本文關(guān)鍵詞： 呼吸道合胞病毒 FHRA-HRB片段 載體構(gòu)建 蛋白表達(dá)　出處：《暨南大學(xué)學(xué)報(bào)(自然科學(xué)與醫(yī)學(xué)版)》2017年04期 　論文類(lèi)型：期刊論文

【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,構(gòu)建體外的原核表達(dá)載體,對(duì)FHRA-HRB蛋白進(jìn)行表達(dá)純化,為進(jìn)一步研究FHRA-HRB蛋白構(gòu)效關(guān)系及抗RSV活性提供實(shí)驗(yàn)依據(jù).方法:用Trizol法從RSV感染的Hep-2細(xì)胞中提取病毒RNA,逆轉(zhuǎn)錄得c DNA,使用Primer 6.0軟件設(shè)計(jì)特異性引物,聚合酶鏈反應(yīng)(PCR)擴(kuò)增得到FHRA-HRB片段,測(cè)序成功后,將FHRA-HRB插入p ET-15b脫磷載體中,轉(zhuǎn)化入Rosettagami表達(dá)宿主,異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)FHRA-HRB的體外表達(dá),Ni-NTA親和層析法純化蛋白.用Western-blot法鑒定FHRA-HRB蛋白表達(dá).結(jié)果:成功擴(kuò)增了大小約1100 bp的FHRA-HRB片段,克隆進(jìn)p ET-15b載體后,菌液PCR驗(yàn)證與測(cè)序分析結(jié)果表明FHRA-HRB片段序列與預(yù)期一致且讀碼框插入正確,表達(dá)載體構(gòu)建成功.工程菌經(jīng)IPTG誘導(dǎo)后成功表達(dá)大小約43 000的FHRA-HRB蛋白,經(jīng)Ni-NTA agarose純化后FHRA-HRB蛋白純度達(dá)95%以上.純化后的FHRA-HRB蛋白經(jīng)Western-blot鑒定其大小正確.結(jié)論:成功構(gòu)建p ET-15b-FHRA-HRB表達(dá)載體,經(jīng)表達(dá)純化后,得到了純度較高的FHRA-HRB蛋白,純化蛋白經(jīng)Western-blot鑒定正確.本研究為進(jìn)一步研究其抗RSV活性和開(kāi)發(fā)以F蛋白為靶點(diǎn)的抗RSV藥物提供實(shí)驗(yàn)依據(jù).
[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.
【作者單位】： 暨南大學(xué)藥學(xué)院中藥與天然產(chǎn)物藥物研究所;中藥藥效物質(zhì)基礎(chǔ)及創(chuàng)新藥物研究廣東省高校重點(diǎn)實(shí)驗(yàn)室;暨南大學(xué)藥學(xué)院基因組藥物研究所;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(81473116) 高等學(xué)校學(xué)科創(chuàng)新引智計(jì)劃項(xiàng)目(B13038) 廣東省自然科學(xué)基金重點(diǎn)項(xiàng)目(S2013020012864) 廣東省教育廳科技創(chuàng)新項(xiàng)目(2012KJCX0017)
【分類(lèi)號(hào)】：R373.14
【正文快照】： 呼吸道合胞病毒(respiratory syncytial virus,RSV)是一種易引起嬰幼兒肺炎的病原體.該病毒感染正常細(xì)胞后會(huì)導(dǎo)致細(xì)胞間發(fā)生融合,因此被命名為呼吸道合胞病毒[1-4].臨床上用于治療RSV的藥物有兩種:利巴韋林(Ribavirin)是第一個(gè)被用作治療RSV的藥物,但因利巴韋林對(duì)機(jī)體毒副作用，

本文編號(hào)：1465684