【摘要】 目的:构建刚地弓形虫(简称弓形虫)主要表面抗原P30基因的2个不同片段的克隆并进行表达、鉴定。方法:根据已发表的弓形虫P30基因序列,参考有关文献设计合成两对引物,用PCR从弓形虫RH株基因组DNA中扩增出P30基因的两个不同片段,分别构建T-A克隆,经PCR扩增及酶切鉴定后,进行测序,再亚克隆入pMAL-p2质粒并转化DH5α感受态细胞,于氨苄青霉素LB平板上初步筛选阳性克隆,再经酶切及PCR扩增鉴定重组子。将重组子转入表达菌BL21,用IPTG进行诱导表达,并对所表达的蛋白以western blot鉴定。结果:成功构建相应的T-A克隆及pMAL-p2亚克隆,经诱导、表达和鉴定,证实2个表达产物均为目的蛋白,其分子量分别为70和73Ku。结论:成功获得弓形虫主要表面抗原P30的2个表达产物,为其进一步的纯化及应用奠定了基础。更多还原

【Abstract】 Objective: To clone, express and identify two different fragments of the major surface antigen P30 of Toxoplasma gondii (Tg). Methods: According to P30 gene sequence of toxoplasma and some references published, two pairs of primers were designed, syntheszied and used to amplify the two different fragments of P30 gene from the genomic DNA of RH strain of Tg by PCR , and then the corresponding T-A clones were constructed with the two fragments respectively. After being identified by PCR amplification and enzyme digestion, they were sequenced. The two fragments were subcloned into pMAL-p2 plasmid and then transformed into susceptible E. coli DH5α. After identification by PCR and enzyme digestion, the positive clones from the ampicillin positive LB plate were selected. The expression was induced by IPTG and the expressed proteins were analyzed by western blotting. Results: The corresponding T-A clones and pMAL-p2 sub-clones have been successfully constructed, and the two protein fragments of P30, with the molecular weights of 70 and 73Ku, respectively, were expressed successfully. Conclusion: The two protein fragments of the major surface antigen P30 of Tg were obtained, which established the foundation for their purifications and applications.更多还原