人Calnexin基因慢病毒载体及稳定表达Calnexin蛋白的宫颈癌细胞株构建  

Construction of human Calnexin lentiviral vector and cervical cancer cell line with stable expression of Calnexin protein

作　　者：李宏帅 王鑫廷[1] 王媛[1] 杨洁[1] LI Hongshuai;WANG Xinting;WANG Yuan;YANG Jie(Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学,天津300070

出　　处：《山东医药》2018年第35期46-49,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(31370749);国家青年科学基金资助项目(31300709)

摘　　要：目的构建人Calnexin基因慢病毒载体及稳定表达Calnexin蛋白的人宫颈癌Hela细胞株,为进一步探讨Calnexin基因对宫颈癌潜在的调控作用提供细胞模型。方法采用TRIzol提取宫颈癌细胞Hela细胞株总RNA,RT-PCR法逆转录出含有目的基因Calnexin的cDNA序列;根据Calnexin的cDNA序列,选择EcoR1、Bam H1限制性酶切位点作为目的位点,设计FLAG-Calnexin基因的PCR引物;利用PCR法从Hela细胞cDNA中扩增出FLAG-Calnexin基因片段,连接到慢病毒载体pLVX-IRES-puro中,得到重组慢病毒pLVX-FLAG-Calnexin质粒。将慢病毒包装质粒与pLVX-FLAG-Calnexin重组质粒共转染到人肾上皮293T细胞中,获得携带FLAG-Calnexin基因序列的重组慢病毒。将慢病毒感染Hela细胞24 h,在细胞培养基中加入1.0μg/m L嘌呤霉素,药筛48 h。最后筛选出稳定表达FLAG-Calnexin蛋白的Hela细胞,采用Western blotting法、Real-time PCR法检测该细胞内的Calnexin蛋白及mRNA。结果构建完成的重组质粒经菌落PCR、重组质粒的双酶切、质粒PCR验证后,条带位置对应片段大小与目的基因大小一致,且经质粒测序后与Calnexin基因片段比对一致。经过慢病毒感染、药物筛选后得到的Hela细胞中的Calnexin蛋白及mRNA的相对表达量分别为2.14±0.25、6.15±0.21,高于野生型Hela细胞及转入空载的对照细胞(P均<0.01)。结论成功构建了人Calnexin基因慢病毒载体,同时建立、筛选出了能够稳定表达Calnexin蛋白的Hela细胞株,为进一步探讨Calnexin在宫颈癌发生发展中的作用提供了细胞模型。Objective To construct human lentiviral vector of Calnexin gene and human cervical cancer Hela cell line stably expressing Calnexin protein,which provides a cell model for investigating the potential regulation effect of Calnexin gene on cervical cancer.Methods Total RNA of cervical cancer Hela cells was extracted by TRIzol.RT-PCR was used to reverse transcribe the cDNA sequence containing the target gene Calnexin.According to the Calnexin cDNA sequence,EcoR1 and BamH1 restriction sites were chosen as the target site and PCR primer of FLAG-Calnexin gene;FLAG-Calnexin gene fragment was amplified from Hela cell cDNA by PCR and ligated into lentiviral vector pLVX-IRES-puro to obtain recombinant lentiviral pLVX-FLAG-Calnexin plasmid.The lentiviral packaging plasmid and the pLVX-FLAG-Calnexin recombinant plasmid were co-transfected into 293T cells to obtain a recombinant lentivirus carrying the FLAG-Calnexin gene sequence.Hepa cells were infected with lentivirus for 24 h,1.0μg/mL puromycin was added to the cell culture medium,and the drug sieve was for 48 h.Finally,Hela cells with stable FLAG-Calnexin protein were screened out,and the Calnexin protein and mRNA in cells were detected by Western blotting and real-time PCR.Results After the constructed recombinant plasmid was verified by colony PCR,double digestion of the recombinant plasmid,and plasmid PCR,the size of the fragment corresponding to the position of the fragment was the same as the size of the target gene,and the sequence alignment with the Calnexin gene fragment after plasmid sequencing was identical.The expression levels of Calnexin protein and mRNA in Hela cells obtained by lentivirus infection and drug screening were 2.14±0.25 and 6.15±0.21,respectively,which were higher than those of the wild-type Hela cells and control cells(all P<0.01).Conclusion We successfully construct the human Calnexin gene lentiviral vector and establish and screen out the Hela cell line stably expressing Calnexin protein,which provides a cell model for further exploratio

关 键 词：Calnexin基因 慢病毒载体 宫颈癌细胞 HELA细胞 

分 类 号：Q591.2[生物学—生物化学] Q784