摘要
目的克隆弓形虫RH株次黄嘌呤—黄嘌呤—鸟嘌呤磷酸核糖转移酶(HXGPRT)基因,构建原核表达载体,并表达该重组蛋白。方法收集、纯化弓形虫RH株速殖子,提取总RNA,在设计合成的引物中引入BamH和Xho酶切位点。应用RT-PCR扩增弓形虫HXGPRT基因片段,插入克隆载体pMD18-T,提取重组质粒,双酶切获得目的基因,插入原核表达质粒pET28a,重组子双酶切、PCR和测序鉴定,转化大肠杆菌BL21并以IPTG诱导表达。结果从弓形虫RH株cD-NA中扩增出693bp的HXGPRT基因片段;含pET28a/HXGPRT的宿主菌经诱导后,获得与预期分子量相符的表达产物,Western blotting提示重组蛋白能够被弓形虫患者血清中的IgG抗体识别,获得纯化的重组蛋白。结论成功地从弓形虫RH株基因组DNA中获取HXGPRT基因,构建pET28a/HXGPRT重组质粒,并高效表达,为进一步研究提供了条件。
Objective To clone and express hypoxanthiae-xanthiae-guanine phosphoribosyltransferase（HXGPRT） gene from Toxoplasma gondii RH strain. Methods RH strain tachyzoites,by laboratory mouse passage, were harvested from ascites of mice and genomic RNA was prepared. A fragment of HXGPRT encoding gene was obtained by RT-PCR amplification from genomic RNA of T. gondii. The PCR products were ligated to pMD18-T. The recombinants were confirmed by BamHI/XhoI digestion, PCR,and DNA sequencing and were cloned into expression vector pET28a. The recombinants were transformed into E. coli BL21 （DE3） followed by identification of the fusion expression with Western blotting. Results T. gondii HXGPRT encoding gene with a molecular size of 693 bp, which is highly homologous to the sequence previously reported,was obtained. The expression was confirmed by SDS-PAGE and Western blotting. Conclusion The recombinant construction of T. gondii HXGPRT was generated and expression was induced.
出处
《临床输血与检验》
CAS
2007年第2期97-101,共5页
Journal of Clinical Transfusion and Laboratory Medicine
基金
国家高技术研究发展计划863计划资助(No.2004AA2Z3570)
