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目的构建pcDNA3.1-叶酸结合蛋白1(Folbp1)基因真核表达载体,检测其在肺腺癌A549细胞中的表达。方法采用反转录-PCR技术从肺腺癌A549细胞中扩增Folbp1基因的完整编码框,将其克隆到真核表达载体pcDNATM3.1/myc-His B,脂质体转染肺腺癌A549细胞,通过G418筛选,建立稳定转染的肺腺癌A549细胞株,并利用Western blot法鉴定其表达。结果 PCR扩增Folbp1基因全长编码区,电泳显示其分子大小与823 bp预期值一致;连接产物pMD-Folbp1转化感受态细菌,经氨苄西林筛选,提取质粒鉴定表明插入pMD-18T载体的目的片段大小和方向正确;重组质粒扩增Folbp1基因连入pcDNATM3.1/myc-His B载体,酶切鉴定显示切出约5.1 kb符合载体大小的条带及符合插入片段大小的特异条带;转染pcDNA3.1-Folbp1重组质粒的肺腺癌A549细胞株经抗生素G418筛选,Western blot分析显示与预期大小一致的特异性条带。结论 pcDNA3.1-Folbp1真核表达载体构建成功,并在肺腺癌A549细胞内过表达,这将为研究Folbp1基因的功能及其肺发育中奠定了良好的基础。
Objective To construct the eukaryotic expression vector pcDNA3.1 - Folbp1 and determine its expression in lung adenocarcinoma A549 cells. Methods The complete coding sequence of Folbp1 gene was amplified from lung adenocarcinoma A549 cells by RT-PCR and cloned into eukaryotic expression vector pcDNATM3.1 / myc-His B. The liposome was transfected into lung adenocarcinoma A549 Cells were screened by G418 to establish a stable transfected lung adenocarcinoma A549 cell line and identified by Western blot. Results The full-length coding region of Folbp1 gene was amplified by PCR and its molecular size was consistent with the expected value of 823 bp by electrophoresis. The pMD-Folbp1 transformed competent cell was identified by ampicillin. The plasmid was extracted and identified. Size and orientation were correct. Folbp1 gene was amplified by PCR and ligated into pcDNATM3.1 / myc-His B vector. The restriction endonuclease digestion showed that the vector was about 5.1 kb in size and the specific band was in accordance with the size of insert. The lung adenocarcinoma A549 cell line with pcDNA3.1-Folbp1 recombinant plasmid was screened by antibiotic G418 and Western blot analysis showed the same specific bands as the expected size. Conclusion The eukaryotic expression vector pcDNA3.1-Folbp1 was successfully constructed and overexpressed in lung adenocarcinoma A549 cells, which will lay a good foundation for studying the function of Folbp1 gene and its lung development.