CN113845575A - Porcine circovirus type 2 Cap protein and coding gene and application thereof - Google Patents

Abstract

The invention belongs to the field of genetic engineering in the biotechnology pharmaceutical industry, and particularly relates to a porcine circovirus type 2 Cap protein, and a coding gene and application thereof. The amino acid sequence is shown as SEQ ID NO. 2. The invention further provides application of the porcine circovirus type 2 Cap protein in preparation of porcine circovirus type 2 vaccines. The porcine circovirus type 2 Cap protein disclosed by the invention well utilizes the advantages of Cap and Pichia pastoris, the coding gene of the protein can be efficiently expressed in the Pichia pastoris, the production cost is reduced, and the application of the recombinant protein in the preparation of porcine circovirus vaccines has high economic value and great social benefit.

Description

Porcine circovirus type 2 Cap protein and coding gene and application thereof

Technical Field

The invention belongs to the field of genetic engineering in the biotechnology pharmaceutical industry, and particularly relates to a porcine circovirus type 2 Cap protein, and a coding gene and application thereof.

Background

Currently, the most effective prevention means against PCV2 is an injection vaccine, and most of the commercial PCV2 vaccines are inactivated vaccines. Although the application of the commercial inactivated vaccine plays an important role in effectively preventing and controlling PCV2 infection, the inactivated vaccine has low virus titer in antigen preparation, long production period, high production cost and difficult virus culture. Inactivated vaccines do not completely block PCV2 transmission and do not completely eliminate infected viruses in the body. Therefore, subunit vaccines developed based on the structural protein Cap of PCV2 attract attention of the subject group due to the characteristics of controllable quality, safety, stability, effectiveness and the like.

At present, commercial subunit vaccines based on Cap are available at home and abroad, but the commercial subunit vaccines are expressed by using baculovirus or escherichia coli expression vectors. The subunit vaccine produced by the baculovirus expression system has high operation difficulty and high production cost, and the escherichia coli expression system has little post-translational modification on protein, is easy to form inclusion bodies and does not utilize later purification. The pichia pastoris expression system used by the subject group has the advantages of high protein yield, high secretion efficiency, low culture cost and the like, and has complete processing capacity on translated protein. The yeast expresses extracellular protein, which greatly reduces the difficulty of purifying protein and improves the yield of protein.

The porcine circovirus type 2 Cap protein successfully expressed by the pichia pastoris eukaryotic expression system provides a certain theoretical basis for the development of a porcine circovirus genetic engineering subunit vaccine.

Disclosure of Invention

The invention provides a porcine circovirus type 2 Cap protein and a coding gene and application thereof by utilizing the advantages of the porcine circovirus type 2 Cap and Pichia pastoris.

The invention is realized by the following technical scheme: the amino acid sequence of the porcine circovirus type 2 Cap protein is shown as SEQ ID NO 2.

The invention further provides application of the porcine circovirus type 2 Cap protein in preparation of porcine circovirus type 2 vaccines.

The invention further provides a preparation method of the porcine circovirus type 2 Cap protein, which comprises the following steps:

construction of expression vector: designing restriction enzyme cutting sites on the gene sequence of the artificially synthesized Cap, connecting the gene sequence of the Cap and the expression plasmid pwPICZalpha after enzyme cutting by the same restriction enzyme, and detecting and screening the clone containing the gene sequence of the Cap by double enzyme cutting;

construction of the Yeast expression System: integrating the gene sequence of Cap into the genome of the yeast by an electrotransformation method by using a restriction enzyme linearized recombinant expression plasmid; coating the transformed thallus suspension on a YPD plate containing Zeocin, and culturing at 30 ℃; picking a single colony, carrying out small-dose induced expression, and detecting expression supernatant by SDS-PAGE to confirm positive clone;

purification and activity detection of recombinant proteins: selecting positive clone colonies for large-dose induced expression, purifying the expression supernatant by adopting Protein Pure Ni-NTA resin, and detecting the purified recombinant Protein by SDS-PAGE and Western blot.

The invention also provides a coding gene of the porcine circovirus type 2 Cap protein, and the base sequence is shown as SEQ ID NO. 1.

In the invention, the coding gene of the porcine circovirus type 2 Cap protein consists of a cDNA sequence for coding the porcine circovirus type 2 Cap protein, a 6 histidine cDNA sequence, cDNA sequences of restriction enzymes Xho I and EcoRI sites and a gene sequence conforming to yeast expression preference.

The porcine circovirus type 2 Cap protein disclosed by the invention well utilizes the advantages of Cap and Pichia pastoris, the coding gene of the protein can be efficiently expressed in the Pichia pastoris, the production cost is reduced, and the application of the recombinant protein in the preparation of porcine circovirus vaccines has high economic value and great social benefit.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is an SDS-PAGE and Western blot analysis of the recombinant protein Cap. In the figure, M: protein molecular weight standards; 1: SDS-PAGE analysis; 2: and (5) carrying out Western blot analysis.

FIG. 2 is an SDS-PAGE analysis of the purified recombinant protein Cap from the Ni-NTA Resin column. Marker in the figure: protein molecular weight standards; sample: expressing the supernatant; flow: flowing through the liquid; washing: washing liquid; elution: and (4) eluting the solution.

FIG. 3 is a diagram of PCV2 specific antibody titer analysis after mice are immunized with the recombinant protein Cap.

FIG. 4 is a graph of neutralizing antibody titer analysis after the immunization of mice with recombinant protein Cap and PCV2 challenge.

FIG. 5 is a graph of PCV2 load analysis in lymph nodes after the challenge of recombinant protein Cap immune mice and PCV 2.

FIG. 6 is a graph of PCV2 specific antibody titer analysis of an immunized mouse after the recombinant protein Cap is used in combination with an immunoadjuvant.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

1. The reagent and its preparation

(1) LB (Luria-Bertani) liquid medium: dissolving peptone 10g, yeast powder 5g, and NaCl 5g in 800mL ddH₂Adjusting pH to 7.0 with 10M NaOH, diluting to 1000mL, and autoclaving (121 deg.C, 1.034 × 10)⁵Pa) for 20min, and storing at 4 ℃.

(2) LB (Luria-Bertani) solid Medium: per 1000mL ddH₂Adding 15g of agar powder into O, adding the agar powder into an LB liquid culture medium, sterilizing at high pressure, and storing at 4 ℃.

(3) Storage solution:

10 XYNB (13.4%, ammonium sulfate-containing without amino acids): 34g YNB and 100g ammonium sulfate were hot dissolved in 1000mL ddH₂And O, filtering and sterilizing, and storing at 4 ℃.

500 × biotin (0.02%): 20mg Biotin dissolved in 100mL ddH₂And O, filtering and sterilizing, and storing at 4 ℃.

10 × glucose (20%): 200g glucose dissolved in 1000mL ddH₂Sterilizing at high pressure in O, and storing at 4 deg.C.

10 × methanol (5% methanol): 5mL methanol with 95mL ddH₂Mixing, filtering, sterilizing, and storing at 4 deg.C.

10 × glycerin (10% glycerin): 100mL Glycerol and 900mL ddH₂Mixing, filtering, sterilizing or autoclaving, and storing at room temperature.

1M dipotassium phosphate buffer, pH = 7.0: 132mL 1M K₂HPO₄868mL of 1M KH₂PO₄Mixing, autoclaving, and storing at room temperature.

10 x acid hydrolyzed casein (10% acid hydrolyzed casein): 100g acid hydrolyzed Casein dissolved in 1000mL ddH₂Sterilizing at high pressure in O, and storing at 4 deg.C.

(4) YPD medium (1.0% yeast extract, 2.0% peptone, 2.0% glucose): 10g of yeast extract and 20g of peptone were dissolved in 900mL of ddH2O, and 20g of agar-agar YPD plate was added thereto, followed by autoclaving, 100mL of 10 Xglucose was added thereto, and the mixture was stored at 4 ℃ until use.

(5) YPG medium (1.0% yeast extract, 2.0% peptone, 1.0% glycerol): 10g yeast extract, 20g peptone in 900mL ddH₂In O, autoclaving, adding 100mL of 10 Xglycerol, and storing at 4 deg.C.

(6) BMMYC medium (1.34% YNB, 4X 10)^-5% biotin, 0.5% methanol, 1.0% yeast extract, 2.0% peptone, 1.0% casamino acid): 10g yeast extract, 20g peptone in 600mL ddH₂O, autoclaved, cooled to room temperature, added with 100mL of 1M dipotassium hydrogen phosphate buffer, mixed well with pH =7.0, 100mL of 10 XYNB, 2mL of 500 Xbiotin, 100mL of 10 Xmethanol, and 100mL of 10 Xacid hydrolyzed casein, and stored at 4 ℃.

(7) Buffer solution: buffer

(50 mM sodium phosphate, 0.3M sodium chloride, 10mM imidazole and 10mM Tris-HCl pH 8.0); buffer

(50 mM sodium phosphate, 0.3M sodium chloride, 500mM imidazole and 10mM Tris-HCl pH 8.0).

2. Optimization and acquisition of Cap Gene sequences

According to the sequence of Cap gene (DQ235696.1) logged in by NCBI and codon preference of yeast, the sequence of Cap gene is optimized, for easy purification, 6 histidines (6 XHis tag) are added at C end, and restriction enzymes Xho I and EcoR I sites are introduced at two ends of the sequence. The optimized Cap gene sequence is synthesized by Invitrogen company, and the nucleotide sequence is shown as SEQ ID NO. 2.

3. Construction of expression vectors

And carrying out double enzyme digestion on the synthesized Cap and plasmid pwPICZalpha by using two restriction enzymes Xho I and EcoR I, carrying out agarose electrophoresis on the product after enzyme digestion, and recovering a DNA fragment by using a gel recovery kit and connecting.

Mixing 7 mu L of double-enzyme-digested target gene fragment, 1 mu L of double-enzyme-digested plasmid, 1 mu L T4 ligase and 1 mu L of ligase buffer solution in a sterile Eppendorf tube, and connecting for 12h at 4 ℃; adding 10 μ L of the ligation product into 90 μ L of competent Escherichia coli Trans 10, mixing, transforming by heat shock at 42 ℃ for 90s, adding 400 μ L of LB liquid medium, culturing at 37 ℃ for 1h by shaking, spreading the bacterial liquid on LB solid medium with Zeocin resistance, and culturing at 37 ℃ overnight. Single colonies were picked from the transformed plates and inoculated into 5mL of LB liquid medium containing Zeocin, and cultured overnight at 37 ℃. Plasmids were extracted using a plasmid extraction kit. Xho I and EcoR I are used for carrying out double enzyme digestion identification on the ligation product, the correct insertion of the gene fragment is verified, and the recombinant plasmid is named as pwPICZalpha-Cap.

4. Expression of recombinant proteins

1) Small dose induced expression

Linearizing 5-10 mu g of recombinant plasmid pwPICZalpha-Cap by using restriction enzyme SacI, recovering linearized pwPICZalpha-Cap by using a PCR product purification kit, adding into 90 mu L of competent Pichia pastoris X-33, uniformly mixing, carrying out electric shock transformation after ice bath for 5min, adding 1mL of sorbitol, standing at 30 ℃ for 2h, coating a bacterial liquid on a Zeocin-resistant YPD solid culture medium, and culturing at 30 ℃ for 3-4 d. Single colonies were picked from the transformed plates and inoculated into 5mL YPD liquid medium containing Zeocin, incubated at 30 ℃ 250rpm for 24h, centrifuged at 3500rpm, the supernatant was discarded, 5mL YPG liquid medium was added, incubated at 30 ℃ 250rpm for 24h, centrifuged at 3500rpm for discarding the supernatant, 2mL BMMYC liquid medium was added, incubated at 27 ℃ 225rpm for 48h, and methanol was supplemented every 12h to maintain the methanol concentration at 0.5%. After centrifugation at 3500rpm, the supernatant was collected and analyzed by SDS-PAGE.

2) Large dose induced expression

Selecting a positive colony to inoculate into YPD liquid culture medium, culturing at 30 ℃ and 250rpm for 24h as seed culture solution, inoculating 13mL of seed culture solution into 250mL of YPD liquid culture medium, culturing at 30 ℃ and 250rpm for 24h, centrifuging at 3500rpm, discarding supernatant, adding 250mL of YPG liquid culture medium, culturing at 30 ℃ and 250rpm for 24h, centrifuging at 3500rpm, discarding supernatant, adding 125mL of BMMYC liquid culture medium, culturing at 27 ℃ and 225rpm for 48h, and supplementing methanol every 12h to maintain the methanol concentration at 0.5%. After centrifugation at 3500rpm, the supernatant was collected and analyzed by SDS-PAGE.

5. Recombinant protein Ni-NTA Resin column purification

To the supernatant of the induced expression, 50mM sodium phosphate, 0.3M sodium chloride, 10mM imidazole and 10mM Tris-HCl pH8.0 were added and filtered through a 0.45 μ M filter. Loading 10mL Ni-NTA Resin column into XK16/20 chromatography column, connecting with constant flow pump, and using 10 times volume of Buffer

Balancing the column, loading after balancing, and using Buffer with 8 times volume after loading

The column was washed and the desired protein was eluted with 8 volumes of Buffer II and collected in 8 different tubes at a flow rate of 5mL/min throughout the process. The purified protein was analyzed by SDS-PAGE, and the SDS-PAGE result in FIG. 2 shows that the objective protein was concentrated in Buffer

Tubes No.2 and No. 3. Buffer II containing the target protein was mixed, packed into a 3.5kDa dialysis bag, and dialyzed against a dialysate containing 20mM Tris-HCl pH8.0, 1mM EDTA pH8.0, 5% glycerol at 4 ℃ for 8 hours, with the dialysate being changed every 4 hours.

6. Mouse immune protection test

6.1 mouse test grouping and design

The purified porcine circovirus type 2 Cap protein is taken as an antigen and is grouped as follows:

(1) positive group I: porcine circovirus type 2 baculovirus vector inactivated vaccine, purchased from blinker haggivenh animal health (usa) ltd;

(2) positive group II: porcine circovirus type 2 genetic engineering subunit vaccine 40 mug/only (Yiyuan), purchased from Qingdao Yibang bioengineering GmbH;

(3) 40 μ g test group: 40 mu g/piece of purified Cap protein;

(4) 10 μ g test group: 10 mu g/piece of purified Cap protein;

(5) 2.5 μ g test group: 2.5 mu g/purified Cap protein;

(6) CD40L test group: 40 μ g/pig of purified porcine-derived CD40L protein (this group was designed to verify the specificity of the expressed Cap protein, and not any one protein could produce this effect);

(7) challenge test group: equal volume of PBS/mouse;

(8) PBS test group: equal volume of PBS/mouse;

(9) blank group: feeding in blank, without any administration.

180 female SPF Kunming mice with the age of 6-8 weeks and the weight of 18-20 g are selected, are all subjected to open-air culture for one week before the test, and are randomly divided into 9 groups, and each group comprises 20 mice. 1-8 groups are subjected to intraperitoneal injection of 100 mu L per mouse according to the grouping, the 9 th group is a blank group, 14d after first immunization, 7 mice in each group are subjected to orbital venous blood sampling, and serum is separated to detect the level of PCV2 specific antibodies; carrying out secondary immunization according to the same mode and dosage; at 14d after the second immunization, 5 mice are killed in each group, the level of PCV2 specific antibody is detected, and spleen lymphocytes are separated from the spleen to carry out lymphocyte proliferation test; at the same time, the remaining 15 mice of each group of 1-7 groups were inoculated with PCV2 venom (TCID 50= 10-5.12) by intraperitoneal injection of 0.5 mL, nasal drip of 50 μ L, and dorsal subcutaneous injection of 0.3 mL at multiple sites, and 8 groups were injected with an equal amount of PBS in the same manner, and clinical symptoms of each mouse were observed and recorded.

6.2 ELISA antibody detection

Separating mouse serum at 14d, 28d, 42 d, 49 d and 56d after primary immunization, diluting the serum at a ratio of 1:800, determining the antibody level of the serum of each group of immunized mice according to a method of a reference document, taking Cap protein expressed by a yeast expression system as a coating antigen, detecting the concentration of target protein by using a BCA kit, diluting the concentration of the target protein to 0.1 mg/mL by using 0.05 mol/L sodium carbonate-sodium bicarbonate buffer solution (pH 9.6) as a coating solution, coating an ELISA plate, coating 100 muL/well, incubating at 37 ℃ for 2h, and coating the antigen at 4 ℃ overnight (muiti 12 h); discarding the coating solution, washing with PBST for 3 times (300 μ L/well) for 5min each time, and blocking with 1% BSA-PBST at 37 deg.C for 3 h (200 μ L/well); discard the blocking solution, wash the plate 3 times with PBST for 5min each time, thenDiluting the collected mouse serum with a sealing solution according to the proportion of 1:800, incubating for 1h at 37 ℃ with 100 mu L/hole; discarding mouse serum, washing PBST again, diluting enzyme-labeled anti-mouse secondary antibody with confining liquid according to a ratio of 1:10000, incubating at 100 mu L/hole for 0.5 h at 37 ℃; discarding the secondary antibody, washing with PBST for 3 times (300 μ L/well) for 5min each time; adding 100 mu L of TMB substrate developing solution into each hole, and incubating for 15 min at 37 ℃; finally, 50. mu.L of 2 mol/L H was added to each well₂SO₄The reaction was stopped, and the OD450nm value was detected by microplate reader within 15 min, the specific results are shown in FIG. 3.

6.3 detection of neutralizing antibodies

At 14d post challenge, sera were inactivated at 60 ℃ for 30 min, and different dilutions of sera were mixed with 100 TCID50/100 μ L PCV2 in equal volumes, incubated for 1h at 37 ℃ and then seeded onto 96-well plates of PK15 cells that had grown into monolayers, and incubated for 3 days at 37 ℃. Cells were fixed with fixative (acetone: methanol =1: 1) at-20 ℃ for 30 min, blocked with 3% BSA for 1h, incubated with anti-Cap monoclonal antibody for 2h, then incubated with fluorescently labeled secondary antibody for 1h, and observed with a fluorescence microscope. The neutralizing antibody titer was the reciprocal of the highest serum dilution that completely prevented PCV2 from infecting PK15 cells, see figure 4 for specific results.

6.4 qPCR detection of viral load in lymph nodes

At 14d after challenge, lymph node tissues of mice in each treatment group were taken to extract viral DNA according to the tiamamp Genomic DNA Kit instructions, and 20 μ L of the reaction system: DNA is less than or equal to 100ng, 10 xSYBR Green qPCR Master Mix with ROX 10 muL, F-Primer 1 muL, R-Primer 1 muL, water is added to supplement to 20 muL. An ABI 7500 Real-Time PCR system is used, pre-denaturation at 95 ℃ is adopted for 30 s, denaturation at 95 ℃ is adopted for 5 s, annealing and extension at 60 ℃ is adopted for 34 s, 40 cycles are carried out, and amplification reaction is carried out after annealing at 95 ℃ for 15 s, annealing at 60 ℃ for 1 min, annealing at 95 ℃ for 30 s and annealing at 60 ℃ for 15 s. The virus load in the lymph nodes of mice of each treatment group was detected by absolute fluorescence quantification, and the specific results are shown in fig. 5.

7 adjuvant combination immunoassay

7.1 preparation of the vaccine

The vaccine is prepared by fusing the ISA206 adjuvant and GEL01 adjuvant specifications provided by SEPPIC France and the Cap protein of porcine circovirus type 2 according to a certain proportion.

(1) 10 + ISA206 vaccine: heating ISA206 adjuvant to 50 ℃ in a water bath kettle, then slowly adding equal volume of Cap protein, stirring for 5min on a magnetic stirrer at 350 rpm, then placing a beaker in a cold bath at 20 ℃ and standing for 1h to avoid moving and stirring, and forming the water-in-oil-in-water two-phase oil emulsion vaccine.

(2) 10 + GEL01 vaccine: under the condition of room temperature, adding 3 times of GEL01 adjuvant into 10 volumes of Cap protein, and continuously stirring for 10 min at the rotating speed of 200 rpm to form the stable water-soluble vaccine.

7.2 mouse test grouping and design

The purified porcine circovirus type 2 Cap protein is taken as an antigen and is grouped as follows: (adjuvants ISA206, GEL01 are supplied by SEPPIC, France)

(1) 10 μ g test group: 10 mu g/piece of purified Cap protein;

(2) test group of 10 + ISA 206: purified Cap protein 10 μ g/mouse (antigen: ISA206=1:1, volume ratio);

(3) 10 + GEL01 abdominal test group: purified Cap protein 10 μ g/mouse (antigen: GEL01 adjuvant =10:3, volume ratio, i.p.);

(4) 10 + GEL01 subcutaneous test group: purified Cap protein 10 μ g/mouse (antigen: GEL01 adjuvant =10:3, volume ratio, subcutaneous injection);

(5) 40 μ g test group: 40 mu g/piece of purified Cap protein;

25 female SPF Kunming mice with the age of 6-8 weeks and the weight of about 18-20 g are randomly divided into 5 groups, and each group comprises 5 mice. The 10 + GEL01 subcutaneous group is injected subcutaneously, the other groups are injected intraperitoneally with 100 mu L per mouse according to the grouping, the 14d after the first immunization and the second immunization are carried out according to the same mode and dosage, the mouse serum of the 14d, 28d, 56d, 70d, 84d and 98d after the first immunization is diluted in a ratio of 1:800, and the PCV2 indirect ELISA antibody detection is carried out, and the specific result is shown in figure 6.

8 statistical analysis of data

The test data adopts GraphPad Prism^TMThe software performs the analysis of the sample to be analyzed,all data were in the form of Mean + -SEM (a, b, c, d indicate significant differences between groups,Pis less than 0.05). All experiments of this experiment were conducted in at least 3 independent experiments and 3 replicates each time.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

<110> Shanxi university of agriculture

<120> porcine circovirus type 2 Cap protein, and coding gene and application thereof

<160>2

<210>1

<211>747

<212>DNA

<213> Artificial sequence

<220>

<221>CDS

<223> coding gene of porcine circovirus type 2 Cap protein

<222>(1)…(747)

<400>1

CTC GAG AAG AGA GAG GCT GAA GCT ACT TAC 30

CCA AGA AGA AGA TAC AGA AGA AGA AGA CAC 60

AGA CCA AGA TCT CAC TTG GGT CAA ATT TTG 90

AGA AGA AGA CCA TGG TTG TTG CAC CCA AGA 120

CAC AGA TAC AGA TGG AGA AGA AAG AAC GGT 150

ATT TTC AAC ACT AGA TTG TCT AGA ACT TTC 180

GGT TAC ACT ATT AAG AGA ACT ACT GTT AAG 210

ACT CCA TCT TGG GCT GTT GAC ATG ATG AGA 240

TTC AAC ATT AAC GAC TTC TTG CCA CCT GGT 270

GGT GGT TCT AAC CCA AGA TCT GTT CCA TTC 300

GAG TAC TAC AGA ATT AGA AAG GTT AAG GTT 330

GAG TTC TGG CCA TGT TCT CCA ATT ACT CAA 360

GGT GAC AGA GGT GTT GGT TCT TCT GCT GTT 390

ATT TTG GAC GAC AAC TTC GTT ACT AAG GCT 420

ACT GCT TTG ACT TAC GAC CCA TAC GTT AAC 450

TAC TCT TCC AGA CAC ACT ATT ACT CAA CCA 480

TTC TCT TAC CAC TCT AGA TAC TTC ACT CCA 510

AAG CCA GTT TTG GAC TCT ACT ATT GAC TAC 540

TTC CAA CCA AAC AAC AAG AGA AAC CAA TTG 570

TGG TTG AGA TTG CAA ACT GCT GGT AAC GTT 600

GAC CAC GTT GGT TTG GGT ACT GCT TTC GAG 630

AAC TCT ATT TAC GAC CAA GAG TAC AAC ATT 660

AGA GTT ACT ATG TAC GTT CAA TTC AGA GAG 690

TTC AAC TTG AAG GAC CCA CCT TTG AAC CCA 720

CAC CAC CAC CAC CAC CAC TAA GAA TTC 747

<210>2

<211>238

<212>PRT

<213> Artificial sequence

<223> porcine circovirus type 2 Cap protein

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TYPRRR YRRRRH RPRSHL GQILRR RPWLLH PRHRYR 36

WRRKNG IFNTRL SRTFGY TIKRTT VKTPSW AVDMMR 72

FNINDF LPPGGG SNPRSV PFEYYR IRKVKV EFWPCS 108

PITQGD RGVGSS AVILDD NFVTKA TALTYD PYVNYS 144

SRHTIT QPFSYH SRYFTP KPVLDS TIDYFQ PNNKRN 180

QLWLRL QTAGNV DHVGLG TAFENS IYDQEY NIRVTM 216

YVQFRE FNLKDP PLNPHH HHHH 238

Claims (4)

1. The amino acid sequence of the porcine circovirus type 2 Cap protein is shown as SEQ ID NO 2.

2. The use of the porcine circovirus type 2 Cap protein of claim 1 in the preparation of a porcine circovirus type 2 vaccine.

3. The method for preparing the porcine circovirus type 2 Cap protein of claim 1, which is characterized by comprising the following steps:

construction of expression vector: designing restriction enzyme cutting sites on the gene sequence of the artificially synthesized Cap, connecting the gene sequence of the Cap and the expression plasmid pwPICZalpha after enzyme cutting by the same restriction enzyme, and detecting and screening the clone containing the gene sequence of the Cap by double enzyme cutting;

construction of the Yeast expression System: integrating the gene sequence of Cap into the genome of the yeast by an electrotransformation method by using a restriction enzyme linearized recombinant expression plasmid; coating the transformed thallus suspension on a YPD plate containing Zeocin, and culturing at 30 ℃; picking a single colony, carrying out small-dose induced expression, and detecting expression supernatant by SDS-PAGE to confirm positive clone;

purification and activity detection of recombinant proteins: selecting positive clone colonies for large-dose induced expression, purifying the expression supernatant by adopting Protein Pure Ni-NTA resin, and detecting the purified recombinant Protein by SDS-PAGE and Western blot.

4. The base sequence of the coding gene of the porcine circovirus type 2 Cap protein is shown as SEQ ID NO 1.

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