【摘要】 为构建弓形虫主要表面抗原1(SAG1)的原核重组表达质粒并获得重组表达蛋白,采用聚合酶链反应(PCR)从弓形虫RH株基因组DNA中扩增SAG1基因,经克隆和测序后,亚克隆至原核表达载体pGEX-4T-1,转化大肠杆菌BL21,PCR和双酶切鉴定阳性菌株,表达产物用SDS-PAGE和Western blot进行鉴定观察其诱导的抗体应答.结果表明,PCR扩增出SAG1基因的特异片段,获得的阳性克隆序列与GenBank中的SAG1基因序列一致,SAG1基因被亚克隆到表达载体pGEX-4T-1,在BL21中表达了SAG1,表达的蛋白能被弓形虫感染兔血清识别.以上结果说明,已成功构建了弓形虫SAG1的重组表达质粒,在大肠杆菌中表达的SAG1具有良好的免疫学活性.更多还原

【Abstract】 To clone and express Toxoplasma gondii major surface antigen 1 truncated fragment and analyze its immunological activity.The gene encoding Toxoplasma gondii major surface antigen 1 truncated fragment was amplified by PCR from Toxoplasma gondii genomic DNA,and was cloned into pMD18 Tvector.Positive clones were identified by PCR and sequencing with ABIPRISMTM 377XL DNA sequencer;The insert digested with BamHⅠ and SalⅠwas subcloned into pGEX-4T-1 which was also digested with BamHⅠ and SalⅠ;There combinant plasmids were transformed into E.coli BL21 and were identified by PCR and double enzyme digestion;The recombinant clone was induced with IPTG to express target protein and characterized by SDS-PAGE and immunoblot.The results showed,the gene fragment encoding Toxoplasma gondii major surface antigen 1 truncated fragment was amplified by PCR from from Toxoplasma gondii genomic DNA.The inser of positive clone was subcloned into pGEX-4T-1 correctly;Recombinant protein was expressed in the positive recombinant clone when induced with IPTG and reacted with the rabbit sera infected with Toxoplasma gondii.The findings indicated that the recombinant plasmid expressing Toxoplasma gondii major surface antigen 1 truncated fragment was successfully constructed and the recombinant protein showed good immunologica lactivity.更多还原