作　　者： ; ; ; ; ; ; ; ;

机构地区： 南方医科大学公共卫生与热带医学学院病原生物学系

出　　处： 《寄生虫与医学昆虫学报》 2013年第1期7-11,共5页

摘　　要： 本文构建了刚地弓形虫RH株致密颗粒蛋白7（GRA7）基因原核表达重组质粒，进行蛋白表达并分析重组蛋白的免疫反应性。设计合成引物，从弓形虫RH株基因组DNA中特异扩增出GRA7基因片段并进行序列分析。目的片段用EcoRI和BamHI双酶切后分别构建重组质粒pET32a-GRA7和pET28a-GRA7，而后转人大肠埃希菌BL21（DE3）中诱导表达，Westernblotting分析表达蛋白。结果显示，从弓形虫RH株中成功扩增出大小约710bp的GRA7基因片段，构建的重组质粒经双酶切和PCR鉴定及测序，目的基因片段与预期相符。重组质粒pET32a-GRA7经IPTG诱导后，可高效表达重组蛋白，Westernblotting分析显示重组GRA7蛋白能被弓形虫慢性感染血清识别，而重组质粒pET28a-GRA7未能诱导表达出目的蛋白。原核表达的重组GRA7抗原具有特异的免疫反应性，为弓形虫的诊断应用和疫苗研究奠定了基础。 To construct prokaryotic expression plasmid pET28a-GRA7 and pET32a-GRA7 containing the dense granulesantigen 7 （GRAT） gene of T. gondii RH strain. The gene fragment GRA7 was amplified by PCR from genomic DNA of T. gondii RH strain. The GRA7 gene was digested by the restriction endonuclease EcoR I and BamH I , and then was further snbcloned into plasmid pET28a （ ＋ ） and pET32a （ ＋ ） , respectively. The plasmid pET28a-GRA7 and pET32a-GRA7 were then transformed into BL21 （DE3） to express the recombinant protein with IPTG induction. The expression product was identified by SDS-PAGE and Western blotting. The plasmid pET28a-GRA7 and pET32a-GRA7 were confirmed with Enzyme digestion and PCR analysis, respectively. With IPTG induction, the recombinant protein GRA7 was expressed in a fusional form in pET32a-GRA7/BL21 （DE3） , while pET28a-GRA7/BL21 （DE3） was fail to express the recombinant protein. Western blotting showed that the recombinant protein could be specifically recognized by sera from mice chronically infected by T. gondii, which shows the recombinant GRA7 expresses a specific immunoreactivity, those built a foundation for the further studies on T. gondii diagnosis and vaccine.

关 键 词： 刚地弓形虫 致密颗粒蛋白 基因克隆 原核表达 免疫反应性

领　　域： [生物学]