中文摘要：

目的克隆刚地弓形虫活化蛋白激酶C受体1（TgRACKl）基因，原核表达、纯化TgRACKl，并分析其抗原性。方法制备弓形虫RH株速殖子总RNA，根据TgRACKl基因全长编码序列（GenBank登录号：AY547291）的开放阅读框设计引物并进行逆转录-聚合酶链式反应（RT—PCR）扩增，扩增产物经双酶切后连接入pGEX-6p-1载体，重组质粒转化大肠埃希菌DH5a，阳性菌落经菌液PCR、双酶切及DNA测序进行鉴定。将重组质粒pGEX-6p-1-TgRACKl转化至大肠埃希菌BL21（DE3）并加入异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）结合考马斯亮蓝染色检测表达产物；分别以抗GST标签抗体和兔抗弓形虫血清为一抗，免疫印迹（Westernblotting）分析鉴定重组蛋白及其抗原性。重组融合蛋白经GST琼脂糖凝胶亲和纯化，纯化蛋白经SDS—PAGE分离，考马斯亮蓝染色结合凝胶成像系统分析其纯度。结果RT—PCR扩增产物约为970bp，重组质粒pGEX-6p-1-TgRACKI构建成功。经IPTG诱导获得相对分子质量约63kDa的可溶性重组蛋白。诱导表达的蛋白质为带GST标签的重组融合蛋白，且能被兔抗弓形虫血清识别。亲和纯化后获得纯度大于95％的重组TgRACKl蛋白质。结论克隆得到弓形虫活化蛋白激酶C受体1基因，原核表达并纯化出该基因编码的蛋白质，且该重组蛋白具有抗原性。

英文摘要：

Objective To explore the cloning,prokaryotic express and purification of the receptor for activated C kinase 1 （RACKI） gene of RH strain of Toxoplasma gondii,and to analyze the antigenicity of recombinant protein. Methods Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgRACKI gene was amplified with a pair of specific primers which was designed according to the coding sequence of TgRACK1 gene （GenBank accession No： AY547291） ,the product of RT-PCR was digested with double restrict enzyme and ligated into a pGEX-6p-1 vector. The recombinant pGEX-6p-I-TgRACK1 plasmid was transferred into E. coli DH5α and the positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing. The successful pGEX-6p-1-TgRACK1 construct was transformed into E. coli BL21 （DE3） and induced with IFFG to express. The products of expression were analyzed through SDS-PAGE followed by Coomassie blue staining and the recombinant TgRACK1 was purified by using glutathione-triethyleneglycocyl-sepharose 6B. Western blotting assay with GST primary antibody and rabbit anti-T.gondii serum were used to confirm the expression of rTgRACK1 and analyze its antigenic properties. Results The product of RT- PCR was 970 bp. The recombinant plasmid pGEX-6p-I,TgRACK1 was confirmed successfully by colony-PCR,double restrict enzyme digestion and sequencing. A recombinant protein with a rough molecular weight of 63 kDa was analyzed via SDS- PAGE followed by Coomassie blue staining. The GST tag in rTgRACK1 and the antigenicity of rTgRACK1 were detected efficiently by Western blotting analysis with the GST primary antibody and with the prepared antiserum against TgRACK1 respectively. The isolated protein was water soluble and showed 95% purity based on SDS-PAGE and gel imaging analysis. Conclusion The TgRACK1 gene is cloned,the recombinant protein TgRACK1 is produced in E. coli and purified and maintained the specific antigenicity.