摘要
目的原核表达并纯化人呼吸道合胞病毒(Human respiratory syncytial virus,HRSV)兰州株黏附蛋白(G)片段。方法利用PCR技术从HRSV兰州株(A亚型)中扩增G基因AA75-225片段,克隆至原核表达载体pET-42b(+)中,构建重组表达质粒pET-42b-G,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经镍离子亲和层析柱纯化后,进行SDS-PAGE和Western blot分析。结果 PCR扩增获得453 bp的DNA片段;重组表达质粒pET-42b-G经双酶切及测序鉴定证明构建正确;表达的重组蛋白相对分子质量为50 230,表达量占菌体总蛋白的25%,以包涵体和可溶性两种方式存在;纯化后重组蛋白纯度可达95%以上,可与抗RSV-G蛋白单克隆抗体特异性结合。结论已成功地在大肠杆菌BL21(DE3)中表达了HRSV兰州株G片段,为后续RSV疫苗的研制、RSV感染的血清学诊断及试剂盒的研发提供了材料。
Objective To express the attachment protein （G） fragment of human respiratory syncytial virus （HRSV） in prokaryotic cells and purify the expressed product. Methods AA75-225 fragment of G gene was amplified from HRSV Lanzhou strain （subtype A） by PCR and cloned into prokaryotic expression vector pET-42b （＋）. The constructed recombinant plasmid pET- 42b-G was transformed to E. coli BL21 （DE3） and induced with IPTG. The expressed product was purified by nickel ion affinity chromatography, and identified by SDS-PAGE and Western blot. Results DNA fragment at a length of 453 bp was amplified by PCR. Restriction analysis and sequencing proved that recombinant plasmid pET-42b-G was constructed correctly. The expressed recombinant protein, with a relative molecular mass of 50 230, contained 25% of total somatic protein and existed in both inclusion body and soluble forms. The purified recombinant protein reached a purity of more than 95%, and showed specific binding to anti- RSV-G monoclonal antibody. Conclusion The G fragment of HRSV Lanzhou strain was successfully expressed in E. coli BL21 （DE3）, which provided a material for further preparation of RS~ vaccine, the serological diagnosis of RS~ infection and the development of diagnostic kit.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第4期389-392,共4页
Chinese Journal of Biologicals
关键词
呼吸道合胞病毒
黏附蛋白
原核细胞
基因表达
纯化
Respiratory syncytial virus （RSV）
Attachment protein
Prokaryotic cells
Gene expression
Purification
