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

摘要/Abstract

摘要： 为进一步探究猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)S蛋白的抗原表位及其功能,本试验通过优化S1D基因的密码子,构建了S1D基因未优化的重组原核表达质粒pET-S1D和已优化的pET-ΔS1D,并进行了诱导表达和纯化。使用SDS-PAGE和Western blotting方法验证S1D、ΔS1D蛋白在大肠杆菌内得到正确表达,利用Image J软件对S1D、ΔS1D蛋白表达量进行灰度扫描,通过t检验分析两者差异性。将纯化的ΔS1D 蛋白免疫 BALB/c小鼠,通过细胞融合、筛选及亚克隆,获得单克隆细胞株。利用体内诱生法制备抗PEDV S1D蛋白的单克隆抗体腹水,使用ELISA、Western blotting、间接免疫荧光试验3种方法对腹水效价及特异性进行检测和验证。SDS-PAGE和Western blotting结果显示,表达S1D、ΔS1D蛋白的样品均在34 ku处出现正确的目的条带。t检验结果表明, S1D、ΔS1D两者蛋白表达量差异极显著(P<0.01)。ELISA结果显示, 腹水的抗体效价达到了1∶1 000 000,腹水与PEDV病毒粒子和纯化后的ΔS1D蛋白反应均呈阳性,与PEDV N蛋白、pET-32a(+)空载体蛋白和猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)、猪瘟病毒(Classical swine fever virus,CSFV)、猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)和猪急性腹泻综合征冠状病毒(Swine acute diarrhea syndrome coronavirus,SADS-CoV) 5种病毒反应均呈阴性。Western blotting结果显示,腹水与ΔS1D蛋白和PEDV S蛋白分别在34和180 ku处有特异性条带出现,与pET-32a(+)空载体蛋白、正常Vero细胞蛋白均无特异性条带出现。间接免疫荧光试验结果显示,腹水及阳性对照组均能使细胞出现特异性绿色荧光信号,而空白及阴性对照组均未见绿色荧光信号。密码子优化可在原核表达系统中显著提高重组蛋白的表达水平,本研究基于高效表达的ΔS1D蛋白,成功制备了1株能稳定分泌与 PEDV S蛋白特异性结合的单克隆抗体的细胞株,为进一步探究 PEDV S蛋白抗原表位及蛋白功能的研究奠定了基础。

关键词: 猪流行性腹泻病毒(PEDV); 原核表达; S1D蛋白; 单克隆抗体

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

中图分类号:

S852.65⁺1

李洁森, 孙荣航, 邝燕齐, 陈路漫, 刘青, 郭霄峰. 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[J]. 中国畜牧兽医, 2021, 48(12): 4641-4651.

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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