Objective To prepare gene overexpressing cell model of human wild-type DJ-1 and its L166P mutant, and to investigate the role of lentiviral vector in gene overexpressing cell model .Methods Wild type DJ-1 and L166P mutant DJ-1 lentiviral vector plasmids were respectively constructed .After sequencing and comparing cor-rectly, the plasmid was amplified and transfected into HEK 293T cell line.Expression of WT DJ-1 and L166P mu-tant DJ-1 in cell lines was detected by fluorescence and Western blot .After determining the accurate expression of the target protein, a large amount of HEK293T cells was transfected and packaged to produce lentiviral particles. The PC12 cells were infected with the titer of virus supernatant.The fluorescence intensity of GFP and the expres-sion of target protein were observed by fluorescence microscope and Western blot method ,and the infection effi-ciency of the virus was determined .Results Lentiviral vectors carrying wild type DJ-1 and its mutants were suc-cessfully constructed .The virus vector can be transfected into HEK 293T cells and the target protein can be correctly expressed.The viral titers of LV-DJ-1 and LV-DJ-1/L166P were 2×109 TU/mL and 2×108 TU/mL, respectively. Virus supernatant can efficiently infect PC 12 cells, and most cells can express target proteins .The protein expres-sions of exogenous wild-type DJ-1 and L166P mutants were 315% and 285% of endogenous content ,respectively. Conclusions Lentivirus vector can infect cells efficiently , and it is a good way to prepare gene over expressing cell model.A cell model overexpressing DJ-1 or its L166P mutant is successfully prepared .The model can be used for subsequent DJ-1 function research .%目的 构建人野生型DJ-1及其L166P突变体的慢病毒载体并探讨慢病毒载体在构建基因过表达细胞模型中的作用.方法 分别构建野生型DJ-1与L166P突变型DJ-1慢病毒载体质粒.进行测序确定比对正确后,进行质粒的大量扩增与制备并转染包装细胞系HEK293T细胞,荧光法和Western blot检测野生型DJ-1与L166P突变型DJ-1在细胞系中的表达.在确定目的蛋白正确表达之后,大量转染HEK293T细胞进行包装并生产携带目的基因的慢病毒颗粒.测定病毒上清滴度后感染PC12细胞,荧光显微镜和Western blot观察GFP荧光强度以及目的蛋白的表达,确定病毒的感染效率.结果 成功构建携带DJ-1野生型及其突变体的慢病毒载体.该病毒载体可以转染进入HEK293T细胞内且目的蛋白能够正确表达.LV-DJ-1与LV-DJ-1/L166P的病毒滴度分别为2×109 TU/mL与2×108 TU/mL.病毒上清可以高效感染PC12细胞,绝大多数细胞可表达目的蛋白.外源野生型DJ-1和L166P突变体的蛋白表达量分别是内源性含量的315%和285%.结论 慢病毒感染细胞效率很高,是很好的制备基因过表达细胞的方法.通过慢病毒载体介导,本研究获得了DJ-1及其突变体的过表达细胞模型.该模型可以用于后续DJ-1功能研究.
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