PRDM5过表达慢病毒载体的构建及稳定转染Neuro-2a细胞的建立

doi:10.13481/j.1671-587X.20250101

吉林大学学报(医学版) ›› 2025, Vol. 51 ›› Issue (1): 1-8.doi: 10.13481/j.1671-587X.20250101

• 基础研究 •

PRDM5过表达慢病毒载体的构建及稳定转染Neuro-2a细胞的建立

吴钊淳^1,²,李友^1,²,何嘉文^1,²,廖科棋^1,²,李胜男^1,²()

^1.广东医科大学广东省衰老相关心脑疾病重点实验室，广东湛江 524002
^2.广东医科大学附属医院神经病学研究所，广东湛江 524002

收稿日期:2023-08-12 接受日期:2023-10-10 出版日期:2025-01-28 发布日期:2025-03-06
通讯作者: 李胜男 E-mail:nancylee@gdmu.edu.cn
作者简介:吴钊淳（1997-），女，广东省潮州市人，医学硕士，主要从事脑血管病发病机制方面的研究。
基金资助:
国家自然科学基金项目(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);广东省卫健委医学科研基金项目(A2022139);广东医科大学百项青年研究项目资助计划项目(GDMUD2022010)

Construction of PRDM5 over-expression lentivirus vector and establishment of stably transfected Neuro-2a cells

Zhaochun WU^1,²,You LI^1,²,Jiawen HE^1,²,Keqi LIAO^1,²,Shengnan LI^1,²()

^1.Guangdong Key Laboratory of Age Related Cardiac and Cerebral Diseases，Guangdong Medical University，Zhanjiang 524002，China
^2.Institute of Neurology，Affiliated Hospital，Guangdong Medical University，Zhanjiang 524002，China

Received:2023-08-12 Accepted:2023-10-10 Online:2025-01-28 Published:2025-03-06
Contact: Shengnan LI E-mail:nancylee@gdmu.edu.cn

摘要/Abstract

摘要：

目的构建PR锌指区域蛋白（PRDM）5基因的过表达慢病毒载体，建立稳定转染的小鼠神经瘤母细胞Neuro-2a，为探讨PRDM5在缺血性脑卒中（IS）发病机制中的作用奠定基础。方法在NCBI上搜索PRDM5的序列并设计引物，PCR法扩增获取PRDM5基因序列，将其与BamHⅠ和AgeⅠ限制性内切酶双酶切后的慢病毒载体GV492进行连接，构建GV492-PRDM5过表达重组质粒，采用PCR法筛选并鉴定出的与目的基因片段长度相近的阳性克隆送生工生物工程（上海）股份有限公司测序。将测序正确的GV492-control质粒和GV492-PRDM5过表达重组质粒分别转染至人胚胎肾细胞HEK293T中，转染48 h后离心收集慢病毒，分别为GV492-control慢病毒和GV492-PRDM5过表达慢病毒，采用慢病毒滴度测定法检测上述2种慢病毒滴度。将Neuro-2a细胞分为GV492-control组和GV492-PRDM5组，分别使用GV492-control慢病毒和GV492-PRDM5过表达慢病毒感染Neuro-2a细胞，慢病毒感染复数（MOI）为100，感染72 h后使用嘌呤霉素（10 mg·L^-1）对成功感染GV492-control慢病毒和GV492-PRDM5过表达慢病毒的Neuro-2a细胞进行筛选，通过荧光显微镜观察GV492-control组和GV492-PRDM5组Neuro-2a细胞的生长状态及绿色荧光蛋白的表达情况。采用实时荧光定量PCR（RT-qPCR）法检测2组Neuro-2a细胞中PRDM5 mRNA表达水平；Western blotting法检测2组Neuro-2a细胞中PRDM5蛋白表达水平。结果 PCR法，GV492-PRDM5重组质粒阳性转化子的长度约为684 bp，GV492-PRDM5过表达重组质粒基因序列与设计合成的PRDM5过表达序列一致；GV492-control慢病毒和GV492-PRDM5过表达慢病毒的滴度均为2.5×10⁸ TU·mL^-1。荧光显微镜，GV492-control组和GV492-PRDM5组Neuro-2a细胞的生长状态均良好，且能观察到绿色荧光蛋白的表达。RT-qPCR法，与GV492-control组比较，GV492-PRDM5组Neuro-2a细胞中PRDM5 mRNA表达水平明显升高（P<0.01）。Western blotting法，GV492-control组和GV492-PRDM5组Neuro-2a细胞在相对分子质量为75 000附近出现特异性条带；与GV492-control组比较，GV492-PRDM5组Neuro-2a细胞中PRDM5蛋白表达水平升高（P<0.01）。结论成功构建PRDM5过表达慢病毒载体，建立了稳定转染的GV492-PRDM5-Neuro-2a细胞。

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

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 BamHⅠ and AgeⅠ 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 mg·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.

Key words: PRDI-BF1 and RIZ domain proteins, Over-expression lentivirus vector, Stable expression, Neuro-2a cells

中图分类号:

R543.5

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.

Zhaochun WU,You LI,Jiawen HE,Keqi LIAO,Shengnan LI. Construction of PRDM5 over-expression lentivirus vector and establishment of stably transfected Neuro-2a cells[J]. Journal of Jilin University(Medicine Edition), 2025, 51(1): 1-8.

图/表 3

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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

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