黑色素转铁蛋白重组慢病毒载体的构建及其在宫颈癌HeLa细胞中的功能验证

Construction of recombinant expression lentivirus vector carrying melanotransferrin and identification of its role in cervical cancer HeLa cells

目的:构建黑色素转铁蛋白(melanotransferrin,MFI2)基因嵌入的慢病毒载体,上调宫颈癌HeLa细胞中MFI2表达水平,并探讨MFI2对HeLa细胞增殖的影响。方法:以HeLa细胞cDNA为模板复制扩增MFI2相应片段,并将该单边对应序列嵌入到慢病毒载体pHAGE-CMV-MCS-IzsGreen(pHAGE)质粒中构建pHAGE-MFI2重组质粒;将重组质粒与包膜质粒pMD2.G、包装质粒psPAX2协同转染入人胚肾上皮293T细胞,制备MFI2融合病毒,通过梯度稀释法检测病毒滴度。以MFI2融合病毒感染HeLa细胞,通过免疫印迹法检测MFI2标签在细胞中的表达量,采用CCK-8法及划痕实验评价上调MFI2表达量对HeLa细胞扩增能力的影响。结果:质粒双酶切鉴定以及核酸测序比对结果均证实pHAGE-MFI2质粒构建成功;通过慢病毒三质粒协同包装系统成功构建MFI2基因融合慢病毒,病毒液滴度高达6×10^7TU/mL;MFI2慢病毒感染HeLa细胞后,其MFI2的蛋白表达水平明显增高,而细胞增殖活力则显著降低。结论:MFI2高表达可有效抑制HeLa细胞增殖。

Objective:To construct the recombinant lentivirus vector carrying melanotransferrin(MFI2)for upregulating MFI2 expression level in HeLa cells and investigating its effect on HeLa cells proliferation.Methods:The sequences of MFI2 were cloned from cDNA of HeLa cells and were inserted into lentiviral plasmid pHAGE-CMV-MCS-IzsGreen(pHAGE)to construct the recombinant plasmid p HAGE-MFI2,followed by cotransfection of the package plasmid ps PAX2 and the envelope plasmid pMD2.G into 293 T cells.The titer of the generated MFI2-expressing lentivirus was determined by gradient dilution.The MFI2-expressing lentivirus was transduced into the HeLa cells and the expression of MFI2 was determined by Western blotting,and then the proliferation of the treated HeLa cells was further studied via CCK-8 assay and wound healing.Results:Both the identification of plasmid double enzyme digestion and nucleic acid sequencing comparison results confirmed the successful construction of pHAGE-MIF2 plasmid.MFI2 gene fusion lentivirus was successfully constructed by lentivirus triplasmid collaborative packaging system,and the virus titer was up to 6×10^7 TU/mL.After the infection of HeLa cells with MFI2 lentivirus,the expression level of MFI2 was significantly increased,while the cell proliferation activity was significantly decreased.Conclusion:The overexpression of MFI2 could inhibit the proliferation of HeLa cells with high efficiency.