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作者采用PCR方法克隆了我国海南省FCC1/HN株P190抗原两个保守区基因,分别定名为P190CRI和P190CRV。基因片段经纯化后连接到pUC18载体中进行DNA序列分析,结果显示:除了P190CRV中有5个碱基变换外,其余序列均与MAD20型序列一致。经序列分析的两个基因片段分别与pGEX-2T载体连接,经双酶切鉴定后转化感受态JM109(DE_3)大肠杆菌进行高效融合表达,并且用Sepharose 4B-谷胱甘肽层析柱进行亲和纯化,结果为:两个插入基因片段均得到高效融合表达,经一步亲和纯化后就取得高纯度的重组蛋白。
The authors cloned the two conserved regions of the P190 antigen of FCC1 / HN strain of Hainan Province in China by PCR and named P190CRI and P190CRV respectively. After purification, the gene fragment was ligated into pUC18 vector for DNA sequence analysis. The results showed that except for the 5 base transitions in P190CRV, all other sequences were identical with MAD20 sequence. The two gene fragments were respectively linked with pGEX-2T vector and identified by double enzyme digestion. The recombinant plasmid was transformed into competent E. coli JM109 (DE_3) for high-performance fusion expression, and the recombinant protein was expressed by Sepharose 4B-glutathione chromatography As a result, both of the inserted gene fragments were highly expressed and fused, and the high-purity recombinant protein was obtained after one-step affinity purification.