Preparation of Monoclonal Antibody Based on VP1 Protein Against Senecavirus A (SVA) and Trial Production of Colloidal Gold Test Strips for SVA Detection
ZHANG Tao1,2, WANG Hui-Bao1,2, SUN Yan-Yan3, ZHAI Guo-Yuan2, CUI Yan1,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 China Agricultural Vet. Bio. Science and Technology CO., Ltd., Lanzhou 730046, China; 3 Lanzhou Animal Research Biotechnology Co., Ltd., Lanzhou 730046, China
Abstract：Senecavirus A (SVA), an emerging pathogen in recent years, is posing a severe danger to the healthy development of pig (Sus domesticus) industry in China. Viral structural protein 1 (VP1), the main structural protein of SVA, can induce the body to produce specific antibodies. In order to prepare monoclonal antibodies (McAbs) against SVA VP1 and establish a rapid, efficient and simple method for the detection of SVA, the SVA VP1 gene was amplified by RT-PCR and cloned into the expression vector pET-28a (+). After induction, expression and purification, the recombinant SVA VP1 protein was obtained. BALB/c mice (Mus musculus) were immunized with recombinant SVA VP1 protein, 2 hybridoma cell lines (2E10 and 3E4) stably secreting monoclonal antibodies against SVA VP1 protein were obtained. Ascites type McAbs were obtained and purified by immunizing mice with cell lines 2E10 and 3E4. McAbs were identified by indirect immunofluorescence assay (IFA) and Western blot. The prepared 2E10 McAbs were used as capture antibody, Goat anti-mouse IgG and 3E4 McAbs were used as quality control band (C band) and detection band (T band) to produce SVA colloidal gold test strip. The results showed that the recombinant SVA VP1 protein was mainly expressed in the form of inclusion bodies, with a molecular weight of about 43 kD. The results of antibody subtype identification showed that the heavy chain subtypes of 2E10 and 3E4 were IgG2b and IgG2a, respectively, and the light chains were both κ chain. The results of IFA and Western blot showed that the obtained McAbs were specific bound to SVA. The test of strip showed that colloidal gold test strips could specifically detect SVA, and had no cross reaction with Foot and mouth disease virus (FMDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Classical swine fever virus (CSFV) and Porcine circovirus type 2 (PCV2); The minimum detected viral content was 5×101.13 TCID50/mL;The positive coincidence rate between this method and qPCR was 96.55%. The above results indicate that the prepared colloidal gold test paper based on VP1 monoclonal antibody in this study can provide a new method for the point-of-care testing of SVA.
张涛, 王会宝, 孙燕燕, 翟国元, 崔燕. A型塞内卡病毒VP1蛋白单克隆抗体制备及胶体金试纸条的试制[J]. 农业生物技术学报, 2022, 30(11): 2255-2266.
ZHANG Tao, WANG Hui-Bao, SUN Yan-Yan, ZHAI Guo-Yuan, CUI Yan. Preparation of Monoclonal Antibody Based on VP1 Protein Against Senecavirus A (SVA) and Trial Production of Colloidal Gold Test Strips for SVA Detection. 农业生物技术学报, 2022, 30(11): 2255-2266.
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