Eukaryotic expression plasmid of B602L protein, B602L protein, application and detection system

Abstract

The invention belongs to the field of biology, and discloses a eukaryotic expression plasmid of African swine fever B602L protein, the plasmid comprises a vector and a modified AFSV B602L gene connected in the vector; the nucleotide sequence of the modified AFSV B602L gene is as shown in SEQ ID NO. 1 (sequence identifier number 1). A target gene of the plasmid is subjected to codon optimization, a melittin signal peptide sequence (MKFLVNVALVFMVVYI SYIYA) is designed at the N end of the B602L protein, a (6-10) * His tag is designed at the C end of the B602L protein, a flexible linker (3-5 GGGGS repetitive sequences) is connected with the target gene, the purity of the protein obtained by expression of the plasmid is high, and the sensitivity of the protein can reach 1: 25600 after the protein is used for indirect ELISA detection. Meanwhile, the invention further provides the B602L protein, application and a detection system.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a eukaryotic expression plasmid of a B602L protein, a B602L protein, a use, and a detection system. Background Art

[0002] African swine fever is an important swine infectious disease caused by African swine fever virus (ASFV), characterized by rapid onset and high mortality. Pigs are the only natural host of ASFV. Currently, there are no specific drugs or effective vaccines on the market for the treatment and prevention of African swine fever. Perfect biosafety protection measures and rapid detection technology are the main means of prevention and control of the disease. The clinical symptoms of African swine fever are similar to those of classical swine fever. The development of specific and sensitive serological detection technology has broad application value and is helpful for the differential diagnosis, prevention and control of African swine fever.

[0003] ASFV is an enveloped, double-stranded linear DNA virus with a complex viral genome structure, encoding 151 to 167 open reading frames. Among them, the reported proteins such as P72, P54, P30, pp62, CD2v, and B602L have strong immunogenicity and are the main targets for the development of subunit vaccines and serological detection technologies. Among them, the non-structural protein B602L is efficiently expressed in the cytoplasm in the late infection period. It is generally considered to be a molecular partner of the P72 protein and plays an important role in the assembly of the viral nucleocapsid. Wang Pengfei used the pGEX-6P-1 prokaryotic expression vector to express the B602L protein in prokaryotes and established an indirect ELISA detection method based on the B602L recombinant protein. After comparison, the consistency rate of this method with the detection results of the blocking ELISA kit based on the p72 protein was 95%, indicating that B602L is one of the main antigenic proteins of ASFV, but the prokaryotic expression system lacks a post-translational modification mechanism and often cannot form a complete three-dimensional conformation.

[0004] Compared with prokaryotes (such as E. coli), insect cells can perform correct post-translational modifications such as glycosylation, phosphorylation, and nitrosation, which are important in protein function research and biopharmaceutical production. In addition, the Baculovirus expression system has the advantages of high yield, flexible operation, and easy purification.

[0005] The application of B602L protein in detecting African swine fever can be found in the following literature:

[0006] CN118388665A A tandem recombinant protein of dominant antigenic epitopes of African swine fever virus and its application;

[0007] CN117031039A Indirect ELISA detection method for ASFV antibodies based on double antigens;

[0008] CN115032387A An indirect ELISA antibody detection method for African swine fever virus, a kit and its application;

[0009] The sensitivity of the above scheme is shown in Table 1:

[0010] Table 1 Sensitivity statistics

[0011]

[0012] Note: There is no correlation between antigen coating concentration and sensitivity. The above data is only to clearly show the parameter data of different schemes.

[0013] It can be seen from the above scheme that the sensitivity does not exceed 1:6400.

[0014] The problem that this program needs to solve: How to improve the detection sensitivity of African swine fever. Summary of the invention

[0015] The invention aims to provide a eukaryotic expression plasmid of a B602L protein. The target gene of the plasmid is codon-optimized, a bee venom signal peptide sequence (MKFLVNVALVFMVVYI SYIYA) is designed at the N-terminus of the B602L protein, a (6-10)×His tag is designed at the C-terminus of the B602L protein, and the plasmid is connected to the target gene with a flexible linker (3-5 GGGGS repeating sequences). The plasmid has high purity of protein obtained after expression, and the sensitivity of the protein after indirect ELISA detection can reach 1:25600.

[0016] At the same time, the present invention also provides B602L protein, use and detection system.

[0017] To achieve the above objectives, the present application discloses a eukaryotic expression plasmid of African swine fever B602L protein, the plasmid comprising a vector and a modified AFSV B602L gene connected to the vector; the nucleotide sequence of the modified AFSV B602L gene is shown in SEQ ID NO.1.

[0018] B602L gene sequence before optimization (SEQ ID NO.3):

[0019]

[0020] B602L gene optimized sequence (SEQ ID NO.1):

[0021]

[0022] In the above-mentioned eukaryotic expression plasmid of African swine fever B602L protein, the vector is a baculovirus shuttle vector pFastBac1; the modified AFSV B602L gene is located between EcoR I and Sal I restriction endonucleases of the baculovirus shuttle vector pFastBac1.

[0023] At the same time, the present invention also discloses an African swine fever B602L protein, whose amino acid sequence is shown in SEQ ID NO.2.

[0024] Amino acid sequence of B602L protein (SEQ ID NO.2):

[0025] AEFNIDELLKNVLEDPSTEISEETLKQLYQRTNPYKQFKNDSRVAFCSFTNLREQYIRRLIMTSFIGYVFKALQEWMPSYSKPTHTTKTLLSELITLVDTLKQETNDVPSESVVNTILSIADSCKTQTQKSK EAKTTIDSFLREHFVFDPNLHAQSAYTCADTNVDTCASMCADTNVDTCASMCADTNVDTCASTCTSTEYTDLADPERIPLHIMQKTLNVPNELQADIDAITQTPQGYRAAAHILQNIELHQSIKHMLENPRA FKPILFNTKITRYLSQHIPPQDTFYKWNYYIEDNYEELRAATESIYPEKPDLEFAFIIYDVVDSSNQQKVDEFYYKYKDQIFSEVSSIQLGNWTLLGSFKANRERYNYFNQNNEIIKRILDRHEEDLKIGKE ILRNTIYHKKAKNIQETGPDAPGLSIYNSTFHTDSGIKGLLSFKELKNLEKASGNIKKAREYDFIDDCEEKIKQLLSKENLTPDEESELIKTKKQLDNALEMLNVPDDTIRVDMWVNNNNKLEKEILYTKAEL

[0026] In the above-mentioned African swine fever B602L protein, its preparation method is:

[0027] The eukaryotic expression plasmid as described above was transfected into eukaryotic cells, and the protein expressed by the eukaryotic cells was collected and identified as the African swine fever B602L protein.

[0028] In the above-mentioned African swine fever B602L protein, the eukaryotic cell is a Sf9 insect cell.

[0029] In addition, the present invention also discloses the use of a kit or vaccine for preparing an African swine fever B602L protein as described above.

[0030] In the above-mentioned use, the kit is an indirect ELISA kit for detecting African swine fever.

[0031] Finally, the present invention also discloses an African swine fever detection system, comprising any of the African swine fever B602L proteins described above.

[0032] In the above detection system, the detection system is an indirect ELISA detection system.

[0033] The beneficial effects of this application are:

[0034] The expression level of AFSV B602L recombinant protein in insect cells can reach 0.6 mg / mL. The purified B602L recombinant protein was coated on a 96-well ELISA plate to establish an indirect ELISA detection method. This method only requires a protein coating amount of 25 μg / well and has the same or even better detection sensitivity and specificity as commercial ELISA kits, and the compliance rate reaches 93.47%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the result of PCR amplification of B602L gene;

[0036] Figure 2 This is a picture showing the results of western blots to identify the B602L recombinant protein;

[0037] Figure 3 This is the result of SDS-PAGE identification of B602L recombinant protein;

[0038] Figure 4 This is a graph showing the specificity detection results of the B602L indirect ELISA method;

[0039] Figure 5 This is the sensitivity test result of the B602L indirect ELISA method. DETAILED DESCRIPTION

[0040] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that, if no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0041] Example 1

[0042] 1.1 Construction of B602L baculovirus transfer vector

[0043] The B602L gene sequence of ASFV Pig / HLJ / 2018 strain (MK333180) was downloaded from the NCBI website, and its signal peptide sequence was analyzed using an online tool (https: / / services.healthtech.dtu.dk). The melittin signal peptide sequence (MKFLVNVALVFMVVYISYIYA) was designed at the N-terminus of the B602L protein, and a 6×His tag was designed at the C-terminus of the B602L protein, which was connected to the target gene with a flexible linker (G4S) 3. Codon optimization strategy Codon optimization was performed according to the preference of Sf9 insect cells.

[0044] The gene sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0045] Using the synthetic gene as a template, primers were designed for PCR amplification. The primer sequences are shown in Table 2. The B602L gene was cloned between the EcoR I and Sal I restriction endonucleases of the baculovirus transfer plasmid pFastBac1. The results are shown in Figure 1 As shown, a target band of about 1700 bp can be amplified, which is consistent with expectations, and sequencing confirmed that there is no frameshift and mutation, and it was named pFastBac1-B602L.

[0046] Table 2 Primer information

[0047]

[0048] Figure 1 This is a diagram of the PCR amplification results of the B602L gene; the meanings of the numbers are: M: DL8000 DNA Marker; 1: PCR amplification results of the B602L gene.

[0049] 1.2 Rescue of recombinant baculovirus

[0050] The constructed baculovirus donor plasmid pFastBac-B602L was transformed into DH10Bac competent cells, and blue-white screening was performed on antibiotic plates containing 50 μg / mL kanamycin, 10 μg / mL tetracycline, 7 μg / mL gentamicin, 125 μg / mL IPTG, and 100 μg / mL X-gal. Single white colonies were identified by PCR using B602L-F / B602L-R primers.

[0051] The recombinant bacmid B602L was extracted according to the instructions of the BAC / PAC DNA mini-extraction kit, and the nucleic acid concentration was measured and stored in a -20°C refrigerator for later use.

[0052] Sf9 insect cells in the logarithmic growth phase were inoculated into 6-well cell culture plates and allowed to stand for 4 hours before bacmid transfection. The steps were as follows: 5 μg of Bacmid-B602L bacmid and 8 μL of Cellfectin II transfection reagent were added to 125 μL of Opti medium respectively, and premixed at room temperature for 5 minutes; the two were mixed and allowed to stand at room temperature for 5 minutes; then slowly added to Sf9 cells. The cells were blindly propagated for 3 generations in Sf9 cells until Sf9 cells showed obvious cytopathic effects. The Sf9 cells with obvious lesions were frozen and thawed 3 times, and the supernatant samples were separated by centrifugation, which was the recombinant baculovirus expressing B602L protein.

[0053] 1.3 Expression and identification of B602L recombinant protein

[0054] Western blots were used to detect whether the exogenous protein B602L was expressed in Sf9 cells. The western blots test method was briefly as follows: Sf9 cells with obvious lesions were harvested, and after freezing and thawing three times, the supernatant and cell sediment samples were separated by centrifugation, and 12% SDS-PAGE electrophoresis was performed. After NC membrane transfer, anti-His monoclonal antibody (1:5000 dilution) and ASFV standard positive serum (1:500 dilution) were used as primary antibodies and incubated overnight at 4°C, washed with PBST three times, and then incubated with HPR-labeled goat anti-mouse IgG secondary antibody (1:10000 dilution) and HPR-labeled rabbit anti-swine IgG secondary antibody (1:10000 dilution) at room temperature for 1 hour, PBST was used to wash off the residual secondary antibody, and imaging was performed using the Azure Biosystems C600 multifunctional molecular imaging system.

[0055] The results are as follows Figure 2 As shown in the figure, both the Sf9 cell precipitate and the supernatant can develop specific binding bands, indicating that the B602L recombinant protein can be expressed in insect cells and secreted into the cell supernatant. The supernatant samples after Sf9 cell lysis were taken for western blots identification, and the African swine fever standard positive serum was used as the primary antibody (1:500 dilution), and specific binding bands can still be developed, indicating that the B602L recombinant protein expressed by insect cells has good antigenicity and can be recognized by the African swine fever positive serum.

[0056] Figure 2 This is a picture showing the results of western blots to identify the B602L recombinant protein; Figure 2 The meanings of the numbers in the figure are as follows: M: protein molecular weight standard; 1: Sf9 cell pellet, incubated with His monoclonal antibody as the primary antibody; 2: Sf9 cell supernatant, incubated with His monoclonal antibody as the primary antibody; 3: Sf9 cell supernatant, incubated with African swine fever positive serum as the primary antibody.

[0057] 1.4 Purification of B602L recombinant protein

[0058] After western blot identification, the recombinant baculovirus expressing B602L protein was inoculated into Sf9 cell culture medium at a ratio of 1:10, incubated at 27°C for 72 hours, and then ultrasonically disrupted after 3 freeze-thaw cycles, with a power of 35W, a working time of 5 minutes, an interval of 2 minutes, and a total time of 10 minutes. Centrifuged at 7000g for 10 minutes at room temperature, and the supernatant was collected. The B602L recombinant protein was purified by His nickel affinity chromatography.

[0059] The SDS-PAGE analysis showed that Figure 3 As shown, the size of the B602L recombinant protein purified by His nickel strain is about 70 ku, and the protein concentration of B602L after purification is 0.6 mg / mL as determined by the BCA method.

[0060] Figure 3 This is the result of SDS-PAGE identification of B602L recombinant protein; Figure 3 The meanings of the symbols are as follows: M: protein molecular weight standard; 1: purified B602L recombinant protein.

[0061] 1.5 Establishment of indirect ELISA test method based on B602L protein

[0062] The optimal antigen coating concentration and the optimal dilution of the serum to be tested for the B602L indirect ELISA detection method were determined by the square matrix method. The antigen gradient coating concentrations were 400, 200, 100, 50, 25, 12.5, and 6.25 ng / well, respectively. The dilutions of ASFV negative and positive sera were 1:50, 1:100, 1:200, 1:400, and 1:800, respectively. The optimal antigen coating concentration and serum dilution were screened according to the ratio of positive serum OD450nm (P) to negative serum OD450nm (N), i.e., the P / N value. The optimal antigen coating concentration and serum dilution were identified to be 25 ng / well and 1:400, respectively.

[0063] After determining the optimal antigen coating mass concentration and serum dilution, the optimal HRP-labeled rabbit anti-pig secondary antibody dilution (1:5000, 1:10000, 1:20000, 1:40000), the incubation time of the serum to be tested (30min, 45min, 60min, 90min), the optimal incubation time of the HRP-labeled rabbit anti-pig secondary antibody (30min, 45min, 60min), and the optimal substrate color development time (5min, 10min, 15min, 20min) were optimized to determine the reaction conditions of indirect ELISA. The results showed that the optimal incubation time of the serum to be tested was 60min; the optimal HRP-labeled rabbit anti-pig secondary antibody dilution was 1:10000; the optimal incubation time of the HRP-labeled rabbit anti-pig secondary antibody was 60min; and the optimal substrate color development time was 15min;

[0064] According to the indirect ELISA method established above, 40 ASF-negative sera with clear background were tested and OD was determined. 450nm The values ​​were statistically analyzed and the mean OD value (X) and standard deviation (SD) were calculated. 450nm ≥X+3SD, it is considered positive. 450nm ≤X+2SD is considered negative. The OD of 40 ASF-negative sera with clear background was determined by the established indirect ELISA method. 450nm After statistical analysis, the mean value (X) of the serum samples was calculated to be 0.095, and the standard deviation (SD) was 0.029. 450 When the value is ≥0.182, the sample is judged to be positive; if the OD 450 When OD ≤0.153, the sample is judged as negative; 450 If the value is between the two, the sample is considered suspicious.

[0065] 1.6 Specificity, sensitivity and repeatability test of indirect ELISA

[0066] To determine whether the B602L indirect ELISA detection method has cross-reactions with common swine disease pathogens, positive sera (2 copies each) of four common swine disease pathogens, including classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine epidemic diarrhea virus (PEDV), were selected, and African swine fever positive and negative sera were used as controls. Indirect ELISA tests were repeated 3 times for each serum to evaluate the specificity of the method.

[0067] refer to Figure 4The results shown in the figure indicate that the B602L indirect ELISA method does not react with the positive sera of four common swine disease pathogens (CSFV classical swine fever virus, PRV pseudorabies virus, PRRSV porcine blue ear virus, and PEDV porcine epidemic diarrhea virus), and has good specificity.

[0068] To determine the sensitivity of the B602L indirect ELISA detection method, the standard positive serum of African swine fever (China Veterinary Drug Administration, batch 202101) was diluted 2 times (1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600) and tested by ELISA. The test was repeated 3 times. The maximum dilution factor of the ELISA method for ASFV standard positive serum was determined to evaluate the sensitivity of the method.

[0069] refer to Figure 5 The results showed that the maximum dilution factor of the B602L indirect ELISA method for detecting ASFV standard positive serum was 1:25600, with good sensitivity.

[0070] To determine the repeatability of the B602L indirect ELISA detection method, three ELISA plates from the same batch and three from different batches were used to perform inter-batch and intra-batch repeat tests with eight samples (four positive and four negative), and the average OD of each serum sample was calculated. 450nm (X), standard deviation (SD) and coefficient of variation (CV = SD / X). The results showed that the inter-assay and intra-assay coefficients of variation of the B602L indirect ELISA method were 4.12% and 8.75%, respectively, both less than 10%, indicating that the ELISA method had good repeatability.

[0071] The established indirect ELISA antibody detection method was used to test 92 clinical pig serum samples and compared with the Zhaoqing Dahuanong African swine fever virus ELISA antibody detection kit to compare the consistency of the two methods. The results showed that the consistency rate of the B602L-ELISA method and the commercial ELISA detection kit was 93.47%.

Claims

1. A eukaryotic expression plasmid of African swine fever B602L protein, characterized in that: The plasmid includes a vector and a modified AFSV B602L gene connected to the vector; the nucleotide sequence of the modified AFSV B602L gene is shown in SEQ ID NO.

1.

2. The eukaryotic expression plasmid of African swine fever B602L protein according to claim 1, characterized in that: The vector is a baculovirus shuttle vector pFastBac1; the modified AFSV B602L gene is located between EcoR I and Sal I restriction endonucleases of the baculovirus shuttle vector pFastBac1.

3. An African swine fever B602L protein, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

4. The African swine fever B602L protein according to claim 3, characterized in that Its preparation method is: The eukaryotic expression plasmid as described in claim 1 or 2 is transfected into eukaryotic cells, and the protein expressed by the eukaryotic cells is collected and identified as African swine fever B602L protein.

5. The African swine fever B602L protein according to claim 4, characterized in that The eukaryotic cells are Sf9 insect cells.

6. Use of the African swine fever B602L protein according to any one of claims 3 to 6 to prepare a kit or vaccine.

7. The use according to claim 6, characterized in that The kit is an indirect ELISA kit for detecting African swine fever.

8. An African swine fever detection system, characterized in that: Comprising the African swine fever B602L protein as described in any one of claims 3 to 6.

9. The detection system according to claim 8, characterized in that: The detection system is an indirect ELISA detection system.

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