5种分子伴侣对水貂阿留申病毒核衣壳蛋白VP2部分基因可溶性表达的影响  

Effects of five molecular chaperones on the soluble expression of part gene of the nucleocapsid VP2 protein of Aleutian disease virus

作　　者：柏玲 朱言柱[1] 潘悦[2] 吕甜甜 张蕾[1] BAI Ling;ZHU Yan-zhu;PAN Yue;LYU Tian-tian;ZHANG Lei(Ministry of Agriculture and Rural Affairs Key Laboratory of Special Animal Epidemic Disease,Institute of Special Animal and Plant Sciences,Chinese Academy of Agricultural Sciences,Changchun 130112,China;Key Laboratory of Zoonosis Research,Ministry of Education,Institute of Zoonosis,Jilin University,Changchun 130112,China)

机构地区：[1]中国农业科学院特产研究所,农业部经济动物疫病重点实验室,吉林长春130112 [2]吉林大学人兽共患病研究所,人兽共患病教育部重点实验室,吉林长春130112

出　　处：《中国兽医学报》2020年第9期1699-1706,共8页Chinese Journal of Veterinary Science

基　　金：中国农业科学院特产研究所科技创新工程协同创新资助项目(2018XTCX001);宠物疾病诊疗与防控新技术研究资助项目(2016YFD0501000)。

摘　　要：为实现水貂阿留申病毒(Aleutian disease virus,ADV)部分VP2基因的可溶性表达,进一步探究目的蛋白的反应原性,本研究利用基因工程技术克隆ADV衣壳蛋白VP2部分基因,并亚克隆至原核表达载体pET30a(+)中,获得重组表达载体pET-30a-M,经诱导表达后,SDS-PAGE确定蛋白表达形式,尝试降低诱导温度及诱导剂浓度来增加可溶性蛋白表达量,但效果不显著;随后,为增加可溶性表达,尝试将pET-30a-M分别与pG-KJE8、pGro7、pKJE7、pG-Tf2和pTf165种伴侣蛋白共表达。通过SDS-PAGE筛选有效的伴侣蛋白,将伴侣蛋白pG-KJE8、pGro7、pKJE7、pG-Tf2和pTf165种质粒分别转入感受态细胞BL21(DE3),将pET-30a-M质粒分别转入含各种伴侣蛋白的感受态细胞。各种共表达菌株经双抗性筛选,诱导剂诱导表达,SDS-PAGE分析蛋白的表达情况。并从诱导温度和伴侣蛋白诱导剂L-Arabinose剂量两个方面筛选最佳表达条件。SDS-PAGE和Western blot分析结果表明重组表达载体pET-30a-M诱导表达后,蛋白主要以包涵体形式存在,与伴侣蛋白pKJE7共表达后,上清中的可溶性目的蛋白的含量增加,相对分子质量约为70000,且具有反应原性;与pG-KJE8、pGro7、pG-Tf2和pTf16伴侣蛋白共表达,未见可溶性蛋白增加。随着温度的降低,30℃与25℃比37℃上清中的可溶性蛋白表达量有所增加,诱导剂浓度对蛋白表达量影响不大。pKJE7伴侣分子可以促进pET-30a-M可溶性表达,且表达量不受L-Arabinose诱导剂量影响,随着温度降低,可溶性蛋白表达量有所增加,表达的蛋白具有反应原性,本研究为进一步探究该蛋白的生物学功能奠定基础。To achieve the soluble expression of part of the VP2 gene of Aleutian disease virus(ADV)to further explore the reactogenicity of the target protein.In this study,a part of the VP2 gene of ADV capsid protein was cloned by genetic engineering and subcloned into the prokaryotic expression vector pET30 a(+)to obtain the recombinant expression vector pET-30 a-M.After pET-30 a-M induced expression,the expression pattern of the protein was determined by SDS-PAGE.Try to reduce the induction temperature and inducer concentration to increase the soluble protein expression,but the effect was not significant.Subsequently,to increase soluble expression,pET-30 a-M was attempted to co-express with pG-KJE8,pGro7,pKJE7,pG-Tf2 and pTf16,respectively.The effective chaperone proteins were screened by SDS-PAGE,and the five proteins of chaperones pG-KJE8,pGro7,pKJE7,pG-Tf2 and pTf16 were transformed into competent cell BL21(DE3).Then,the pET-30 a-M plasmid was transformed into competent cells containing various chaperones.Each co-expressing strain was screened by double resistance and induced by an inducer,and the expression of the protein was analyzed by SDS-PAGE.Secondly,the optimal expression conditions were selected from the induction temperature and the dose of the chaperone L-Arabinose.The results of SDS-PAGE and Western blot analysis indicated that co-expressed with the chaperone protein pKJE7,the content of soluble protein in the supernatant was increased,the relative molecular quality was about 70000,and it was reactive;co-expression with pG-KJE8,pGro7,pG-Tf2 and pTf16 chaperones showed no increase in soluble protein.With the decrease of temperature,the expression of soluble protein in the supernatant of 30℃and 25℃was higher than that of37℃,and the concentration of the inducer had little effect on the expression of protein.pKJE7 chaperone can promote the soluble expression of pET-30 a-M,and the expression level is not affected by L-Arabinose induction dose.As the temperature decreases,the soluble protein expression incr

关 键 词：水貂阿留申病毒 伴侣蛋白 可溶性表达 

分 类 号：S852.65[农业科学—基础兽医学]