弓形虫GRA1基因的克隆与原核表达  

Cloning and prokaryotic expression of GRA1 gene of Toxoplasma gondii

作　　者：孙玲[1] 刘慧颖[1,2] 李淑红[1] 宫鹏涛[3] 张西臣[3] 宁静[1] 

机构地区：[1]吉林大学白求恩医学院病原生物学教研室,吉林长春130021 [2]吉林省长春市绿园区疾病预防控制中心,吉林长春130062 [3]吉林大学畜牧兽医学院预防兽医学系,吉林长春130062

出　　处：《吉林大学学报（医学版）》2011年第4期631-635,共5页Journal of Jilin University：Medicine Edition

基　　金：国家科技支撑计划资助课题(2008BAD96B11-3);吉林省发改委基金资助课题(200603)

摘　　要：目的:克隆并构建PET-28a-GRA1重组表达载体,转化入大肠杆菌E.coli BL21,诱导表达并鉴定,为进一步研究GRA1的生物学特性和免疫保护作用奠定实验基础。方法:根据GenBank中编码GRA1的已知基因序列设计并合成一对引物,应用PCR技术扩增GRA1基因,插入原核表达载体PET-28a中,构建重组表达质粒PET-28a-GRA1,转化E.coli BL21,IPTG诱导后,进行SDS-PAGE和Western blotting鉴定。结果:克隆的GRA1基因与GenBank收录的基因序列同源性为100%。对重组质粒进行酶切和PCR鉴定,获得573bp大小的目的基因片段,与预期结果相符,成功地构建了重组质粒PET-28a-GRA1。SDS-PAGE电泳分析表明重组蛋白条带的相对分子质量约为24 000,Western blotting显示重组蛋白能被鼠抗弓形虫血清识别。结论:成功获取GRA1基因,构建了PET-28a-GRA1重组质粒并获得了高效表达。Objective To clone the GRA1 gene and construct the prokaryotic expression vector PET-28a-GRA1,and to transform the expression vector into E.coli BL21,then induce and identiy,and to lay a foundation for the study on the biological characteristics and immune protection of GRA1.Methods A pair of primers were designed according to the sequence of GRA1 from GenBank.The GRA1 gene was amplified by PCR.The amplified GRA1 gene was inserted into prokaryotic expression vector PET-28a,and the constructed recombinant plasmid PET-28a-GRA1 was transformed to E.coli BL21 for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blotting.Results The sequencing result showed the homology of 100% of the cloned GRA1 gene to that reported in GenBank.The positive recombinant plasmids PET-28a-GRA1 were identified by restriction endonuclease digestion and PCR,the objective gene fragment with the size of 573 bp was acquired,in accordance with the expected results,and the recombinant plasmid PET-28a-GRA1 was constructed successfully.The results of SDS-PAGE revealed that the molecular weight of recombinant protein PET-28a-GRA1 was 24 000 and Western blotting proved that the recombinant protein was recognized by murine antisera against Toxoplasma gondii.Conclusion The GRA1 gene has been successfully cloned,the recombinant plasmid PET-28a-GRA1 is generated and expressed highly in prokaryote.

关 键 词：GRA1基因 原核表达载体 弓形虫 克隆 GRAl SDS-PAGE BLOTTING 重组表达载体 

分 类 号：R349.64[医药卫生—基础医学]