吉林大学学报（医学版）2025，Vol.51Issue(1)：1-8,8.DOI:10.13481/j.1671-587X.20250101
PRDM5过表达慢病毒载体的构建及稳定转染Neuro-2a细胞的建立
Construction of PRDM5 over-expression lentivirus vector and establishment of stably transfected Neuro-2a cells
摘要
Abstract
Objective:To construct the over-expressed lentivirus vector of PRDM5 gene and establish the Neuro-2a cells stably transfected PRDM5,and to provide the basis evidence for exploring the effect of PRDM5 in pathogenesis of ischemic stroke(IS).Methods:The sequence of PRDM5 was searched and designed based on NCBI.The PRDM5 gene was amplified by PCR and ligated with the lentiviral vector GV492 digested by BamH Ⅰ and Age Ⅰ restriction enzymes to form the GV492-PRDM5 over-expression recombinant plasmid.The positive clones with similar length and size to the target gene fragment were screened by PCR and sent to Shenggong Bioengineering(Shanghai)Co.Ltd.for identification.The correctly-sequenced GV492-control plasmid and GV492-PRDM5 over-expression recombinant plasmid were transfected into the HEK293T cells,respectively.After 48 h of transfection,the lentiviruses were collected by centrifugation,and they were GV492-control lentivirus and GV492-PRDMS over-expression lentivirus;the titers of these two lentiviruses were determined by lentiviral titer assay.The Neuro-2a cells were divided into GV492-control group and GV492-PRDM5 group,and then infected with GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus,respectively,with a lentivirus multiplicity of infection(MOI)of 100.The Neuro-2a cells successfully infected with GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus were screened with puromycin(10 mng-L-1)after 72 h of infection.The growth status and the expression of green fluorescence protein of Neuro-2a cells in GV492-control group and GV492-PRDM5 group were observed by fluorescence microscope.The expression levels of PRDM5 mRNA and PRDM5 protein in the Neuro-2a cells in two groups were detected by real-time fluorescence quantitative RCR(RT-qPCR)and Western blotting methods.Results:The PCR results showed that the length of the positive transformant of GV492-PRDM5 recombinant plasmid was about 684 bp,and the gene sequence of GV492-PRDM5 over-expression recombinant plasmid was consistent with the designed and synthesized PRDM5 over-expression sequence.The titers of GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus were both 2.5×108TU·mL-1 The Neuro-2a cells in GV492-control group and GV492-PRDM5 group grew well,and the expressions of green fluorescence protein were found under fluorescence microscope.The RT-qPCR results showed that the expression level of PRDM5 mRNA in the Neuro-2a cells in GV492-PRDM5 group was significantly increased compared with GV492-control group(P＜0.01).The Western blotting results showed that the specific bands appeared in the Neuro-2a cells in GV492-control group and GV492-PRDM5 group with a relative molecular weight of 75 000;compared with GV492-control group,the expression level of PRDM5 protein in the Neuro-2a cells in GV492-PRDM5 group was increased(P＜0.01).Conclusion:The over-expression lentivirus vector of PRDM5 gene is successfully constructed,and the stably transfected GV492-PRDM5-Neuro-2a cells are established.关键词
PR锌指区域蛋白/过表达慢病毒载体/稳定表达/Neuro-2a细胞Key words
PRDI-BF1 and RIZ domain proteins/Over-expression lentivirus vector/Stable expression/Neuro-2a cells分类
临床医学引用本文复制引用
吴钊淳,李友,何嘉文,廖科棋,李胜男..PRDM5过表达慢病毒载体的构建及稳定转染Neuro-2a细胞的建立[J].吉林大学学报（医学版）,2025,51(1):1-8,8.基金项目
国家自然科学基金项目(81571157) （81571157）
广东省基础与应用基础研究基金委员会自然科学基金面上项目(2023A1515012750) （2023A1515012750）
广东省卫健委医学科研基金项目(A2022139,A2023193) （A2022139,A2023193）
广东医科大学百项青年研究项目资助计划项目(GDMUD2022010) （GDMUD2022010）
