人呼吸道合胞病毒FHRA-HRB片段的表达纯化与鉴定

发布时间：2018-01-26 13:02

  本文关键词： 呼吸道合胞病毒 FHRA-HRB片段 载体构建 蛋白表达　出处：《暨南大学学报(自然科学与医学版)》2017年04期 　论文类型：期刊论文

【摘要】：目的:克隆呼吸道合胞病毒融合F蛋白(RSV-F)中FHRA-HRB基因片段,构建体外的原核表达载体,对FHRA-HRB蛋白进行表达纯化,为进一步研究FHRA-HRB蛋白构效关系及抗RSV活性提供实验依据.方法:用Trizol法从RSV感染的Hep-2细胞中提取病毒RNA,逆转录得c DNA,使用Primer 6.0软件设计特异性引物,聚合酶链反应(PCR)扩增得到FHRA-HRB片段,测序成功后,将FHRA-HRB插入p ET-15b脱磷载体中,转化入Rosettagami表达宿主,异丙基-β-D-硫代半乳糖苷(IPTG)诱导FHRA-HRB的体外表达,Ni-NTA亲和层析法纯化蛋白.用Western-blot法鉴定FHRA-HRB蛋白表达.结果:成功扩增了大小约1100 bp的FHRA-HRB片段,克隆进p ET-15b载体后,菌液PCR验证与测序分析结果表明FHRA-HRB片段序列与预期一致且读码框插入正确,表达载体构建成功.工程菌经IPTG诱导后成功表达大小约43 000的FHRA-HRB蛋白,经Ni-NTA agarose纯化后FHRA-HRB蛋白纯度达95%以上.纯化后的FHRA-HRB蛋白经Western-blot鉴定其大小正确.结论:成功构建p ET-15b-FHRA-HRB表达载体,经表达纯化后,得到了纯度较高的FHRA-HRB蛋白,纯化蛋白经Western-blot鉴定正确.本研究为进一步研究其抗RSV活性和开发以F蛋白为靶点的抗RSV药物提供实验依据.
[Abstract]:Objective: to clone the FHRA-HRB gene fragment of respiratory syncytial virus (RSV-FV) fusion F protein, construct prokaryotic expression vector in vitro, and express and purify FHRA-HRB protein. In order to further study the structure-activity relationship of FHRA-HRB protein and its anti-#en1# activity, the virus RNA was extracted from Hep-2 cells infected with RSV by Trizol method. The specific primers were designed by Primer 6.0 software. The FHRA-HRB fragment was amplified by polymerase chain reaction (PCR) and sequenced successfully. FHRA-HRB was inserted into p ET-15b dephosphorization vector and transformed into Rosettagami expressing host. Isopropyl 尾 -Dthiogalactoside (IPTG) induced the expression of FHRA-HRB in vitro. The protein was purified by Ni-NTA affinity chromatography. The expression of FHRA-HRB protein was identified by Western-blot. Results: the size of FHRA-HRB was about 1100. BP FHRA-HRB fragment. After cloned into p ET-15b vector, the results of PCR verification and sequencing analysis showed that the sequence of FHRA-HRB fragment was the same as expected and the reading frame was inserted correctly. The expression vector was successfully constructed and the recombinant strain was induced by IPTG to express about 43 000 FHRA-HRB protein. Via Ni-NTA. The purity of FHRA-HRB protein purified by agarose was more than 95%. The purified FHRA-HRB protein was confirmed by Western-blot to be of correct size. The expression vector of p ET-15b-FHRA-HRB was successfully constructed. After expression and purification, high purity FHRA-HRB protein was obtained. The purified protein was identified correctly by Western-blot. This study provides the experimental basis for further study of its anti- RSV activity and the development of anti-RSV drugs targeting F protein.
【作者单位】： 暨南大学药学院中药与天然产物药物研究所;中药药效物质基础及创新药物研究广东省高校重点实验室;暨南大学药学院基因组药物研究所;
【基金】：国家自然科学基金项目(81473116) 高等学校学科创新引智计划项目(B13038) 广东省自然科学基金重点项目(S2013020012864) 广东省教育厅科技创新项目(2012KJCX0017)
【分类号】：R373.14
【正文快照】： 呼吸道合胞病毒(respiratory syncytial virus,RSV)是一种易引起婴幼儿肺炎的病原体.该病毒感染正常细胞后会导致细胞间发生融合,因此被命名为呼吸道合胞病毒[1-4].临床上用于治疗RSV的药物有两种:利巴韦林(Ribavirin)是第一个被用作治疗RSV的药物,但因利巴韦林对机体毒副作用，

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