【摘要】 目的:克隆、表达SVV基因,并探讨其在肿瘤预后中的作用。方法:从人白血病细胞系HL60提取总RNA,用RT-PCR方法扩增出SVV基因片段。将SVV基因片段插入载体pGEM-TEasy中,经全自动序列分析仪测序正确后用NdeⅠ/XhoⅠ双酶切,亚克隆到原核表达载体pRSET-C中,构建重组表达载体pRSET-c-SVV,并转化大肠杆菌BL21菌株。取工程菌,经IPTG诱导表达,对表达产物进行SDS-PAGE鉴定并初步纯化。结果:经RT-PCR,测序和酶切鉴定,成功地克隆了人SVV基因;经IPTG诱导的重组质粒pRSET-C-SVV表达出相对分子质量约为16500u的蛋白,与预期的结果相符。结论:成功克隆了人肿瘤细胞的抗凋亡基因SVV,并在大肠杆菌BL21中表达出SVV蛋白。该基因的高效表达为进一步探讨以SVV为基础的肿瘤的防治及预后判断提供工具。更多还原

【Abstract】 AIM:To clone and express the apoptosis inhibiting gene Suivivin(SVV),and to explore its role in tumor prognosis. METHODS:The total RNA was extracted from human leukemia cell line HL60,and then the cDNA fragment of SVV gene was amplified by RT PCR.The SVV gene was inserted into pGEM T Easy,and after sequenced by fully automatic sequencer the SVV gene fragment was sub cloned into the prokaryotic expression vector pRSET C with NdeⅠand XhoⅠto construct the recombinant expression vector pRSET c SVV which was used to transform BL21 strain of E.coli.The engineered strain was induced with IPTG,and then the expressed product was identified by SDS PAGE and primarily purified. RESULTS:The sequencing and endonucleases digestion analysis showed that the fragment of SVV gene was cloned successfully after RT PCR.The recombinant plasmid induced by IPTG expressed relative molecular mass with 16 500 u proteins,which accorded with the expected results. CONCLUSION:The apoptosis inhibiting gene SVV is cloned successfully in human tumor cells,and the SVV protein is expressed in the BL21 strain of E.coli.The high efficient expression of the SVV gene fragment offers the new medium for the further study on prevention,treatment and prognosis judgment of tumors.更多还原