Prokaryotic expression and preparation of the polyclonal antibodies of E protein and domain Ⅲ in Zika virus
DING Chen-xi1, 2, ZHU Xu-hui2, AI Le-le2, YE Fu-qiang2, TAN Wei-long2, HU Dan1, 2, CHEN Jia-feng2, GUO Xiao-lu2, PAN Xiu-zhen2, WANG Chang-jun1, 2
1. College of Preventive Medicine, the Third Military Medical University, Chongqing 400038, China; 2. Institute of Military Medical Sciences, Nanjing Military Region, Nanjing 210002, China
Abstract:To clone, express and purify the E Protein and EDⅢ of Zika virus in E. coli and prepare two kinds of polyclonal antibodies, the virus was amplified by Vero-E6 cell culture. Total RNA was extracted by RT-PCR and reverse transcribed into cDNA. The prokaryotic expression vectors pET32a/E and pET28a/EDⅢ were constructed by cDNA sequence of E and EDⅢ gene. Then, recombinant plasmids were transformed into E. coli BL21 and induced by IPTG, and purified by Ni+ column affinity chromatography. BALB/C mice were immunized with purified recombinant proteins. Antiserum was collected and titer was determined by indirect ELISA. Western blot was used to detect the specificity. Results showed that the recombinant proteins were successfully expressed and purified. The titer of the polyclonal antibodies both reached 1:409 600. Western Blot analysis showed that the polyclonal antibodies could specifically recognize the recombinant proteins. Thus, the specific polyclonal antibody were successfully prepared, laying a foundation for further study on the pathogenesis, detection methods and immune strategies of Zika virus.
丁晨曦, 朱旭辉, 艾乐乐, 叶福强, 谭伟龙, 胡丹, 陈家锋, 郭晓璐, 潘秀珍, 王长军. 寨卡病毒E蛋白及第三结构域的原核表达和多克隆抗体制备[J]. 中国人兽共患病学报, 2018, 34(1): 23-28.
DING Chen-xi, ZHU Xu-hui, AI Le-le, YE Fu-qiang, TAN Wei-long, HU Dan, CHEN Jia-feng, GUO Xiao-lu, PAN Xiu-zhen, WANG Chang-jun. Prokaryotic expression and preparation of the polyclonal antibodies of E protein and domain Ⅲ in Zika virus. Chinese Journal of Zoonoses, 2018, 34(1): 23-28.
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