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目的 克隆人RECK基因,构建其原核表达重组体,并进行初步表达。方法 从人的胎盘组织中提取总RNA, 经逆转录聚合酶链反应扩增出RECK基因;将RECK基因克隆入pGEM T载体中,构建克隆载体pGEM RECK,阳性克隆经酶 切、电泳鉴定,测定RECK基因序列并确认后,构建原核表达重组体pBluescriptSK+ RECK,在工程菌BL21(DE3)中进行IPTG 诱导表达。结果 PCR扩增得到了特异性的4.4kb基因片段。基因片段的大小与预期一致。所获得的RECK基因测序正确, 原核表达载体表达重组蛋白,经SDS PAGE及Western blot分析,相对分子量为110kD。结论 成功地克隆了人RECK基因并 构建了原核表达重组体pBluescriptSK+ RECK,此重组体能高效表达RECK蛋白。
Objective To clone human RECK gene and construct its recombinant prokaryotic expression vector. METHODS: Total RNA was extracted from human placenta tissue and RECK gene was amplified by reverse transcription polymerase chain reaction. The RECK gene was cloned into pGEM T vector to construct the cloning vector pGEM RECK. The positive clones were identified by enzyme digestion and electrophoresis RECK gene sequence and confirmed, the prokaryotic expression recombinant pBluescriptSK + RECK was constructed and induced by IPTG in the engineering strain BL21 (DE3). Results The specific 4.4kb gene fragment was obtained by PCR amplification. The size of the gene fragment is as expected. The obtained RECK gene was sequenced correctly. The prokaryotic expression vector expressed the recombinant protein, and its relative molecular weight was 110kD by SDS PAGE and Western blot analysis. Conclusion The human RECK gene was successfully cloned and the prokaryotic expression recombinant pBluescriptSK + RECK was constructed. The RECK protein was highly expressed in this recombinant.