Satb2基因慢病毒载体的构建及其鉴定

Construction and identification of Satb2 lentiviral expression vector.

目的构建特殊富含AT序列结合蛋白2 (Satb2)基因慢病毒表达载体并通过感染293T细胞检测目的基因的表达。方法采用PCR技术扩增目的基因Satb2,并在目的基因的上下游分别添加酶切位点PacⅠ和AscⅠ,将其PCR产物和目的载体用PacⅠ和AscⅠ分别进行双酶切,通过T4DNA ligase连接酶切后的PCR产物及目的载体,构建pLenti6.3-Satb2-IRES-EGFP慢病毒表达载体,通过菌液PCR、酶切及测序鉴定其结果;将阳性克隆质粒与包装质粒Mix共转染293T细胞,收集、浓缩病毒液并测定其浓度;将重组病毒液感染293T细胞,观察其荧光表达情况,通过q PCR检测目的基因表达。结果菌液PCR结果得到一条约2 202 bp的亮带,与目的基因大小相符;重组载体经过双酶切得到一条9.1 kb大片段及一条2 202 bp小片段;通过测序验证重组载体中Satb2基因序列与GenBank报道的序列完全一致,表明慢病毒表达载体构建成功。经过包装,重组慢病毒滴度达到7.9×107ifu/mL,该病毒液感染293T细胞后可观察到大量荧光,感染率约为70%,通过qPCR检测结果显示实验组Satb2 mRNA的表达量增加了约106倍(P<0.05)。结论本实验成功构建pLenti6.3-Satb2-IRES-EGFP慢病毒表达载体并包装出具有感染能力的慢病毒,为进一步研究该基因在牙周支持组织再生中的作用奠定基础。

Objective To construct a lentiviral eukaryotic expression vector with special AT-rich sequence-binding protein 2(Satb2)gene and test the expression of the target gene by infecting 293T cells.Methods The aim gene Satb2 was amplified from plasmids by PCR.PacⅠand AscⅠwere added in the upstream and downstream of the target fragment,and the target gene PCR product and vector were digested by PacⅠand AscⅠ,respectively.Then the T4 DNA ligase was used to connect the PCR product and the target vector after enzyme digestion,and an pLenti6.3-Satb2-IRES-EGFP lentivirus expression vector was constructed,and the results were identified by PCR,enzyme digestion and sequencing.The positive clone plasmid and package plasmid Mix were transfected into 293T cells,and the virus venom was collected,concentrated,and the concentration was determined.293T cells were infected with recombinant virus venom and their fluorescence expression was observed.The expression of the target gene was detected by qPCR.Results A bright band of 2202 bp was obtained by PCR,which was consistent with the size of the target gene.The recombinant vector was digested by double enzyme to obtain the large fragment vector(9.1 kb)and the desired target band(2202 bp).The sequence of Satb2 gene in the recombinant vector was verified to be the same as that reported by GenBank,indicating that the lentivirus expression vector was successfully constructed.After packaging,the virus titer reached 7.9×107 ifu/mL.A large amount of fluorescence was observed after 293T cells were infected with virus,and the infection rate was about 70%.The results of qPCR test showed that the expression of Satb2 mRNA in the experimental group increased by about 106 times(P<0.05).Conclusion In this study,pLenti6.3-Satb2-IRES-EGFP lentivirus expression vector was successfully constructed and the lentivirus with infective ability was packaged,laying a foundation for further study on the function of this gene in periodontal tissue regeneration.