【摘要】 目的:从人胚胎肝、肾和胰腺组织RNA中钓取α1微球蛋白/bikunin前体(AMBP)cDNA序列,构建其重组克隆载体。方法:采用Trizol法提取人胚胎肝、肾和胰腺组织RNA,以此为模板,应用RT-PCR技术扩增目的基因,构建重组载体pMD18-T-ambp。采用蓝白筛选,经限制性内切酶消化和DNA测序进行鉴定。结果:RT-PCR显示,仅从肝组织RNA中扩增出ambp基因片段,而肾和胰腺组织中均未检测出其 mRNA的转录。构建的重组载体经SalⅠ/EcoRⅠ双酶消化得到1098bp大小的片段,与人AMBP cDNA片段大小一致。序列分析结果显示,克隆到载体中的目的基因与GenBank上登录的人AMBP cDNA序列完全一致。结论:从人胚胎肝组织RNA中成功钓取了AMBP cDNA序列,并正确构建其克隆载体pMD18-T-ambp。更多还原

【Abstract】 Objective To obtain the cDNA sequence encoding human α1-microglobulin/bikunin precursor (AMBP),and construct its recombinant cloning vector.Methods RNA was extracted from human embryo tissues of liver,kidney and pancreas with Trizol reagent.The sequence of human AMBP cDNA was cloned by RT-RCR from those RNAs,and inserted to pMD18-T vector.The recombinant vector pMD18-T-ambp was identified by restriction endonucleases SalⅠ/ EcoRⅠand DNA sequencing.Results The ambp gene fragment was obtained by RT-PCR only from liver RNA,but not from kidney and pancreas.A DNA fragment of 1 098 bp,consistent with the size of ambp gene fragment,was generated by digesting the recombinant plasmid with the restriction enzymes SalⅠ/EcoRⅠ.The results of sequence analysis showed that the constructed AMBP cDNA sequence was identical to the published one in GenBank.Conclusion The human AMBP cDNA is obtained successfully and the recombinant cloning vector pMD18-T-ambp is constructed correctly.更多还原