Porcine circovirus type 3 cap protein recombinant protein, preparation method, kit and use thereof

Abstract

The application belongs to the technical field of biology, discloses a porcine circovirus type 3 Cap protein recombinant protein and a preparation method, a kit and an application thereof, and the PCV3 Cap protein recombinant protein is obtained by truncation design of PCV3 Cap protein. The kit prepared from the recombinant protein has a coincidence rate of more than 95% with a commercially available product and has good sensitivity and specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a porcine circovirus type 3 Cap protein recombinant protein, a preparation method, a kit and a use thereof. BACKGROUND

[0002] Porcine circovirus type 3 (PCV3) was first reported in the United States in October 2016, and has a high prevalence rate. Studies have shown that China, Brazil, Germany, Poland and other countries have a prevalence rate of more than 50%, indicating that the virus is a world epidemic virus. At present, the virus is showing a growing trend year by year, and is mixedly infected with other diseases, causing great harm to the pig breeding industry.

[0003] In clinical practice, different pig populations infected with PCV3 can exhibit symptoms of systemic disease, reproductive disease, PDNS, respiratory disease, gastrointestinal disease, pre-weaning growth retardation syndrome (PFTS), and heart and multiple system inflammation. The main clinical manifestations mainly include mummified and dead fetuses, weight loss, dyspnea, rectal prolapse, abdominal breathing, diarrhea, depression, congenital tremor and emaciation.

[0004] The mature virion of PCV is a spherical particle with a diameter of 17-20 nm, and the genome of the virus is wrapped therein, encoding two major open reading frames (ORFs): Cap and Rep (replication-associated enzyme). The Cap protein is the only structural protein and major antigen of the virus, and can induce the body to produce an effective immune protective response. Genetic sequence analysis shows that the PCV3 Cap protein is composed of 214 amino acid residues, which is 19-20 amino acids less than the PCV2 Cap protein, has good specificity, and does not exist cross-reaction. By using its antigenicity, a specific antibody detection kit can provide a powerful tool for early clinical detection.

[0005] At present, there have been reports about PCV3 in China, and the detection methods are mainly PCR detection. ELISA, as a conventional antibody detection method, has the advantages of rapidness and batch detection, and is widely used in clinical practice. At present, there are few PCV3 antibody detection kits on the market, which cannot meet the market demand.

[0006] The technical problem to be solved by the present application is how to provide a detection antigen for a PCV3 antibody detection kit. SUMMARY

[0007] The purpose of the present application is to provide a PCV3 Cap protein recombinant protein, which is designed by truncating PCV3 Cap protein, and a kit prepared using the recombinant protein has a coincidence rate of 95.86% (K=0.92) with a commercially available product, a sensitivity of 97.06%, and a specificity of 94.81%. The recombinant protein has better specificity, sensitivity, repeatability, and correlation when applied to the kit.

[0008] To achieve the above-mentioned purpose, the present application discloses a porcine circovirus type 3 Cap protein recombinant protein, and the amino acid sequence of the porcine circovirus type 3 Cap protein recombinant protein is shown as SEQ ID NO. 1.

[0009] Preferably, the nucleotide sequence of the porcine circovirus type 3 Cap protein recombinant protein is shown as SEQ ID NO. 2.

[0010] In addition, the present application also discloses the use of the porcine circovirus type 3 Cap protein recombinant protein as described above for preparing a porcine circovirus type 3 detection kit.

[0011] Preferably, the porcine circovirus type 3 detection kit is an ELISA antibody detection kit.

[0012] In addition, the present application also discloses an ELISA antibody detection kit, and the porcine circovirus type 3 Cap protein recombinant protein as described above is used as a detection antigen.

[0013] Preferably, the ELISA antibody detection kit comprises a sample diluent, an enzyme-labeled antibody, a TMB substrate solution, a stop solution, a concentrated washing solution, a negative control, a positive control, a plate sealing film, and a dilution plate.

[0014] The positive control is prepared from positive serum.

[0015] The negative control is prepared from negative serum.

[0016] The porcine circovirus type 3 Cap protein recombinant protein is coated into a pre-coated plate as a detection antigen.

[0017] In addition, the present application also discloses a preparation method of the porcine circovirus type 3 Cap protein recombinant protein as described above, and a part of the amino acid sequence of the porcine circovirus type 3 Cap protein shown as SEQ ID NO. 1 is intercepted and nucleotide sequence synthesis is performed.

[0018] Subsequently, the synthesized nucleotide sequence is cloned into a prokaryotic expression vector to construct a recombinant plasmid, and the recombinant plasmid is transfected into E. coli and expressed to obtain the porcine circovirus type 3 Cap protein recombinant protein.

[0019] The application has the beneficial effects that the application designs a PCV3 Cap protein recombinant protein through PCV3 Cap protein truncation, the coincidence rate of the reagent kit prepared from the recombinant protein with the market product is 95.86% (K=0.92), the sensitivity is 97.06%, the specificity is 94.81%, and the recombinant protein applied to the reagent kit has better specificity, sensitivity, repeatability and correlation for PCV3. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 For the sequence codon optimization nucleotide difference chart of SEQ ID NO. 2 in Example 1 (red mark is the optimization site);

[0021] Figure 2 For the amino acid sequence comparison difference of Example 1 and Comparative Examples 1-2;

[0022] Figure 3 For the SDS-PAGE chart of the recombinant Cap protein after purification, wherein M is a protein marker; 1: Cap protein, and the arrow indicates the position of the recombinant protein;

[0023] Figure 4 For the Western blot chart of the recombinant Cap protein after purification, wherein M is a protein marker; 1: Cap protein, and the arrow indicates the position of the recombinant protein;

[0024] Figure 5 For the SDS-PAGE chart of the full-length Cap protein induction expression, wherein M is a protein marker; 1: no obvious target protein expression;

[0025] Figure 6 For the SDS-PAGE chart of the recombinant Cap-2 protein after purification in Comparative Example 1, wherein M is a protein marker; 1: Cap-2 protein; and the arrow indicates the position of the recombinant protein. DETAILED DESCRIPTION

[0026] The application will be described in detail below in combination with the embodiments of the application. In the description of the application, it should be noted that the specific conditions not noted in the embodiments are carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not noted by the manufacturer are all conventional products that can be obtained by market purchase.

[0027] Before the embodiments and comparative examples are described, the sources of the related raw materials involved in the application are introduced:

[0028] IPTG: isopropyl-β-D-thiogalactoside: purchased from Lanjierke, with the item number B541007;

[0029] Imidazole: purchased from Macklin, item number 288-32-4;

[0030] YS\HIS tag protein agarose purification resin: purchased from Yixing Biotech, item number 20502ES10;

[0031] BCA kit (BCA protein concentration determination kit): purchased from Shanghai Biyun Tian Biotech Co., Ltd., item number P0012S;

[0032] PBST: 99.95wt% PBS + 0.05wt% Tween;

[0033] Blocking solution for Western blot: PBST containing 5wt% skimmed milk powder;

[0034] Blocking solution (for ELISA blocking solution): internal patent blocking solution (the blocking solution for coating the antigen-coated reaction plate as recorded in the specification 27 of the applicant's prior application CN116298267A);

[0035] HPR-labeled goat anti-pig secondary antibody: purchased from Jackson; item number 161245;

[0036] ECL chemiluminescence method: Biyun Tian: P1008AS;

[0037] The coating solution is 0.05M carbonate buffer (pH=9.6);

[0038] Sample diluent: company's internal patent protection reagent (the monoclonal antibody protection agent as recorded in the specification 30 of the applicant's prior application CN116239678A);

[0039] The washing solution is 1×PBST;

[0040] The termination solution is 1mol / L HCL;

[0041] Enzyme-labeled antibody: dilute the HPR-labeled goat anti-pig enzyme-labeled antibody with the company's internal antibody protection solution (the monoclonal antibody protection agent as recorded in the specification 30 of the applicant's prior application CN116239678A) to the working concentration.

[0042] Example 1

[0043] 1.1 Synthesis of PCV3 Cap protein gene fragment and construction of prokaryotic expression plasmid

[0044] According to the Cap amino acid sequence SEQ ID No. 4 of PCV3 (GenBank: MF593110.1) and truncation, the PCV3 Cap amino acid sequence is SEQ ID No. 1 and the nucleotide sequence is SEQ ID No. 3. According to the codon bias of E. coli, the obtained gene sequence is codon optimized, as shown in SEQ ID No. 2. Figure 1

[0045] More intuitively, the comparison of the truncated amino acid sequence SEQ ID No. 1 and the full-length Cap amino acid sequence SEQ ID No. 4 is shown in Figure 2 As can be seen from the figure, the difference between SEQ ID No. 1 and SEQ ID No. 4 is the sequence of the nuclear localization region (1 aa-33 aa), the non-antigen core region between the nuclear localization and the non-antigen core region at the back.

[0046] The codon-optimized nucleotide sequence (SEQ ID No. 2) of Example 1 of the present application was sent to Suzhou Hongxun Biotechnology Co., Ltd. for sequence synthesis.

[0047] It should be noted that the full-length amino acid sequence of PCV3 Cap protein is SEQ ID No. 4, and the corresponding full-length nucleotide sequence of PCV3 Cap protein is SEQ ID No. 5.

[0048] The sequence SEQ ID No. 2 of the recombinant Cap protein after codon optimization was sent to Suzhou Hongxun Biotechnology Co., Ltd. for preparation, and the specific preparation method was as follows:

[0049] Through the restriction enzyme cutting site NdeI And XhoI The Cap protein gene sequence (SEQ ID No. 2) was cloned into the expression vector plasmid pET-28a (+) (Suzhou Hongxun Biotechnology Co., Ltd.) to construct the recombinant plasmid pET-28a (+)-Cap protein. The sequencing result is correct, and the Cap protein nucleotide sequence (SEQ ID No. 2) is obtained by sequencing.

[0050] The recombinant plasmid pET-28a (+)-Cap protein was transformed E. coli BL21 (DE3) to obtain the recombinant expression strain BL21-Cap protein, and the transformation was carried out on the LB plate with Kanamycin resistance.

[0051] 1.2 Optimization of the induction expression conditions of the recombinant Cap protein

[0052] ​The recombinant expression strain pET-28a (+) -Cap protein monoclonal bacteria were selected from the transformed plate, and the induction expression conditions were optimized. The bacteria were inoculated into 4 mL of LB medium (the final concentration of kanamycin was 25 μg / ml), and the culture was incubated to OD 600 0.4-0.6, respectively, 0.1, 0.2, 0.5, 0.8, 1.0 mM IPTG was used for induction, respectively, 16 ℃, 25 ℃, 30 ℃, 37 ℃, 220 rpm shaking bed induction for 4 h, 6 h, 8 h, and the bacteria liquid induced without IPTG was compared. After boiling the whole bacteria, identification was performed, and the best expression condition was determined according to the protein expression amount, which was 1 mM IPTG with a final concentration, and 30 ℃ shaking bed induction for 4 h.

[0053] 1.3 Purification of recombinant Cap protein

[0054] The induced bacteria were collected by centrifugation, and the bacterial pellet was resuspended with PBS at pH 7.2; the bacteria were lysed by ultrasonic for 30 min until the bacterial liquid became clear. Centrifugation was performed again at 4 ℃ and 8000 rpm for 10 min to obtain the supernatant and bacterial pellet; the bacterial pellet was resuspended with PBS of the same volume as the supernatant to obtain the suspension of the bacterial pellet;

[0055] The supernatant and the suspension of the bacterial pellet were subjected to electrophoresis, and the loading buffer was added and boiled for 10 min. The supernatant was obtained by centrifugation at 4 ℃ and used for SDS-PAGE electrophoresis. First, electrophoresis was performed at 90 V, and then electrophoresis was performed at 180 V after the indicator of bromophenol blue entered the separation gel. The electrophoresis was stopped when the bromophenol blue band migrated to the bottom of the separation gel. The gel was stained with coomassie brilliant blue staining solution for 30 min, and then transferred to the decolorizing solution until the background was clear. The results showed that the PCV3 recombinant truncated Cap protein mainly existed in the supernatant, which was soluble expression, and then the soluble purification method was used for purification.

[0056] In order to more clearly illustrate the difference of the Cap protein gene sequence of the present application, the sequence of the Cap protein truncated recombinant gene in the PCV3 sequence (GenBank: MF593110.1) is provided, wherein SEQ ID No. 3 is the original nucleotide sequence, and SEQ ID No. 2 is the nucleotide sequence after optimization of codon, and the codon optimization site is shown Figure 1 As shown, the red site is the optimized base substitution, which significantly improves the CAI value (Table 1).

[0057] In order to more clearly illustrate the expression advantage of the Cap protein gene sequence of the present application after optimization, the present application provides the comparison results of GC content analysis and codon adaptability index CAI analysis, and the comparison results are shown in Table 1:

[0058] Table 1: GC content analysis and codon adaptation index CAI analysis

[0059] Nucleotide sequence GC content Codon adaptation index SEQ ID No. 2 55% 0.92 SEQ ID No. 3 45% 0.62 SEQ ID No. 5 47% 0.55 SEQ ID No. 6 53% 0.89 SEQ ID No. 8 38% 0.62 SEQ ID No. 9 50% 0.94

[0060] Example 2

[0061] 2.1 Mass expression, purification, antigenicity identification, and expression quantity statistical analysis of PCV3 recombinant Cap protein

[0062] The recombinant expression strain BL21-Cap protein identified as positive expression was cultured in 2L of liquid LB medium added with Kanamycin, and when the growth reached OD 600 0.4-0.6, 1 mM IPTG was preferably used at the final concentration, and the bacterial body was collected after 4h of induction at 30°C;

[0063] The induced bacterial body, control sample, and protein molecular weight Marker were subjected to SDS-PAGE electrophoresis, the induced bacterial body was collected by centrifugation, and the bacterial body precipitate was resuspended with PBS at pH=7.2; the bacteria were lysed by ultrasonic disrupter until the bacterial solution became clear. The supernatant and bacterial body precipitate were obtained by centrifugation at 8000rpm for 10min; the supernatant was purified by Ni-NTA affinity chromatography.

[0064] After Ni-NTA affinity chromatography purification, different concentrations of eluate were obtained, and the eluate was subjected to SDS-PAGE electrophoresis. The purification results are shown in Figure 3 .

[0065] The eluate with single band was collected and dialyzed with 0.1M PBS, and the dialyzed protein was concentrated with sucrose. The protein concentration was determined by BCA kit, and the protein concentration was about 1.6mg / mL. The protein purity analysis reached more than 95%, and the sample was labeled and stored at -80°C.

[0066] 2.2 Western blot identification of PCV3 recombinant Cap protein

[0067] SDS-PAGE electrophoresis:

[0068] 1) Transferring membrane: after the electrophoresis, the gel was placed in the transferring membrane buffer solution for 10min; the PVDF membrane and filter paper with the same size as the gel were soaked in methanol for 10s, and then placed in the transferring membrane buffer solution for 10min; the transferring membrane instrument was placed horizontally, and the filter paper, PVDF membrane, and electrophoresis gel were placed in turn from bottom to top, and then the power was turned on, and the voltage was adjusted to 10V for 40min of transferring membrane;

[0069] 2) Washing: After the end of the transfer, the PVDF membrane was taken out, and PBST was washed 3 times, each time for 5 min;

[0070] 3) Blocking: The PVDF membrane was placed in a blocking solution (5% skimmed milk powder-PBST) overnight, and the washing step was the same as step 2;

[0071] 4) Primary antibody incubation: PCV3 positive serum was diluted (1:100 dilution), incubated at room temperature for 1 h, and washed according to step 2;

[0072] 5) HRP-labeled goat anti-pig secondary antibody diluted with PBST at a ratio of 1:10000 was added;

[0073] 6) Incubation at 25°C for 1 h, and washing according to step 2;

[0074] 7) Color development: Western blot color development was performed using ECL chemiluminescence method, and the reaction was 1-2 min, and a brown product band appeared.

[0075] As shown in the imaging color development results, a specific band of about 17 kDa was visible. Figure 4 Example 3

[0076] 3.1 Screening of optimal coating concentration of antigen and optimal dilution multiple of serum.

[0077] According to the chessboard method, PCV3 recombinant Cap protein (prepared in Example 2) was diluted with coating solution to a final concentration of 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, 100 μL was added to each well of the enzyme-labeled plate, and it was coated at 4°C overnight; wash the liquid 3 times and dry, add blocking solution and incubate at 25°C for 4 h; wash the liquid 3 times, discard the liquid and dry; dilute PCV3 positive serum and PCV3 negative serum with sample diluent at 1:50, 1:100, 1:200, and 1:400, each dilution is repeated once; add 100 μL to each well, react at 25°C for 1 h, wash 3 times and dry; add 100 μL of diluted enzyme-labeled antibody 80000 times to each well, and react at 25°C for 30 min; wash 3 times and dry. Add 100 μL / well of TMB substrate color developing liquid, react at room temperature 25°C for 15 min in the dark, add 50 μL / well of stop solution, and measure the OD 450 value by an enzyme-labeled instrument, and the results are shown in Table 2.

[0078] Table 2 Screening of optimal coating concentration of antigen and optimal dilution multiple of serum

[0079]

[0080]

[0081] ​The relative OD values of the positive control serum and the negative control serum (P / N values) were calculated. 450 The relative OD values of the positive control serum and the negative control serum (P / N values) were calculated. 450 By comparing the P / N values under different conditions, the antigen coating concentration of 1 pg / mL and the serum dilution of 1:100 were determined as the optimal reaction conditions for ELISA.

[0082] The optimal dilution of the enzyme-labeled antibody was determined. The determined PCV3 recombinant Cap protein was added to the enzyme-labeled plate at a concentration of 1 pg / mL for coating, 100 pL was added to each well of the enzyme-labeled plate, and the plate was coated at 4°C overnight; washed with washing solution for 3 times and dried, 100 pL of blocking solution was added and incubated at 25°C for 4 hours; the solution was discarded and dried; the known PCV3 positive serum and PCV3 negative serum were diluted with sample diluent at 1:100, and each dilution was repeated once; 100 pL was added to each well, and the reaction was carried out at 25°C for 1 hour, washed for 3 times and dried; 100 pL of diluted enzyme-labeled antibody was added to each well at a dilution of 20,000, 40,000, 80,000 and 160,000, and the reaction was carried out at 25°C for 30 minutes; washed for 3 times and dried. 100 pL of TMB substrate color developing liquid was added to each well, the reaction was carried out at room temperature for 15 minutes, 50 pL of stop solution was added to each well, and the OD 450 values were determined by an enzyme-labeled instrument, and the determination results are shown in Table 3.

[0083] Table 3 Screening of optimal dilution of enzyme-labeled antibody

[0084] Enzyme-labeled antibody dilution factor selection 20000 40000 80000 160000 PCV3 positive serum-1 4.212 3.602 2.460 1.463 PCV3 negative serum-1 0.616 0.286 0.121 0.078 P / N 6.838 12.594 20.331 18.756

[0085] The relative OD values of the positive control serum and the negative control serum (P / N values) were calculated. 450 The relative OD values of the positive control serum and the negative control serum (P / N values) were calculated. 450 The dilution of the enzyme-labeled antibody with a relatively large P / N value was determined, and a dilution of 80,000 times was determined as the optimal reaction condition for ELISA.

[0086] 3.2 Determination of sample incubation conditions

[0087] Based on the optimal experimental conditions determined above, the sample incubation time and temperature were screened. After 1:100 dilution of the clinical sample was added to each well, the reaction was carried out at 25°C and 37°C for 30 minutes, 45 minutes and 60 minutes, respectively, and ELISA tests were carried out on the negative serum and the positive serum. The OD 450 values of each well were determined, and the determination results are shown in Table 4.

[0088] Table 4 Selection of serum incubation conditions

[0089]

[0090] The OD 450The ratio (P / N) value of the OD value of the positive sample to the OD value of the negative sample was relatively large. 450 The ratio (P / N) value of the OD value of the positive sample to the OD value of the negative sample was relatively large.

[0091] 3.3 Determination of enzyme-labeled antibody reaction conditions

[0092] Based on the optimal experimental conditions determined above, the incubation time and temperature of the enzyme-labeled antibody were screened, 100 μL of enzyme-labeled antibody was added to each well, and the reaction was carried out at 25°C and 37°C for 30 min, 45 min, and 60 min, respectively, in the dark. ELISA tests were performed on negative serum and positive serum, and the OD 450 value of each well was determined, and the results are shown in Table 5:

[0093] Table 5 Selection of enzyme-labeled antibody incubation conditions

[0094]

[0095] The ratio (P / N) value of the OD value of the positive sample to the OD value of the negative sample was relatively large. 450 The ratio (P / N) value of the OD value of the positive sample to the OD value of the negative sample was relatively large. 450 The ratio (P / N) value of the OD value of the positive sample to the OD value of the negative sample was relatively large.

[0096] 3.4 Determination of TMB color development time

[0097] Based on the optimal experimental conditions determined above, the TMB color development time was screened, 100 μL of TMB was added to each well, and the reaction was carried out at room temperature for 5 min, 15 min, and 25 min, respectively, in the dark. ELISA tests were performed on negative serum and positive serum, and the OD 450 value of each well was determined, and the results are shown in Table 6:

[0098] Table 6 Comparison of color development times

[0099] Color development time Color development 5 min Color development 15 min Color development 25 min Positive serum-1 1.264 2.005 3.095 Negative serum-1 0.082 0.105 0.244 P / N 15.415 19.095 12.684

[0100] The ratio (P / N) value of the OD value of the positive sample to the OD value of the negative sample was relatively large. 450 The ratio (P / N) value of the OD value of the positive sample to the OD value of the negative sample was relatively large. 450 The ratio (P / N) value of the OD value of the positive sample to the OD value of the negative sample was relatively large.

[0101] Example 4

[0102] 200 parts of healthy pig serum samples (PCV3 antibody detection negative samples from Keqian Biotechnology) were determined under the optimal conditions screened, and negative and positive control wells were set to statistically analyze the detection results and calculate the S / P value. The S / P value calculation formula is shown below:

[0103] ;

[0104] According to the average value and standard deviation of S / P value of 200 negative samples, the S / P value greater than or equal to 0.4 is determined as positive, and the S / P value less than 0.4 is determined as negative.

[0105] Example 5

[0106] The PCV3 recombinant Cap protein is diluted to 1 μg / mL with a coating solution, and 100 μL is added to each well of an enzyme-labeled plate, which is incubated at 4°C overnight. After washing 3 times with a washing solution and drying, 200 μL of blocking solution is added to each well, which is incubated at 25°C for 4 hours. After discarding the solution and drying, the plate is dried, and the plate is sealed with a sealing film. Thus, a pre-coated plate for PCV3 Cap protein is obtained, which is stored at 4°C for standby.

[0107] The pre-coated plate for PCV3 recombinant Cap protein, negative control serum, positive control serum, sample diluent, enzyme-labeled antibody, substrate TMB, termination solution and concentrated washing solution are assembled to obtain an indirect ELISA detection kit for PCV3 Cap protein antibody.

[0108] The diluted sample to be tested, negative control serum (PCV3 negative serum) and positive control serum (PCV3 positive serum) are added to the coated plate, 100 μL per well. One well is set for each sample to be tested, and two wells are set for the negative control serum and the positive control serum. The plate is incubated at 25°C for 30 minutes. The plate is washed 3 times with a washing solution. Then, 100 μL of enzyme-labeled antibody is added to each well, which is incubated at 25°C for 30 minutes. The plate is washed 3 times with a washing solution, and the last time is dried on a water-absorbing paper. 100 μL of substrate color developing solution is added to each well, which is color developed at room temperature (25°C) for 15 minutes in the dark. Finally, 50 μL of termination solution is added to each well, and the OD 450 absorption value is measured within 15 minutes by using an enzyme-labeled instrument.

[0109] The typical requirements or empirical value test under the conditions of the experimental system / kits is as follows: the OD 450 average value of the positive control hole must be greater than 1, and the OD 450 average value of the negative control hole must be less than 0.3; the sample S / P is greater than or equal to 0.4, which is determined as positive; and the sample S / P is less than 0.4, which is determined as negative.

[0110] Example 6

[0111] According to the method of Example 4, 68 PCV3 positive sera with positive PCV3 antibody detection by Kefa Biological Co., Ltd. and 77 PCV3 negative samples are tested, and the test results are shown in Table 7.

[0112] Table 7: Conformance rate test of PCV3 recombinant Cap protein ELISA antibody detection of clinical serum

[0113]

[0114] The detection results are as follows: for 68 PCV3 positive sera detected by Kefa Biological, 66 can be detected by PCV3 recombinant Cap protein ELISA antibody detection kit, and 2 are not detected; 77 PCV3 negative sera, 73 are detected negative and 4 are detected positive by PCV3 recombinant Cap protein ELISA antibody detection kit; the sensitivity is 97.06%, the specificity is 94.81%, and the overall coincidence rate is 95.86%.

[0115] According to the method of Example 4, 4 specific sera were detected: reference sera purchased by China Animal Health Inspection and Quarantine: African swine fever positive serum, porcine reproductive and respiratory syndrome virus positive serum, swine fever positive serum and pseudorabies positive serum.

[0116] Results: 4 samples were all negative, and the specificity was good.

[0117] Example 7

[0118] Stability test of PCV3 recombinant Cap protein ELISA antibody detection kit

[0119] PCV3 Cap protein antibody detection kit was prepared and placed in a 37°C incubator for a 15-day heat stability test. At the same time, the kit stored at 4°C was compared, and the degradation rate of the OD 450 value of the clinical sample and the degradation of the S / P value were determined on the 15th day, as shown in Table 8:

[0120] Table 8 Overall stability test of kit

[0121]

[0122] Through the 37°C heat stability test of PCV3 recombinant Cap protein ELISA antibody detection kit, the average degradation rate of the OD 450 value of the positive control and the clinical sample was 18.49%, and the average degradation of the S / P value was 0.075. The results show that the kit has good stability.

[0123] Comparative Example 1

[0124] The PCV3 Cap protein genome (MF593110.1) full-length was codon-optimized to obtain the optimized sequence SEQ ID No. 6.

[0125] The Cap protein gene of Comparative Example 1 was synthesized by Suzhou Hongxun Biological Technology Co., Ltd.;

[0126] The purpose sequence adds enzyme digestion site, recombinant vector, expression and purification method are the same as Examples 1 and 2.

[0127] Results: The full-length sequence of PCV3 Cap protein sequence cannot be expressed.

[0128] SDS-PAGE results show no target band ( Figure 5 ), Western blot did not see specific signal. The results analysis showed that the nuclear localization region (1-33aa) of full-length Cap protein contains high charge disordered structure, leading to the failure of prokaryotic system expression.

[0129] Comparative Example 2

[0130] The truncated gene sequence (SEQ ID No. 1) of PCV3 Cap protein was designed, and the nucleotide sequence corresponding to the amino acid truncated sequence 2 (SEQ ID No. 7) of Cap protein was obtained from the optimized Cap protein full gene. The obtained sequence was codon optimized according to the codon preference, and the sequence SEQ ID No. 9 was obtained. According to the comparison of different truncation methods, it can be seen from the figure that the difference between SEQ ID No. 4 (full length) and SEQ ID No. 7 is that the sequence only removes the front nuclear localization amino acid (1-33aa).

[0131] The Cap protein gene SEQ ID No. 9 of Comparative Example 2 was synthesized by Suzhou Hongxun Biotechnology Co., Ltd.;

[0132] The purpose sequence adds enzyme digestion site, and the expression and purification method of the recombinant vector is the same as that of Example 1 and 2; the purification results of the target protein are as Figure 6 .

[0133] The kit preparation is the same as Example 5, and the corresponding clinical sample detection system is established;

[0134] The effect evaluation is the same as Example 5, and the test results are shown in Table 9:

[0135] Table 9 Clinical serum coincidence rate test of developed kit

[0136]

[0137] The detection results are as follows: for the 68 PCV3 positive sera detected by Kebio, 66 can be detected by the kit of Comparative Example 2, and 2 are not detected; 77 PCV3 negative sera are detected by the kit of Comparative Example 2, 67 are detected negative, and 10 are detected positive; the sensitivity is 97.06%, the specificity is 87.01%, and the overall coincidence rate is 91.72%.

[0138] By comparison, the advantages of the recombinant Cap protein of the application mainly include the following aspects:

[0139] The application carries out a large amount of sequence analysis on the Cap protein sequence, selects the target sequence with better antigenicity and hydrophilicity according to the hydrophilicity and antigenicity prediction of the target sequence, and carries out truncated expression, so that the target sequence has good antigenicity and hydrophilicity.

[0140] After determining the recombinant Cap protein sequence, the application carries out codon optimization according to the codon bias, uses the codon system preferred by the expression host without changing the amino acid sequence, so that the success rate of expression of the recombinant Cap protein is greatly improved.

[0141] The application uses the recombinant Cap protein sequence to shorten the original target gene sequence, which can reduce the gene synthesis cost to a certain extent.

[0142] The kit prepared by the application has good sensitivity and specificity, and meets the market requirements.

[0143] It can be found from the comparison of Comparative Example 1 and the examples that the full-length Cap protein cannot be expressed. Analysis shows that there are a large number of disordered and high-charge regions in the nuclear localization interval, which leads to the failure of protein expression.

[0144] Through Comparative Example 2 and Examples 1 and 2, it can be found that the nuclear localization interval of 1-33 aa stage is considered when selecting the sequence fragment. Compared with Comparative Example 2, the example only retains the region with the strongest antigenicity, which contains three main linear epitopes and surface exposure regions. In this way, the expression length of the target sequence can be shortened, and the protein expression amount can be increased. The results prove that the relative expression amount of the antigen of Example 1 is higher than that of Comparative Example 2.

[0145] Analysis of this sequence shows that the quality of the protein used in the kit is related to at least the following two factors:

[0146] 1. Antigenic nuclear localization amino acid of the sequence;

[0147] 2. Non-antigen core region in the sequence except the position with high antigenicity.

Claims

1. A recombinant protein of porcine circovirus type 3 Cap protein, characterized in that, The amino acid sequence of the porcine circovirus type 3 Cap protein recombinant protein is shown as SEQ ID NO.

1.

2. The recombinant protein of porcine circovirus type 3 Cap protein according to claim 1, characterized in that, The nucleotide sequence of the porcine circovirus type 3 Cap protein recombinant protein is shown as SEQ ID NO.

2.

3. Use of the porcine circovirus type 3 Cap protein recombinant protein according to any one of claims 1-2 for preparing a porcine circovirus type 3 detection kit.

4. Use according to claim 3, characterized in that, The porcine circovirus type 3 detection kit is an ELISA antibody detection kit.

5. An ELISA antibody detection kit characterized in that, The porcine circovirus type 3 Cap protein recombinant protein according to any one of claims 1-2 is used as a detection antigen.

6. The antibody detection kit according to claim 5, characterized in that, The kit further comprises sample diluent, enzyme-labeled antibody, TMB substrate solution, stop solution, concentrated washing solution, negative control, positive control, plate sealing film and dilution plate. The positive control is prepared from positive serum. The negative control is prepared from negative serum. The porcine circovirus type 3 Cap protein recombinant protein is coated into a pre-coated plate as a detection antigen.

7. A method for preparing the porcine circovirus type 3 Cap protein recombinant protein according to any one of claims 1 to 2, characterized by, The amino acid sequence shown as SEQ ID NO. 1 in the amino acid sequence of the porcine circovirus type 3 Cap protein is intercepted and subjected to nucleotide sequence synthesis; Subsequently, the synthesized nucleotide sequence is cloned into a prokaryotic expression vector to construct a recombinant plasmid, and the recombinant plasmid is transfected into E. coli and expressed to obtain the porcine circovirus type 3 Cap protein recombinant protein.

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