香蕉条斑病毒云南河口分离物开放阅读框Ⅱ的原核表达、融合蛋白纯化及抗血清制备  

The prokaryotic expression and purification of ORFⅡof banana streak virus Yunnan isolate(BSV-YN) and the preparation of antiserum against the protein

作　　者：林湛松[1,2] 王健华[1] 刘志昕[1] 

机构地区：[1]中国热带农业科学院热带生物技术研究所农业部热带作物生物技术重点开放实验室,海口571101 [2]海南大学环境与植物保护学院,儋州571737

出　　处：《生物学杂志》2011年第5期50-54,共5页Journal of Biology

基　　金：国家自然基金项目(31070131);中央级公益性科研院所基本科研业务费专项(ITBBZD0754)

摘　　要：以课题组保存的连接在pMD-18T载体上的香蕉条斑病毒河口分离物全基因组为模板,设计一对特异引物,经过PCR扩增,切胶回收,并通过与pET32(b)载体共同双酶切及T4连接酶连接,得到重组载体pET32-ORF2。将获得的重组质粒pET32-ORF2转化表达菌株E.coli BL21(DE3)感受态细胞,得到表达BSV的ORFⅡ的工程菌。经0.1mmol/L的IPTG诱导表达,超声波破碎后,获得的融合蛋白大多为包含体,但仍有少部分为可溶性蛋白可以进行后续的纯化步骤。将上述可溶性融合蛋白通过Ni2+-NTA亲和层析柱纯化,获得纯度较高的融合蛋白。以纯化蛋白作为抗原免疫家兔,得到特异性抗血清。间接ELISA以及western blotting表明,所得抗血清效价和特异性都较高,可以用于检测。为BSV的快速检测以及一些相关后续理论研究工作奠定了一定基础。In the present research,the target segment was firstly generated by PCR amplification by using the whole genome of BSV-Yunnan as the template.Then after double digestion and ligation by T4 ligase with pET-32 plasmid,the recombinant plasmid pET32-ORF2 was generated.The plasmid was thereafter transformed into the competent cell of E.coli BL21（DE3）.Then the E.coli cells were induced by IPTG at the concentration of 0.1mmol/L to express the fusion protein.The result of PAGE showed that the fusion protein was inclusion body in majority.Then the fusion protein was purified by the Ni2＋-NTA column and utilized as the antigen to immunize rabbits in order to generate the antiserum.The results of ELISA and western blotting showed that the antiserum had extremely high tilter and specificity and thus was qualified to be a competent method to detect BSV in Musa.

关 键 词：香蕉条斑病毒 原核表达 ELISA WESTERN BLOTTING 

分 类 号：Q78[生物学—分子生物学] S432.4[农业科学—植物病理学]