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目的克隆人T细胞免疫球蛋白黏蛋白-4(TIM-4)基因cDNA,构建其真核表达载体pEGFP-C2-TIM-4,并转染16HBE细胞。方法根据GeneBank中人TIM-4 cDNA序列(编号:NM-138379.2),设计出带有EcoRI和BamHI的上下游引物,应用RT-PCR技术,从人骨髓中扩增出TIM-4基因编码区序列,克隆到pMD18-T载体,形成pMD18-T-TIM-4重组质粒,经菌落PCR,双酶切,测序鉴定获得人TIM-4基因cDNA片段,然后将其亚克隆入pEGFP-C2绿色荧光载体中,并通过菌落PCR,双酶切,测序鉴定其重组体。将测序正确的pEGFP-C2-TIM-4质粒转染人气道上皮细胞(16HBE),用实时定量PCR检测目的基因的表达。结果成功扩增出人TIM-4 cDNA全长1134 bp,经T-A克隆构建的pMD18-T-TIM-4重组质粒经菌落PCR,双酶切,测序证实载体中含有正确的TIM-4编码区片段。由此构建的真核表达载体pEGFP-C2-TIM-4,经菌落PCR扩增出1134 bp左右的片段,经双酶切后产生4.7 kb和1134 bp左右的2条带,DNA测序显示与GeneBank中人TIM-4 cDNA序列一致。重组质粒转染16HBE细胞在荧光显微镜下可见绿色荧光,经实时定量PCR分析,转染细胞TIM-4基因的表达显著增加。结论首次从人骨髓中扩增出TIM-4基因cDNA全长,构建了其真核表达载体pEGFP-C2-TIM-4,并在16HBE细胞内成功高表达,为进一步研究TIM-4的生物学功能奠定了基础。
Objective To clone human T cell immunoglobulin mucin-4 (TIM-4) cDNA and construct its eukaryotic expression vector pEGFP-C2-TIM-4 and transfect it into 16HBE cells. Methods The upstream and downstream primers with EcoRI and BamHI were designed according to the cDNA sequence of human TIM-4 in GeneBank (ID: NM-138379.2). RT-PCR was used to amplify the coding region of TIM-4 gene from human bone marrow And cloned into pMD18-T vector to form recombinant plasmid pMD18-T-TIM-4. The cDNA fragment of human TIM-4 gene was identified by colony PCR, double enzyme digestion and sequencing, then subcloned into pEGFP-C2 green fluorescent carrier The recombinant was identified by colony PCR, double digestion and sequencing. The pEGFP-C2-TIM-4 plasmid was transfected into human airway epithelial cells (16HBE), and the expression of the target gene was detected by real-time quantitative PCR. Results The full-length cDNA of human TIM-4 was amplified 1134 bp in length. The recombinant plasmid pMD18-T-TIM-4 constructed by TA cloning was confirmed by colony PCR and double enzyme digestion. Sequencing confirmed that the vector contained the correct TIM-4 coding region . The eukaryotic expression vector pEGFP-C2-TIM-4 constructed by this method amplified 1134 bp fragment by colony PCR, and double-digested the two fragments of 4.7 kb and 1134 bp. The DNA sequencing showed that compared with GeneBank Human TIM-4 cDNA sequence is consistent. The 16HBE cells transfected with the recombinant plasmids showed green fluorescence under the fluorescence microscope, and the expression of TIM-4 gene was significantly increased by real-time quantitative PCR analysis. Conclusion The full-length cDNA of TIM-4 gene was amplified from human bone marrow for the first time, and its eukaryotic expression vector pEGFP-C2-TIM-4 was constructed and successfully expressed in 16HBE cells. To further study the biological characteristics of TIM-4 Function laid the foundation.