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目的构建人细胞间黏附分子-1真核表达重组体pcDNA3.1hisB-ICAM-1。方法设计、合成ICAM-1基因序列特异性引物,从肝癌组织中提取总RNA,以此为模板经RT-PCR扩增人ICAM-1的cDNA片段;然后将其克隆到pGEM-TEasyVector,构建中介重组体(pGEM-ICAM-1),再克隆,构建真核表达重组体pcDNA3.1HisB-ICAM-1,经菌落PCR和限制性酶切有目的片段出现,进行DNA序列分析。结果RT-PCR扩增得到人成熟ICAM-1的cDNA片段大小为1622bp,中介重组体(pGEM-ICAM-1),酶切后与真核表达载体连接,根据酶切鉴定和DNA序列分析得到真核表达重组体pcDNA3.1HisB-ICAM-1;结论成功构建了真核表达重组体pcDNA3.1HisB-ICAM-1。
Objective To construct human recombinant eukaryotic expression vector pcDNA3.1hisB-ICAM-1. Methods The sequence-specific primers of ICAM-1 gene were designed and synthesized. Total RNA was extracted from HCC tissues. The cDNA fragment of human ICAM-1 was amplified by RT-PCR. Then, it was cloned into pGEM-TEasyVector The recombinants (pGEM-ICAM-1) were recombined. The eukaryotic expression recombinant pcDNA3.1HisB-ICAM-1 was constructed and sequenced by colony PCR and restriction enzyme digestion. Results The cDNA fragment of human mature ICAM-1 was amplified by RT-PCR. The size of cDNA fragment was 1622bp. The recombinant plasmid pGEM-ICAM-1 was digested with restriction endonuclease and identified by DNA sequencing. The recombinant plasmid pcDNA3.1HisB-ICAM-1 was successfully constructed. The eukaryotic expression recombinant pcDNA3.1HisB-ICAM-1 was successfully constructed.