, so that the method can concentrate the antigen and remove part of the foreign protein to a certain extent, but has the defects of complex operation, high requirement on technical equipment, higher purification and concentration cost, lower antigen recovery rate, lower concentration multiple and the like, therefore, a method capable of purifying and concentrating the vaccine antigen efficiently and cheaply is urgently needed in the production of the circovirus vaccine to solve the problems of insufficient content and low purity of the vaccine antigen.

Disclosure of Invention

The invention aims to provide a porcine circovirus type 2 antigen purification and concentration method, which can remove a large amount of foreign proteins, and has the advantages of high antigen recovery efficiency, high concentration multiple, low requirement on equipment, simple operation and low cost.

The invention also aims to provide the porcine circovirus type 2 vaccine which has high purity of antigen, low production cost and good immune effect, and can reduce immune side reaction and improve the safety of the vaccine.

The purpose of the invention is realized by adopting the following technical scheme:

a method for purifying and concentrating porcine circovirus type 2 antigen comprises the following steps:

(1) adding a linker protein with an amino acid sequence shown as SEQ ID NO. 1 into porcine circovirus type 2 antigen, mixing uniformly, and incubating;

(2) adding a purification carrier, uniformly mixing, and incubating; the purification carrier is a lactococcus lactis framework;

(3) centrifuging and taking the precipitate.

In the present invention, the incubation conditions in step (1) are as follows: shaking in the incubation process, wherein the incubation temperature is 35-37 ℃, and the incubation time is 45-60 min.

In the present invention, the incubation conditions in step (2) are as follows: shaking in the incubation process, wherein the incubation temperature is 20-30 ℃, and the incubation time is 25-35 min.

In the present invention, the linker protein is added in the following ratio: 10^8.1TCID₅₀80-100 mu g of linker protein is added into the PCV2 antigen.

In the present invention, the purification carrier is added in the following ratio: 10^8.1TCID₅₀2.3 × 10 was added to PCV2 antigen⁹～2.7×10⁹And (3) purifying the carrier.

In the invention, the adaptor protein is obtained by inducing and expressing a recombinant bacterium carrying the encoding gene of the adaptor protein.

In the preferred technical scheme, the recombinant strain is obtained by inserting a linker protein coding gene between NdeI and HindIII enzyme cutting sites in an expression vector pET32 and then introducing the linker protein coding gene into escherichia coli; the sequence of the joint protein coding gene is shown as SEQ ID NO:2, respectively.

In a preferred technical scheme, the recombinant bacteria are cultured for 1-2h at 36-37 ℃, then cultured for 2-4h at 18-22 ℃, kept stand for 10-20min at 14-16 ℃, then isopropyl thiogalactoside with the final concentration of 0.05-0.15 mmol/L is added for induction culture for 18-22h at 14-16 ℃, and the adaptor protein is obtained.

In the invention, the lactococcus lactis skeleton is obtained by boiling lactococcus lactis with hydrochloric acid.

The invention also provides a porcine circovirus type 2 vaccine, which contains the precipitate obtained by the purification and concentration method.

Compared with the prior art, the invention has the beneficial effects that: in the PCV2 antigen purification and concentration method, the linker protein can be specifically combined with the PCV2 antigen and the lactococcus lactis skeleton, so that a large amount of foreign proteins can be efficiently removed, the PCV2 antigen can be purified and concentrated, the antigen recovery efficiency is high, the concentration multiple is high, the requirement on equipment is low, the operation is simple, and the cost is low. The recovery efficiency of the method for PCV2 antigen is higher than 99%, and the removal efficiency of foreign protein is higher than 90%. The porcine circovirus type 2 vaccine has low production cost and good immune effect, reduces immune side reaction and improves the safety of the vaccine.

Drawings

FIG. 1 shows the results of restriction enzyme identification of recombinant expression plasmid pET-P L, where M is DNA standard molecular weight, 1 is non-restriction enzyme recombinant plasmid pET-P L, and 2 and 3 are NdeI and HindIII double restriction enzyme products of recombinant plasmid pET-P L.

FIG. 2 shows the result of linker protein identification, and FIGS. 2(A) and 2(B) are the result of SDS-PAGE identification and Western-blot identification for linker protein induced expression, respectively, where lane 1 in FIG. 2(A) is the whole recombinant strain P L/B L21 after induced expression, 2 is the control strain pET/B L21 after induced expression, M is the molecular weight of the pre-stained standard protein, 3 is the supernatant of the lysate of recombinant strain P L/B L21 after induced expression, 4 is the precipitate of the lysate of recombinant strain P L/B L21 after induced expression, FIG. 2(B) M is the molecular weight of the pre-stained standard protein, 1 is the supernatant of the lysate of control strain pET/B L21 after induced expression, and 2 is the supernatant of the lysate of recombinant strain P L/B L21 after induced expression.

FIG. 3 shows the SDS-PAGE identification of the binding and dissociation of the adaptor protein from the purification vector, wherein lane 1 is the adaptor protein supernatant, lane 2 is the supernatant 1, M is the molecular weight of the prestained standard protein, lane 3 is the precipitate 1, lane 4 is the supernatant 2, and lane 5 is the precipitate 2.

FIG. 4 shows the results of the antigen purification and concentration identification of PCV2, wherein FIGS. 4(A), (B) and (C) show the results of PCR, SDS-PAGE electrophoresis and PCV 2-specific Western-blot identification, respectively, wherein M is the molecular weight of a standard protein (or DNA), lane 1 is the precipitate 4, lane 2 is the unpurified PCV2 antigen, lane 3 is the supernatant 3, and lane 4 is the precipitate 3; fig. 4(D) and fig. 4(E) are the results of indirect immunofluorescence and electron microscope observation and identification of purified PCV2 antigen complex, wherein 1 is the purification vector, 2 is the

precipitate

4, and 3 is the precipitate 3, respectively.

Fig. 5 shows the results of antibody detection of PCV2 antigen complex immunized piglets, where fig. 5(a) shows the antibody levels of each group at the fifth week of immunization and fig. 5(B) shows the antibody duration after immunization.

Detailed Description

EXAMPLE 1 construction and characterization of recombinant expression plasmid pET-P L

Designing a linker protein, wherein the amino acid sequence of the linker protein is shown as SEQ ID NO:1, the protein is a fusion protein, the fusion protein contains two functional recognition regions, the linker protein can respectively and specifically recognize PCV2 antigen and lactococcus lactis skeleton, the coding gene of the linker protein is shown as SEQ ID NO:2, in order to identify the linker protein, His tag protein (HHHHHHHHHHHHH) is added at the carboxyl terminal of the sequence shown as SEQ ID NO:1, His tag protein coding sequence is added before the stop codon of the linker protein coding gene to obtain the sequence SEQ ID NO:3, Nde I enzyme cutting sites are designed at the 5 'end, HindIII enzyme cutting sites are respectively designed at the 3' end, the DNA fragments are sent to Kinsys company for synthesis, and the synthesized gene fragments are inserted into pUC vector to obtain the recombinant plasmid pUC-P L.

Double enzyme digestion is carried out on the recombinant plasmid pUC-P L and the prokaryotic expression vector pET32a by adopting endonuclease Nde I and Hind III, a target gene fragment (abbreviated as P L fragment) of the adaptor protein and an expression vector fragment are recovered and are connected by T4 ligase to obtain the recombinant expression vector.

pUC-P L and pET32a plasmid double restriction system (30. mu. L):

wherein 10 × q. cut Buffer, q. cut nde i and q. cut hind iii are available from gangbao bio ltd.

And (2) uniformly mixing the double enzyme digestion systems, acting for 30min at 37 ℃, separating strips through agarose gel electrophoresis, cutting gel, respectively recovering target fragments P L and pET32a vector fragments according to the specification of an AXYGEN gel recovery kit, and connecting through T4 ligase.

The T4 ligase ligation system was as follows (25. mu. L):

and (3) placing the connection system at 16 ℃ for connection for 12-16h to obtain a connection product.

Transforming E.coli DH5 α competent cells by the ligation product, coating a L B plate containing 50mg/m L ampicillin, standing at 37 ℃ for overnight culture, selecting a single clone, inoculating L B liquid culture medium containing 50mg/m L ampicillin for overnight culture, extracting plasmids according to the instruction operation of an AXYGEN plasmid extraction kit, carrying out double-enzyme digestion identification by using Q.cutNde I and Q.cutHind III, and obtaining a graph 1 after electrophoresis of enzyme digestion products, wherein a target band with the size of 5389bp and 1047bp is obtained after double enzyme digestion of the positive recombinant plasmid, and the successfully identified positive recombinant plasmid is named as pET-P L.

Example 2 construction of recombinant expression bacterium P L/B L21 and expression of linker protein

1. Construction of recombinant expression Strain P L/B L21

The recombinant plasmid pET-P L obtained in example 1 was transformed into prokaryotic expression strain B L21 (DE3) to obtain recombinant expression strain P L/B L21, and meanwhile, the empty pET32a plasmid was used as a control for transforming B L21 (DE3) to obtain control strain pET/B L21.

2. Inducible expression and identification of recombinant expression strain P L/B L21

(1) Selecting monoclonals of the strain P L/B L21 and a control strain pET/B L21, respectively inoculating the monoclonals to L B liquid culture media containing 50mg/m L ampicillin, and culturing overnight at 37 ℃ under the condition of 200rmp to obtain mother liquor;

(2) inoculating the mother solution of each strain to fresh L B liquid culture medium (containing 50mg/m L ampicillin) at a ratio of 1:200, culturing at 37 deg.C and 180rmp for 1.5 hr, culturing at 20 deg.C and 180rmp for 3 hr, and standing at 15 deg.C for 15 min;

(3) adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1 mmol/L into the culture obtained in the step (2) for protein induction, wherein the induction temperature is 15 ℃, and the induction culture time is 20 h;

(4) collecting the induced bacteria liquid, centrifuging at 8000g and 4 deg.C for 10min to obtain thallus, washing thallus twice with PBS buffer (pH 7.0-7.4, 0.1M), and then resuspending;

(5) and (3) carrying out high-pressure cracking on the thallus suspension at the temperature of 4 ℃, wherein the cracking conditions are as follows: cracking for 3-5 cycles under the pressure of 800 MPa;

(6) centrifuging the thallus lysate for 15min at 4 ℃ and 12000rpm, respectively collecting lysate supernatant and precipitate, and resuspending the precipitate with PBS buffer solution equal to the supernatant;

all the operations are carried out in a clean environment, all the reagents and containers are subjected to high-pressure sterile treatment, and the treatment conditions are as follows: the temperature is 121 ℃, the pressure is 103.4KPa, and the time is 20 min; sterile feeding and sample collecting pumps are adopted for high-pressure crushing.

(7) In order to analyze and identify the expression condition of the recombinant expression strain P L/B L, the induced P L/B L whole bacteria and the supernatant and the precipitate of the lysate thereof are respectively subjected to SDS-PAGE electrophoresis identification by 80 mu L, wherein the contrast is the induced pET/B L whole bacteria, and a graph (A) shown in FIG. 2 is obtained, compared with the contrast bacteria, the induced P L/B L whole bacteria and the supernatant and the precipitate thereof have obvious adaptor protein bands at about 38KD, and the amount of the target protein soluble in the supernatant of the lysate is about 40-60% of the total target protein.

(8) In order to identify the adaptor protein, Western-blot identification is carried out on supernatant of pET/B L21 lysate after induction and supernatant of P L/B L21 lysate after induction by means of carboxyl-terminal His tag protein, and the method specifically comprises the steps of transferring SDS-PAGE electrophoresis results of samples onto a nitrocellulose membrane, sealing the transfer membrane for 1h at room temperature by TBST sealing buffer solution containing 5% BSA, washing for 3 times by TBST buffer solution, incubating for 1h with 1:5000 diluted mouse His monoclonal antibody (KP L company) at 37 ℃, washing for 3 times by TBST buffer solution, incubating for 45min by goat anti-mouse HRP-IgG secondary antibody (horse radish peroxidase labeled goat anti-mouse IgG antibody, KP L company) at 37 ℃, washing for 3 times by TBST photographing buffer solution after incubation, developing by using a developing kit, storing results are shown in figure 2(B), and a DAB strip of the supernatant of P L/B L21 lysate after induction appears at a KD 38 position, wherein the DAB strip is a DAB strip protein.

The recombinant strain P L/B L21 is adopted to induce and express the adaptor protein according to the method in the embodiment, the recombinant strain after induced expression is subjected to high-pressure lysis, and the supernatant of the lysate is taken as the adaptor protein solution.

His (histidine) tag protein is only used for conveniently identifying expressed protein and does not influence protein expression and function exertion, in the actual production process, the linker protein coding gene is inserted into pET32a, then is introduced into B L21 (DE3), and positive recombinant bacteria are screened, so that the linker protein can be produced.

Example 3 quantitative analysis of linker proteins

1. Preparation of purification vectors

Statically culturing lactococcus lactis I L1403 (The complex Genome Sequence of The L active Acid Bacterium L active bacteria ssp. lactis I L1403, Genome Res., 10.1101/gr.1699701) in fresh GM17 culture medium at 30 ℃ for 16-18h, centrifuging The culture solution at 8000rpm for 5min, collecting thallus, washing The precipitate with PBS buffer solution once, adding 0.1M hydrochloric Acid, boiling for 30min, centrifuging at 8000rpm for 5min, washing The precipitate with PBS buffer solution for 3 times, re-suspending with PBS buffer solution to obtain lactococcus lactis skeleton, namely The purified carrier, counting The number of The hemocytes of The purified carrier, and culturing lactococcus lactis I2.5 × 10 (2.5 ×) at 2.5⁹Each purified vector was designated as 1 unit.

2. Protein dissociation

A linker protein solution was prepared as in example 2 to a 2m L linker protein solution (total protein about 4.6mg) was added an excess of purification vector (10 × 10⁹One), incubation for 30min to allow the linker protein to bind completely to the purification vector, centrifugation at 9000rpm for 3min to obtain supernatant 1 and pellet 1, pellet 1 containing the purification vector and its complex with linker protein, pellet 1 was resuspended in 400. mu. L PBS buffer.

SDS (sodium dodecyl sulfate) was added to the resuspension of the pellet 1 at a final concentration of 1%, boiled at 100 ℃ for 10min to dissociate the adaptor protein from the purification vector, and then centrifuged at 12000rpm for 10min to harvest the supernatant 2 and the pellet 2, and the pellet 2 was resuspended in PBS buffer equivalent to the supernatant 2.

3. Protein dissociation identification

In order to analyze whether the linker protein was completely dissociated from the purified vector, SDS-PAGE analysis was performed on the linker protein solution, the supernatant 1, the precipitate 1, the supernatant 2, the precipitate 2 and the purified vector, and the results are shown in FIG. 3, in which the linker protein was completely dissociated in the supernatant 2.

4. Determination of the amount of adaptor protein

The total protein content of supernatant 2 was measured using BCA protein quantification kit and then converted to the total amount of adaptor protein in the adaptor protein solution 220. mu.g of adaptor protein was obtained after fermentation in 1m L L B broth using the method described in example 2.

The linker protein solution was adjusted to 80-100. mu.g/m L with PBS buffer depending on the protein content.

Example 4 method for purifying concentrated PCV2 antigen

PCV2 antigen preparation

PCV2 NJ strain (E.coli expressing porcine circovirus type 2 Cap protein for producing virus-like particle vaccine, Jiangsu agricultural science, 2015, 48 (5): 976-. The titer of PCV2 antigen was 10^6.5TCID₅₀/mL。

Purification and concentration of PCV2 antigen

(1) A linker protein solution of 90. mu.g/m L (linker protein concentration) was added to the PCV2 antigen to be purified in a ratio of 10^8.1TCID₅₀Of PCV2 (approximately 40m L titer of 10)^6.5TCID₅₀PCV2 antigen of/m L) is added with 1m L linker protein solution and mixed evenly;

(2) placing the mixture in a shaker at 37 ℃ and 150rpm, and performing shaking incubation for 55min to completely combine the PCV2 antigen with the linker protein;

(3) adding a purification carrier in the following proportion: 10^8.1TCID₅₀Of PCV2 (approximately 40m L titer of 10)^6.5TCID₅₀PCV2 antigen of/m L) was added with 2.5 × 10⁹Purifying the carrier (prepared by the method in example 3), fully stirring and uniformly mixing;

(4) placing the mixture in a shaker at 25 ℃ and the rotation speed of 150rpm, and performing shaking incubation for 30min to ensure that the joint protein combined with the PCV2 antigen is completely combined with the purification carrier;

(5) centrifuging at 9000rpm for 5min to obtain supernatant 3 and precipitate 3, wherein the precipitate 3 is PCV2 antigen complex. The PCV2 antigen complex is a complex of PCV2 antigen, linker protein and lactococcus lactis skeleton, and the principle of forming the PCV2 antigen complex is that the linker protein can be specifically combined with PCV2 antigen and lactococcus lactis skeleton at the same time. Therefore, the final product of the PCV2 antigen purification and concentration method of the present example is PCV2 antigen complex, namely precipitate 3.

In the purification process, in order to ensure that the PCV2 antigen is completely recovered, the lactococcus lactis skeleton and the linker protein are added in excess, so that the PCV2 antigen complex also contains a small amount of lactococcus lactis skeleton and its complex with the linker protein.

The above steps are all carried out under aseptic conditions.

Example 5 identification of purified and concentrated PCV2 antigen

To verify that the PCV2 antigen was purified and concentrated by the method of example 4, a series of confirmatory analyses were performed on supernatant 3 and pellet 3 obtained in example 4. Meanwhile, in order to verify the effect of the linker protein, PCV2 antigen was mixed with the purification carrier as in example 4, except that the linker protein solution was not added, and precipitate 4 and supernatant 4 were obtained.

PCR identification of virus DNA in PCV2 antigen, precipitate 3, supernatant 3 and precipitate 4 before purification, diluting 100 times with deionized water and amplifying with PCV2 specific primers, wherein the primers are as follows:

primer 1: 5'-CCGCGGGCTGGCTGAACTT-3' the flow of the air in the air conditioner,

primer 3: 5'-ACCCCCGCCACCGCTACC-3' are provided.

The

primers

1 and 3 are used for specifically amplifying 491-1644 segment gene of PCV2, and the length of the segment is 1154 bp.

The PCR system was as follows (25. mu. L):

2 × Premix ExTaq was purchased from Dalibao Biopsis.

The PCR amplification procedure was: 4min at 94 ℃; 30s at 94 ℃, 30s at 53 ℃, 90s at 72 ℃ and 25 cycles; 10min at 72 ℃.

The PCR product was electrophoresed on agarose gel, and the result is shown in FIG. 4 (A).

SDS-PAGE and Western-blot identification of proteins in PCV2 antigen, precipitate 3, supernatant 3 and precipitate 4 before purification were subjected to SDS-PAGE electrophoretic identification, and FIG. 4(B) was obtained.

And performing PCV2 specific Western-blot identification on the SDS-PAGE electrophoresis result to obtain a graph 4 (C). The Western-blot comprises the following specific operation steps:

(1) transfer printing: after SDS-PAGE electrophoresis is finished, transferring the western blot to a PVDF membrane (polyvinylidene difluoride membrane) by adopting a wet transfer method, wherein the membrane transfer condition is constant pressure of 80V and the time is 60 min;

(2) and (3) sealing: sealing the PVDF membrane with TBST buffer (pH8.0) containing 5% skimmed milk at 4 ℃ overnight;

(3) incubating the primary antibody: washing the PVDF membrane, immersing the PVDF membrane into TBST solution containing 2% BSA (bovine serum albumin) 1:1000 diluted porcine PCV2 positive serum (VRMD company), and incubating for 1h at 37 ℃;

(4) washing: taking out the PVDF membrane, and rinsing with TBST buffer solution for 4 times, 8min each time;

(5) incubating the secondary antibody, namely immersing the PVDF membrane into an HPR-goat anti-pig secondary antibody (goat anti-pig IgG antibody marked by horseradish peroxidase, purchased from KP L company, with the dilution of 1: 10000) diluted by TBST buffer solution containing 2% BSA, and incubating for 45min at 37 ℃;

(6) washing the obtained product in the same way as in the step (4), and performing color development by using a DAB color development kit.

3. Electron microscope observation and indirect immunofluorescence test verification

The purified vector, precipitate 3 and precipitate 4 were subjected to transmission electron microscopy and indirect immunofluorescence assay validation to obtain fig. 4(D) and fig. 4 (E).

The indirect immunofluorescence assay procedure was carried out by washing the sample (purified vector, pellet 3 or pellet 4) three times with PBST buffer (pH7.5) by centrifugation and resuspension, blocking with PBST buffer (pH7.5) containing 5% skim milk for 45min at room temperature, washing with PBST buffer (pH7.5) by resuspension, adding pig PCV2 positive serum (VRMD, USA, dilution 1:1000) diluted with TBST buffer containing 2% BSA, incubating at 37 ℃ for 1h, washing with PBST buffer (pH7.5) by resuspension for 4 times, adding FITC-goat anti-pig secondary antibody (fluorescein isothiocyanate labeled goat anti-pig IgG antibody, purchased from KP L, USA, dilution 1:1000) diluted with PBST buffer (pH7.5) containing 2% BSA, incubating at 37 ℃ for 45min under dark conditions, washing with PBST buffer (pH7.5) for 4 times, smearing, and observing by positive fluorescence microscope.

The results in FIG. 4(A) show that no specific band was detected in pellet 4, a weak band of PCV2 nucleic acid with a size of approximately 27kD was detected in supernatant 3, and the brightness of PCV2 nucleic acid in pellet 3, i.e., the PCV2 antigen complex, was the same as that of PCV2 antigen before purification, indicating that most of PCV2 antigen entered pellet 3.

It can also be seen from fig. 4(B) that the hetero protein band in precipitate 3 was significantly reduced compared to the pre-purified PCV2 antigen, demonstrating that purification removed a significant amount of hetero proteins.

The results in fig. 4(B) and 4(C) show that there are no antigen and linker protein bands in pellet 4, whereas the linker protein band (about 38KD) and the purer PCV2 antigen band (about 27 KD) are visible in pellet 3, i.e., the PCV2 antigen complex lane, again demonstrating that the PCV2 antigen is purified by the enrichment of linker protein on the lactococcus lactis scaffold.

The results of fig. 4(D) and 4(E) show that, under an electron microscope, PCV2 virus particles with a diameter of about 20nm can be observed only on the surface of the vector of the PCV2 antigen complex (sediment 3), and indirect immunofluorescence confirms that the virus particles can react with porcine PCV2 positive serum, so that the enrichment of PCV2 antigen on the surface of the lactococcus lactis skeleton through linker protein is proved again.

The above results confirm that, since the linker protein can be specifically bound to the PCV2 antigen and the purification vector at the same time, the PCV2 antigen can be specifically enriched on the purification vector through the linker protein, most of the foreign proteins in the original PCV2 antigen are removed, and the PCV2 antigen complex is precipitated by centrifugation, thereby being purified and concentrated. Since PCV2 antigen is precipitated by centrifugation, the concentration multiple is very high, and the method can be used for preparing vaccines containing PCV2 antigen with high concentration.

Example 6 PCV2 antigen purification concentration efficiency assay

In order to examine the recovery efficiency of the method in example 4 on the PCV2 antigen, the PCV2 antigen before purification and the antigen content in the supernatant 3 in example 4 were examined by the method of E L ISA and the method of cellular IFA.

The E L ISA is operated as follows, the E L ISA detection is carried out on the PCV2 antigen before purification and the antigen content in the supernatant 3 by adopting SERE L ISA 2 Ag Capture (SYNBIOTICS, Korea), a standard curve is made according to a kit method, the PCV2 antigen before purification is diluted by 0 time, 10 times, 20 times, 40 times, 80 times and 160 times, the supernatant 3 is not diluted, all samples are subjected to two groups of parallel controls, and then the detection is carried out by adopting the kit, the detection results show that the PCV2 antigen detection average OD values before dilution and purification by 0 time, 10 times, 20 times, 40 times, 80 times and 160 times are respectively 2.003, 1.142, 0.793, 0.456, 0.302 and 0.49, the antigen content detection average OD value in the supernatant 3 is 0.296, the purified antigen is diluted by less than 160 times, the residual antigen content after purification is less than (1/160)% 100%, and the residual antigen is less than 1%.

2. The cellular IFA method operates as follows: PCV2 antigen and supernatant 3 before purification were diluted to 10-fold with MEM according to 10-fold dilution method^-1～10^-6TCID₅₀M L, inoculating PK15 cells on a 96-well plate, taking out after the cells grow to a single layer, washing 4 times by PBS buffer solution, adding precooled 80% acetone solution to fix the cells for 30min, then washing 4 times by PBS buffer solution, adding 100 mu L of pig PCV2 positive serum (purchased from VRMD, USA) diluted by 1:1000, shaking and mixing uniformly, incubating for 1h at constant temperature of 37 ℃, washing 4 times by PBS buffer solution after the incubation is finished, then adding 100 mu L of FITC-goat anti-pig secondary antibody (purchased from KP L) diluted by 1:1000 (FITC-goat anti-pig IgG antibody labeled by fluorescein isothiocyanate), shaking and mixing uniformly, incubating for 1h at 37 ℃ in a dark place, washing 4 times by PBS buffer solution after the incubation is finished, observing by an inverted fluorescence microscope, judging the virus titer, and displaying that the detection result of the PCV2 antigen titer before purification is 10^6.5TCID₅₀/m L, IFA assay of supernatant 3 cells 10^4.4TCID50/m L, residual antigen content after purification%＝(10^4.4/10^6.5) 100%, antigen residual less than 1%.

The results of the two antigen content detection methods are consistent, the obtained results are that the residual antigen content is lower than 1%, and the recovery efficiency of the PCV2 antigen purified and concentrated by the method in the embodiment 4 is proved to be more than or equal to 99%, which is obviously higher than that of the prior art.

Example 7 detection of efficiency of PCV2 antigen purification concentration method on removal of hetero proteins

In order to examine the removal efficiency of the purification and concentration method in example 4 for foreign proteins, the PCV2 antigen complex (pellet 3) obtained in example 4 was subjected to protein dissociation by the method of title 2 in example 3 to obtain supernatant 5 and pellet 5, the pellet 5 was resuspended in PBS buffer equal in volume to the supernatant 5, and the total protein content of PCV2 antigen before purification and the total protein content of supernatant 5 were measured by the BCA protein quantification kit, the total protein content of PCV2 antigen before purification was 2.8mg/m L, the protein content of supernatant 5 was 352 μ g/m L, the volume of supernatant 5 was 0.1 times that of PCV2 antigen before purification, and the removal efficiency of foreign proteins was 100% based on the recovery efficiency of antigen being about 100%, and the removal efficiency of foreign proteins was higher than 90%.

Example 8 identification of the immunopotency of purified and concentrated PCV2 antigen

PCV2 antigen complex (precipitate 3 in example 4) prepared in example 4 and PCV2 antigen (prepared under the heading 1 in example 4) before purification were each diluted with PBS buffer (pH 7.0-7.4, 0.1 mol/L) to an antigen concentration of 10⁵TCID₅₀/m L (the amount of the purified carrier in the diluted PCV2 antigen complex is 6.25 × 10⁶M L), then mixed and emulsified with 206 adjuvant (purchased from Seepic, France) at a volume ratio of 46:54 to obtain vaccine A (containing PCV2 antigen complex) and control vaccine 1 (containing PCV2 antigen before purification), respectively, the PCV2 antigen complex prepared in example 4 was diluted with PBS buffer (pH 7.0-7.4, 0.1 mol/L) to an antigen concentration of 2.10⁵TCID₅₀M L, concentration of purification vehicle 2 x 6.25 × 10⁶L per m, and then mixing and emulsifying with adjuvant 206 according to the volume ratio of 46:54 to obtain the vaccine B.

Because the lactococcus lactis skeleton and the linker protein are introduced in the PCV2 antigen purification process,thus, the purification vector was mixed with the linker protein solution and incubated according to the method of example 4, and the precipitate, which was the purification vector and its complex with the linker protein, was centrifuged without adding PCV2 antigen, and the obtained precipitate was adjusted to 6.25 × 10 in PBS buffer⁶And (2) mixing the cells/m L with an adjuvant 206 according to the volume ratio of 46:54, and emulsifying to obtain a control vaccine 2. the control vaccine 2 is used for verifying that the lactococcus lactis skeleton and the adaptor protein introduced after PCV2 antigen is purified have no influence on the immune effect of the PCV2 vaccine and have no obvious toxic or side effect.

(1) Test for immune Effect

The method comprises the steps of selecting healthy piglets of 2-3 weeks, detecting PCV2 and PRRSV antibodies by using PCV2 and PRRSV antibody E L ISA detection kits of proBiotechnology limited company of Wuhan family before testing, and detecting PCV2 and PRRSV nucleic acids by using PCR nucleic acid detection kits of Beijing Shijiheng biotechnology limited company to ensure that each test pig antigen antibody is double negative.

The serum of all test pigs is diluted by 400 times, and the antigen immune group antibody is qualified in the fifth week after the first immunization by using a PCV2 antibody E L ISA test kit of Wuhan pre-biotechnology limited company, the results are shown in FIG. 5(A) and FIG. 5(B), the antigen immune group antibody is qualified in the fifth week after the first immunization compared with a negative control group (G5), wherein the antibody level of a G2 group (immunized with vaccine B containing 2 times of PCV2 antigen complex) is higher than that of other antigen immune test groups, the antibody level of a G1 group (immunized with vaccine A containing 1 time of PCV2 antigen complex) is slightly higher than that of a G8 group (immunized with control vaccine 1 containing PCV2 antigen), the antibody dispersion is smaller in the G1 group and the G2 group, and the antibody level is relatively uniform, and the antibody level is relatively high after the G3 group, the G1 group and the G2 group is relatively high in antibody purification, the PCV 3 level is maintained to be higher than that the PCV 3 antibody level of the PCV 3 antibody is high after the first week, and the PCV 3 antigen immune test group is high purity is maintained and the PCV 465 and the antigen immune test method is consistent with the high purity.

TABLE 1 animal immunization groups and immunization programs

(2) Side reaction test

The immune stress responses of the piglets, including body temperature, food intake, mental state and the like, are observed and recorded immediately after the immunization of the groups G1-G5, and the growth conditions (food intake, body weight, hair covering and the like) of the piglets within 0-35d after the immunization are observed and recorded. The results show that the body temperature and the feed intake of the groups G1, G2, G4 and G5 are normal after immunization, no obvious abnormal reaction exists, the body temperature of one piglet in the group G3 is increased 0.5h after immunization, the piglet is abnormally excited, and the feed intake of the two piglets is reduced. After immunization, the piglets grow well from 0 to 35 days, and no obvious abnormality exists among the groups. The result shows that the vaccine A, B immunized by the G1 and G2 groups has obviously improved purity of PCV2 antigen complex, and the immune stress response of the vaccine is obviously reduced. No obvious abnormal reaction appears after the group G4 is immunized until the detection is finished, which indicates that the introduced purification carrier and the adaptor protein in the purification process can not bring toxic or side effect to the organism and can not influence the normal growth of the organism.

SEQUENCE LISTING

<110> agricultural science and academy of Jiangsu province

<120> method for purifying and concentrating porcine circovirus type 2 antigen

<130>20170126

<160>3

<170>PatentIn version 3.3

<210>1

<211>352

<212>PRT

<213>artificial

<220>

<223> linker protein

<400>1

Met Thr Thr Tyr Thr Val Lys Ser Gly Asp Thr Leu Trp Gly Ile Ser

1 5 10 15

Gln Arg Thr Gly Ser Ala Ser Ser Thr Asn Ser Gly Gly Ser Asn Asn

20 25 30

Ser Ala Ser Thr Thr Pro Thr Thr Ser Val Thr Pro Ala Lys Pro Thr

35 40 45

Ser Gln Thr Thr Val Lys Val Lys Ser Gly Asp Thr Leu Trp Ala Leu

50 55 60

Ser Val Lys Tyr Lys Thr Ser Ile Ala Gln Leu Lys Ser Trp Asn His

65 70 75 80

Leu Ser Ser Asp Thr Ile Tyr Ile Gly Gln Asn Leu Ile Val Ser Gln

85 90 95

Ser Ala Ala Ala Ser Asn Pro Ser Thr Gly Ser Gly Ser Thr Ala Thr

100 105 110

Asn Asn Ser Asn Ser Thr Ser Ser Asn Ser Asn Ala Ser Ile His Lys

115 120 125

Val Val Lys Gly Asp Thr Leu Trp Gly Leu Ser Gln Lys Ser Gly Ser

130 135 140

Pro Ile Ala Ser Ile Lys Ala Trp Asn His Leu Ser Ser Asp Thr Ile

145 150 155 160

Leu Ile Gly Gln Tyr Leu Arg Ile Lys Met Pro Gln Phe Ile Arg Ser

165 170 175

Leu Lys Glu Ile Leu Ser Gly Asp Phe Arg Lys Asn Leu Ala Ala Gln

180 185 190

Leu Leu Gln Ser Arg Leu Gly Ile Ile Tyr Leu Ala Met Glu Leu Val

195 200 205

Ser Leu Lys Phe Lys Val Arg Ile Ile Leu Lys Val Pro Leu Ser Thr

210 215 220

Leu Val Lys Asn Leu Tyr Cys Ile Leu Phe Tyr Gly Ile Ser Val Ala

225 230 235 240

Gln Ile Gln Ser Ala Asn Asn Leu Lys Ser Thr Ile Ile Tyr Ile Gly

245 250 255

Gln Lys Leu Val Leu Asp Thr Cys Asn Glu Ser Thr Ile Tyr Leu Arg

260 265 270

Lys Tyr Gln Ser Lys Val Lys Arg Gln Tyr Gln Ser Glu Val Asp Ile

275 280 285

Ile Arg Asp Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Ser Glu Gly Gly Gln Ser Cys Gly Thr Ile Phe Ser Ile

305 310 315 320

Asn Asp Ile Ser Gly Trp Tyr Glu Arg Glu Leu Val Ala Ile Thr Ser

325 330 335

Asp Gly Thr Ser Tyr Glu Ser Val Phe Thr Ile Ser Arg Asp Asp Lys

340 345 350

<210>2

<211>1059

<212>DNA

<213>artificial

<220>

<223> linker protein coding gene

<400>2

atgactactt ataccgtcaa atctggtgat actctttggg gaatctcaca aagaacaggt 60

tcagcttctt ctacaaattc aggtggttca aacaattccg caagcactac tccaaccact 120

tctgtgacac ctgcaaaacc aacttcacaa acaactgtta aggttaaatc cggagatacc 180

ctttgggcgc tatcagtaaa atataaaact agtattgctc aattgaaaag ttggaatcat 240

ttaagttcag ataccattta tattggtcaa aatcttattg tttcacaatc tgctgctgct 300

tcaaatcctt cgacaggttc aggctcaact gctaccaata actcaaactc gacttcttct 360

aactcaaatg cctcaattca taaggtcgtt aaaggagata ctctctgggg actttcgcaa 420

aaatctggca gcccaattgc ttcaatcaag gcttggaatc atttatctag cgatactatt 480

ttaattggtc agtatctacg aataaaaatg cctcaattca taaggtcgtt aaaggagata 540

ctctctgggg actttcgcaa aaatctggca gcccaattgc ttcaatcaag gcttggaatc 600

atttatctag cgatggaatt agtgtcgctc aaattcaaag tgcgaataat cttaaaagta 660

ccattatcta cattggtcaa aaacttgtac tgcatactat tttatggaat tagtgtcgct 720

caaattcaaa gtgcgaataa tcttaaaagt accattatct acattggtca aaaacttgta 780

ctggatacct gtaacgaaag taccatctat ctgcgtaaat accagtccaa agttaaacgc 840

caataccagt ccgaagtcga catcattcgc gatgaactgg gtggaggagg ttctggaggc 900

ggtggaagtg gtggcggagg tagcgagggt ggccagagct gcggcaccat cttcagcatt 960

aacgacatca gcggctggta cgagcgtgaa ctggtggcga tcaccagcga cggtaccagc1020

tacgaaagcg ttttcaccat tagccgtgac gataaataa 1059

<210>3

<211>1077

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<213>artificial

<220>

<223> His-tagged adaptor protein

<400>3

atgactactt ataccgtcaa atctggtgat actctttggg gaatctcaca aagaacaggt 60

tcagcttctt ctacaaattc aggtggttca aacaattccg caagcactac tccaaccact 120

tctgtgacac ctgcaaaacc aacttcacaa acaactgtta aggttaaatc cggagatacc 180

ctttgggcgc tatcagtaaa atataaaact agtattgctc aattgaaaag ttggaatcat 240

ttaagttcag ataccattta tattggtcaa aatcttattg tttcacaatc tgctgctgct 300

tcaaatcctt cgacaggttc aggctcaact gctaccaata actcaaactc gacttcttct 360

aactcaaatg cctcaattca taaggtcgtt aaaggagata ctctctgggg actttcgcaa 420

aaatctggca gcccaattgc ttcaatcaag gcttggaatc atttatctag cgatactatt 480

ttaattggtc agtatctacg aataaaaatg cctcaattca taaggtcgtt aaaggagata 540

ctctctgggg actttcgcaa aaatctggca gcccaattgc ttcaatcaag gcttggaatc 600

atttatctag cgatggaatt agtgtcgctc aaattcaaag tgcgaataat cttaaaagta 660

ccattatcta cattggtcaa aaacttgtac tgcatactat tttatggaat tagtgtcgct 720

caaattcaaa gtgcgaataa tcttaaaagt accattatct acattggtca aaaacttgta 780

ctggatacct gtaacgaaag taccatctat ctgcgtaaat accagtccaa agttaaacgc 840

caataccagt ccgaagtcga catcattcgc gatgaactgg gtggaggagg ttctggaggc 900

ggtggaagtg gtggcggagg tagcgagggt ggccagagct gcggcaccat cttcagcatt 960

aacgacatca gcggctggta cgagcgtgaa ctggtggcga tcaccagcga cggtaccagc1020

tacgaaagcg ttttcaccat tagccgtgac gataaacacc accaccacca ccactaa 1077

Claims

1. A method for purifying and concentrating porcine circovirus type 2 antigen, wherein the porcine circovirus type 2 antigen is PCV2 NJ strain antigen, and is characterized by comprising the following steps:

(3) centrifuging and taking the precipitate.

2. The method for purifying and concentrating porcine circovirus type 2 antigen according to claim 1, wherein the incubation conditions in step (1) are as follows: shaking in the incubation process, wherein the incubation temperature is 35-37 ℃, and the incubation time is 45-60 min.

3. The method for purifying and concentrating porcine circovirus type 2 antigen according to claim 1 or 2, characterized in that the incubation conditions in step (2) are as follows: shaking in the incubation process, wherein the incubation temperature is 20-30 ℃, and the incubation time is 25-35 min.

4. The method for purifying and concentrating porcine circovirus type 2 antigen of claim 3, wherein the addition ratio of the linker protein is as follows: 10^8.1TCID₅₀80-100 mu g of linker protein is added into the PCV2 antigen.

5. The method for purifying and concentrating porcine circovirus type 2 antigen according to claim 4, wherein the proportion of the added purification carrier is as follows: 10^8.1TCID₅₀2.3 × 10 was added to PCV2 antigen⁹～2.7×10⁹And (3) purifying the carrier.

6. The method for purifying and concentrating porcine circovirus type 2 antigen of claim 5, wherein the adaptor protein is obtained by inducing and expressing a recombinant bacterium carrying the gene encoding the adaptor protein.

7. The method for purifying and concentrating porcine circovirus type 2 antigen as claimed in claim 6, wherein the recombinant strain is obtained by inserting a gene encoding the adaptor protein into an expression vector pET32NdeI andHind III, introducing into escherichia coli between enzyme cutting sites; the sequence of the joint protein coding gene is shown as SEQ ID NO:2, respectively.

8. The method for purifying and concentrating porcine circovirus type 2 antigen as claimed in claim 7, wherein the recombinant strain is cultured for 1-2h at 36-37 ℃, then cultured for 2-4h at 18-22 ℃, kept stand for 10-20min at 14-16 ℃, then added with isopropyl thiogalactoside with the final concentration of 0.05-0.15 mmol/L and induced and cultured for 18-22h at 14-16 ℃ to obtain the adaptor protein.

9. The method for purifying and concentrating porcine circovirus type 2 antigen of claim 8, wherein the lactococcus lactis scaffold is obtained by boiling lactococcus lactis with hydrochloric acid.

10. Porcine circovirus type 2 vaccine characterized by comprising the precipitate obtained by the purification and concentration process of claim 1.