寨卡病毒E蛋白的原核表达及鉴定  

Prokaryotic expression and identification of the E protein of the Zika virus

作　　者：张涵[1,2] 曹亮 鲁会军[2,3] 田明尧[2] 孙文超[4] 郭丹丹[2] 汪伟 金宁一 李太元[1] ZHANG Han;CAO Liang;LU Hui-jun;TIAN Ming-yao;SUN Wen-chao;GUO Dan-dan;WANG Wei;JIN Ning-yi;LI Tai-yuan(Department of Preventive Veterinary Medicine,College of Agriculture,Yanbian University,Yanbian,Jilin 133002,China;Institute of Military Veterinary Medicine,The Academy of Military Medical Sciences;College of Animal Science and Technology,J ilin Agricultural University;Institute of Virology,Wenzhou University)

机构地区：[1]延边大学农学院,预防兽医学吉林延边133002 [2]军事医学科学院军事兽医研究所 [3]吉林农业大学动物科学技术学院 [4]温州大学病毒学研究所

出　　处：《中国病原生物学杂志》2018年第7期681-684,690,共5页Journal of Pathogen Biology

基　　金：国家重点研发计划项目(No.2016YFC1200901,2016YFD0500401); 吉林省科技发展计划项目(No.20160623024TC)

摘　　要：目的应用原核表达载体pET-28a表达ZIKV主要结构蛋白E蛋白,并对反应条件进行优化,为ZIKV亚单位疫苗的研究奠定基础。方法从GenBank检索ZIKV基因组,合成E基因并进行密码子优化,以提高原核表达效率。设计引物,以合成的E基因为模板PCR扩增ZIKV E基因,用BamHI和HindIII进行双酶切后连接表达载体pET-28a,构建原核表达重组质粒pET28a-ZIKV E,转化大肠埃希菌BL21(DE3)进行目的蛋白的表达,并对最适IPTG诱导浓度进行优化。结果 pET28a-ZIKV E经酶切及测序鉴定均构建正确。用重组质粒转化DE3,当加入IPTG终浓度为2mmol/L,成功表达出分子质量单位(Mr)为56×103重组ZIKV E蛋白,与理论分子质量相符合。Western blot检测重组蛋白可被His标签抗体识别。结论成功构建原核重组载体pET-28a-ZIKV E,表达的重组蛋白具有免疫反应性,为ZIKV亚单位疫苗的研制奠定了基础。Objectives To express the E protein,a main structural protein,of the Zika virus in a prokaryotic expression system and to optimize reaction conditions in order to lay the foundation for prevention of Zika virus infection and to facilitate research on subunit vaccines. Methods The DNA sequence of the E gene of the Zika virus was obtained from GenBank,synthesized,and optimized to accommodate a prokaryotic expression vector.The E gene of the Zika virus was amplified with a pair of specific primers and double digested with BamH I and Hind III,and the gene was ligated into pET-28 ato generate the prokaryotic expression vector pET-28 a-ZIKV E.pET-28 a-ZIKV E was transformed into E.coli BL21（DE3）,and the optimal concentration of IPTG for expression was examined. Results The E gene of the Zika virus was successfully amplified（1 575 bp in length）,and the plasmid pET-28 a-ZIKV E was constructed.The final concentration of IPTG was 2 mmol/L.The E protein of the Zika virus was successfully expressed with this concentration.The protein has a molecular mass of 56×103,which is identical to the theoretical molecular mass of ZIKV E.Western blot analysis indicated that the recombinant protein specifically reacted with His tag antibody. Conclusion The prokaryotic expression vector pET-28 a-ZIKV E was constructed,and the target protein ZIKV E was successfully identified using SDS-PAGE and Western blotting.This study has laid the foundation for immunization testing of ZIKV subunit vaccines.

关 键 词：寨卡病毒 E蛋白 原核表达 鉴定 

分 类 号：R373.9[医药卫生—病原生物学]