中文摘要：

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人pET-30a（＋）载体。将重组质粒pET30a—TgACT转化至大肠埃希菌（Ecoli）DH5α，阳性菌落经PCR和双酶切鉴定，并测序。pET30a—TgACT在EcoliBL21（DE3）中用异丙基-B-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-抗进行蛋白质印迹（westernblotting）分析。结果RT—PCR扩增产物约为1100bp。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EcoliBL21（DE3）中以包涵体形式表达，相对分子质量（M）约为49000。通过蛋白的变性和复性处理及纯化．获得可溶性纯化蛋白。Westernblotting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。

英文摘要：

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.