刚地弓形虫肌动蛋白基因的克隆与表达  

Cloning and Expression of Actin Gene of Toxoplasma gondii

作　　者：李润花[1,2] 郝海霞[2] 王海龙[2] 孟晓丽[2] 申金雁[2] 殷国荣[2] 

机构地区：[1]太原师范学院生物系,太原030031 [2]山西医科大学医学寄生虫学研究所,太原030001

出　　处：《中国寄生虫学与寄生虫病杂志》2013年第5期352-355,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.81071374)~~

摘　　要：目的克隆和表达刚地弓形虫肌动蛋白(TgACT)基因,并分析其免疫反应性。方法提取弓形虫RH株速殖子的总RNA。根据TgACT基因编码序列(登录号为XM_002369622.1)设计合成引物,进行逆转录PCR(RT-PCR)扩增,扩增产物经双酶切后连接入pET-30a(+)载体。将重组质粒pET30a-TgACT转化至大肠埃希菌(E.coli)DH5α,阳性菌落经PCR和双酶切鉴定,并测序。pET30a-TgACT在E.coli BL21(DE3)中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物经SDS-PAGE鉴定。分别用抗多聚组氨酸标签(Anti-His)抗体和兔抗弓形虫血清为一抗进行蛋白质印迹(Western blotting)分析。结果 RT-PCR扩增产物约为1 100 bp。菌落PCR、双酶切及测序结果显示,重组质粒pET30a-TgACT构建成功。SDS-PAGE结果表明,目的蛋白在E.coli BL21(DE3)中以包涵体形式表达,相对分子质量(M r)约为49 000。通过蛋白的变性和复性处理及纯化,获得可溶性纯化蛋白。Western blotting结果显示,重组TgACT蛋白能被Anti-His抗体和兔抗弓形虫血清识别。结论成功构建重组质粒pET30a-TgACT,获得刚地弓形虫重组肌动蛋白,且具有免疫反应性。Objective To clone and express the actin gene of Toxoplasma gondii, and analyze the immunoreactivity of the recombinant protein. Methods Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgACT gene was amplified with a pair of specific primers which were designed according to the coding sequence of TgACT gene （Accession No. XM_002369622.1）. The RT-PCR product was cloned into the prokaryotic expression pET-30a （＋） vector. The recombinant pET30a-TgACT plasmid was transformed into E. coli DH5ct. The positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing. The correct pET30a-TgACT plasmid was transformed into E. coli BL21（DE3） and induced by 1PTG. The expressed proteins were analyzed by SDS-PAGE. Western blotting assay was perfoitned with anti-poly-histidine tag （anti-His） antibody or rabbit anti-T, gondii serum. Results The product of RT-PCR was with 1 100 bp. The recombinant plasmid pET30a- TgACT was confirmed by eolony-PCR, double restriction enzyme digestion and sequencing. SDS-PAGE results showed that the target protein was expressed in E. coli BL21（DE3） in the form of inclusion bodies with a rough molecular weight of 49 000. The purified soluble protein was obtained by using denaturation, renaturation and purification. Western blotting revealed that rTgACT can be recognized by anti-His antibody and rabbit anti-T, gondii serum. Conclusion The recombinant plasmid pET30a-TgACT has been successfully constructed, and the recombinant protein TgACT is produced in E. coli and maintains specific immunoreactivity.

关 键 词：刚地弓形虫 肌动蛋白 基因克隆 原核表达 免疫反应性 

分 类 号：R382.5[医药卫生—医学寄生虫学]