pLenti6/V5-DEST-TAZ载体的构建及其对成骨前体细胞MC3T3-E1分化的影响

目的：构建人真核表达的TAZ慢病毒载体pLenti6/V5-DEST-TAZ，转染293FT 细胞获得重组慢病毒颗粒，探讨TAZ对成骨前体细胞MC3T3-E1 分化的调控作用。方法：将已经过测序验证的含有TAZ基因慢病毒入门质粒pENTR(tm)221-TAZ通过LR反应克隆到慢病毒载体pLenti6/V5-DEST中。对重组质粒进行酶切鉴定，并在细胞中验证表达。将该重组载体和其他PackingMix 3 个质粒载体充分混合，用阳离子脂质体转染293FT 细胞，培养和待细胞完全裂解后收集富含TAZ基因的病毒颗粒上清液，取适量上清液感染MC3T3-E1细胞，采用杀稻瘟菌素Blastcidin 筛选，建立稳定过量表达 TAZ 蛋白的 MC3T3-E1/TAZ细胞系。用条件培养基诱导 MC3T3-E1和 MC3T3-E1/TAZ细胞系向成骨细胞定向分化，von Kossa 和茜素红染色观察MC3T3-E1和MC3T3-E1/TAZ细胞的成骨分化。结果：凝胶电泳和测序结果均证明TAZ重组慢病毒载体pLenti6/V5-DEST-TAZ构建正确，并能在细胞中正确表达。与辅助质粒共转包装细胞获得慢病毒颗粒，并成功感染MC3T3-E1细胞。Von Kossa 染色和茜素红染色， MC3T3-E1/TAZ细胞系中生成的磷酸钙比MC3T3-E1细胞系中生成的磷酸钙多。结论：成功构建了人真核表达的质粒载体pLenti6/V5-DEST-TAZ，并能在细胞中正确表达；成功包装了TAZ病毒，并获得过表达TAZ的MC3T3-E1细胞；过表达TAZ促进MC3T3-E1向成骨细胞分化。

Construction of pLenti6/V5-DEST-TAZ vector and its effect on differentiation of MC3T3-E1 preosteoblasts

Objective To construct TAZ expressing lentivirus vector pLenti6/V5-DEST-TAZ and study the regulatory effect of TAZ on the differentiation of MC3T3-E1 preosteoblasts. Methods LR reaction was performed to clone the pENTR(tm)221-TAZ plasmid containing TAZ cDNA into pLenti6/V5-DEST plasmid.The new recombinant plasmid was identified by restriction enzyme.The overexpression of TAZ in cells was validated by Western blotting.The pseudoviral particles containing the expressed construct were generated by lentiviral packaging system in 293FT cells,which were used to infect MC3T3-E1 to select monoclonal cells by using Blastcidin added.MC3T3-E1 wild type cell line and TAZ overexpressing cell lines were induced towards osteoblasts with condition medium.Differentiation and mineralization of two cell lines were verified by assay of Von Kossa staining and Alizarin red staining,respectively.Results Agarose electrophoresis and sequencing examination showed that TAZ cDNA was cloned into lentiviral vector pLenti6/V5-DEST.The recombinant plasmid could be expressed correctly.The pseudoviral particles with TAZ were obtained and the MC3T3-E1 cell lines were infected successfully.The Von Kossa staining and Alizarin red staining results showed that the mineralization of TAZ overexpressing cell lines was higher than that of MC3T3-E1 wild type cell line. Conclusion The pLenti6/V5-DEST-TAZ is constructed successfully,and it can express in the cells correctly.The human TAZ cDNA is cloned into the lentiviral vector pLenti6/V5-DEST. The results indicate that the overexpression of TAZ can promote the differentiation of MC3T3-E1 cell line into osteoblasts.