【摘要】 用6月龄大月份流产的新鲜肝脏组织为材料,提取总RNA,通过反转录得到cDNA,并以此为模板,经PCR得到人Angiostatin基因的AK(1-3)片段.将此PCR产物直接与载体pMD18-T连接,转化大肠杆菌DH5α.经蓝白菌落筛选,PCR和酶切检测,筛选出阳性克隆,命名为pMDA,测序证明克隆序列与GenBank发表序列一致,大小为858bp.用Sal 和Bgl 酶切将该基因插入载体pQE40,并转化宿主菌M15[pREP4].经质粒电泳PCR和酶切检测筛选阳性克隆,得到表达载体pQEA,插入基因与载体上小鼠DHFR基因重组形成嵌合基因.在30℃,2×YT培养基(Kan25μg/mL+Amp200μg/mL),2mmol/LIPTG条件下诱导pQEA/M15[pREP4]表达.所得菌体总蛋白经SDS-PAGE分析,结果表明,在预期的61kD处得到一条特异带,为AK(1-3)cDNA与载体固有基因DHFR-6хHis的重组蛋白表达产物.实验结果为抗肿瘤药物的研制提供了条件.更多还原

【Abstract】 Human angiostatin k(1-3) cDNA has been obtained by RT-PCR from total RNA of an abortive foetus of six months. The AK(1-3) cDNA product of RT-PCR was inserted directly into cloning vector pMD18-T. Positive clones were identified by PCR and restriction enzyme cleavage test.One of the positive clones has been sequenced and compared with the corresponding sequence in GenBank.The result confirmed that the AK(1-3) gene(858 bp)was obtained.The clone is named pMDA.An expression vector has been constructed and named pQEA in which AK(1-3) gene was recombined to mouse DHFR plus 6X His gene in order to yield fusion protein.The positive clones were identified by test of PCR and restriction enzyme. Cleavage pQEA was induced in E.coli M15[pREP4],under the culture condition of 30 ℃,2 mmol/L IPTG,2×YT (Kan25 μg/mL, Amp200 μg/mL).SDS-PAGE showed that the fused protein of interest has been specifically expressed as 61 kD.The results provide new information for the further study and application of anticancer drugs.更多还原