猪流行性腹泻病毒S1D蛋白的优化表达及其单克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2021.12.032

Abstract

Abstract: To investigate the epitope and function of Porcine epidemic diarrhea virus (PEDV) S protein, by optimizing the codon of S1D gene, the recombinant prokaryotic expression plasmids pET-S1D which was not optimized and pET-ΔS1D which was optimized were constructed.The expression of S1D protein was respectively induced and ultimately purified.SDS-PAGE and Western blotting were used to verify the correct expression of S1D and ΔS1D proteins in Escherichia coli.The expression levels of S1D and ΔS1D proteins were scanned by the software Image J, and the difference was analyzed by t-test.The purified ΔS1D protein was used as antigen to immunize BALB/c mice.Monoclonal cell strain was obtained by cell fusion, screening and subcloning.Induction method in vivo was used to prepare ascites with anti-PEDV S1D protein.ELISA, Western blotting and IFA were used to detect the titer and specificity of ascites.SDS-PAGE and Western blotting results showed that both S1D and ΔS1D proteins had the correct target band at 34 ku.t-test result showed that the protein expression levels of S1D and ΔS1D were extremely significantly different (P<0.01).ELISA result showed that the antibody titer of ascites reached 1∶1 000 000.The monoclonal antibody reacted positively with PEDV and purified ΔS1D protein, but negatively with PEDV N protein, pET-32a(+) empty vector protein and Porcine reproductive and respiratory syndrome virus (PRRSV), Transmissible gastroenteritis virus (TGEV), Classical swine fever virus (CSFV), Porcine deltacoronavirus (PDCoV) and Swine acute diarrhea syndrome coronavirus (SADS-CoV).Western blotting result showed that ascites and ΔS1D protein, ascites and PEDV S protein had specific bands at 34 and 180 ku, respectively, but no band with pET-32a(+) empty vector protein and normal Vero cell protein.IFA indicated that the ascites and the positive control group could make the cells show a specific green fluorescent signal, while the blank and negative control groups had no green fluorescent signal.Codon optimization could significantly increase the expression level of the recombinant protein in the prokaryotic expression system.Based on high efficiency expression of ΔS1D protein, a monoclonal antibody capable of stable secretion and specific response to PEDV S1 protein was successfully prepared in this study.The preparation of S protein monoclonal antibody provided a good basis for further research on the PEDV S protein antigen epitope and protein function.

Key words: Porcine epidemic diarrhea virus (PEDV); prokaryotic expression; S1D protein; monoclonal antibody

CLC Number:

S852.65⁺1

LI Jiesen, SUN Ronghang, KUANG Yanqi, CHEN Luman, LIU Qing, GUO Xiaofeng. Optimal Expression of Porcine Epidemic Diarrhea Virus S1D Protein and Preparation of Its Monoclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4641-4651.

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Optimal Expression of Porcine Epidemic Diarrhea Virus S1D Protein and Preparation of Its Monoclonal Antibody

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