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目的 在大肠杆菌中高效表达ROP1抗原。方法 采用聚合酶链反应 (PCR)从弓形虫RH株cDNA文库中扩增得到编码ROP1的基因 ,经DNA序列分析后导入表达载体pGEX - 5x - 3 ,然后在大肠杆菌BL2 1中进行表达 ,用亲和层析柱纯化表达产物 ,并用SDS -PAGE和Westernblotting进行鉴定。 结果 ( 1)在我们比较的 12 4 9个碱基中 ,我们得到的ROP1基因在Ossorio报道的基因的 4 4 7位与 4 4 8位之间有一 96bp的插入片段 ,另有 14个碱基不同 ,造成 3个氨基酸的改变 ,两基因之间有 91.19%的同源性 ,( 2 )得到一分子量约为 70kDa的融合蛋白。结论 1,我们获得了一与Ossorio报道的基因有较大差异的rop1基因 (GeneBank登录号 :AF3 5 0 2 61) ( 2 ) ,ROP1基因在大肠杆菌中得到了ROP1融合蛋白的高效表达。
Objective To express ROP1 antigen efficiently in E. coli. Methods The gene encoding ROP1 was amplified from the cDNA library of RH strain Toxoplasma gondii by polymerase chain reaction (PCR). After DNA sequence analysis, the gene was introduced into expression vector pGEX - 5x - 3 and then expressed in E. coli BL21. The expressed products were purified by affinity chromatography and identified by SDS-PAGE and Western blotting. Results (1) Among the 1294 bases that we compared, the ROP1 gene we obtained had a 96bp insert between positions 477 and 448 of Ossorio reported genes, and another 14 bases Different, resulting in three amino acid changes, 91.19% homology between the two genes, (2) to obtain a molecular weight of about 70kDa fusion protein. Conclusions 1. We obtained a rop1 gene (GeneBank accession number: AF3 5 0 2 61) (2), which is significantly different from that reported by Ossorio. (2) The ROP1 gene was highly expressed in E. coli.