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目的克隆刚地弓形虫活化蛋白激酶C受体1(TgRACK1)基因,原核表达、纯化TgRACK1,并分析其抗原性。方法制备弓形虫RH株速殖子总RNA,根据TgRACK1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DH5α,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-TgRACK1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(SDSPAGE)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(Western blotting)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970 bp,重组质粒pGEX-6p-1-TgRACK1构建成功。经IPTG诱导获得相对分子质量约63 kDa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TgRACK1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。
Objective To clone the TgRACK1 gene of Toxoplasma gondii, prokaryotic it, purify TgRACK1 and analyze its antigenicity. Methods Toxoplasma gondii RH strain tachyzoite total RNA was prepared and primers were designed according to the open reading frame of TgRACK1 gene full-length coding sequence (GenBank accession number: AY547291) and amplified by reverse transcription-polymerase chain reaction (RT-PCR) The amplified product was double digested and ligated into pGEX-6p-1 vector. The recombinant plasmid was transformed into Escherichia coli DH5α. The positive colonies were identified by bacterial PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pGEX-6p-1-TgRACK1 was transformed into Escherichia coli BL21 (DE3) and induced with isopropyl-β-D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-poly The expressed products were detected by SDS PAGE and Coomassie brilliant blue staining. Anti-GST-tagged antibody and rabbit anti-Toxoplasma gondii serum were used as primary antibodies respectively. The recombinant proteins and their antigenicity were identified by Western blot analysis. The recombinant fusion protein was affinity purified by GST agarose gel. The purified protein was separated by SDS-PAGE and analyzed by Coomassie brilliant blue staining and gel imaging system. Results The amplified product of RT-PCR was about 970 bp. The recombinant plasmid pGEX-6p-1-TgRACK1 was successfully constructed. Inducible IPTG to obtain the molecular weight of about 63 kDa soluble recombinant protein. The protein that induced expression was a GST tagged recombinant fusion protein and was recognized by rabbit anti-Toxoplasma serum. After affinity purification, a recombinant TgRACK1 protein with a purity greater than 95% is obtained. Conclusion Toxoplasma gondii activated protein kinase C receptor 1 gene was cloned and prokaryotic expressed. The protein encoded by this gene was purified and the recombinant protein was antigenic.