摘要
目的克隆并表达人呼吸道合胞病毒(Human respiratory syncytial virus,HRSV)兰州株的融合蛋白(F)基因片段。方法利用PCR技术扩增HRSV兰州株的融合蛋白基因片段,克隆于原核表达载体pET-42b(+),转化大肠杆菌(Rosetta),经IPTG诱导表达,镍离子亲和层析柱纯化,SDS-PAGE和Western-blot分析重组蛋白的表达及其反应原性。结果 PCR扩增得到951 bp的DNA片段,重组质粒pET42b-F经酶切鉴定和测序分析,表明质粒构建正确。表达的重组蛋白的相对分子质量为68 710,表达的重组蛋白占总菌体蛋白的7%,纯化后蛋白纯度达80%。经Western-blot分析,重组蛋白与抗RSV的单抗呈专一性强阳性反应。结论成功构建了HRSV兰州株F基因片段原核表达载体,并在大肠杆菌Rosetta中获得了表达,表达的重组蛋白具有反应原性和特异性,为HRSV感染引起的疾病血清学诊断以及试剂盒的研发提供了材料。
Objective The study aims to clone and express the fragment of the gene encoding fusion protein（F） from Lanzhou strain of human respiratory syncytial virus（HRSV） and to purify and identify the expressed product.Methods PCR technique was employed to amplify the gene fragment of F protein from Lanzhou strain of RSV.The amplicon was ligated with pET-42b（＋） and the plasmid was transformed into E.coli Rosetta for expression.The transformants were induced by IPTG to express the recombinant protein,which was subjected to purification via ProbBondTM Ni-chelating resin column.The expressed protein was analyzed by SDS-PAGE and its reactogenicity was identified by Western-blot analysis.Results The DNA fragment with length of 951 bp was amplified.Both restriction enzyme analysis and sequencing revealed that the recombinant plasmid was correctly constructed.The molecular weight of the expressed protein was 68 710,its content was as high as 7%.After purification,the purity of the recombinant protein reached 80%.Conclusion The expressing vector expressing F protein from Lanzhou strain of RSV was constructed successfully.The recombinant protein was expressed and purified and used for diagnosis of sera and development of kit used for detection of HRSV infection.
出处
《微生物学免疫学进展》
2012年第1期15-19,共5页
Progress In Microbiology and Immunology
关键词
呼吸道合胞病毒
兰州株
融合蛋白(F)
表达
Human respiratory syncytial virus（HRSV）
Lanzhou strain
Fusion protein（F）
Expression
