稳定表达T7 RNA聚合酶BHK-21细胞株的构建

doi:10.16431/j.cnki.1671-7236.2022.04.006

摘要/Abstract

摘要： 【目的】通过建立过表达T7 RNA聚合酶(T7 RNA polymerase,T7 RNAP)的慢病毒系统,构建可稳定表达T7 RNAP的BHK-21细胞株。【方法】根据GenBank中公布的大肠杆菌BL21(DE3)基因组中T7 RNAP基因序列(登录号:NZ_CP081489.1)设计引物,并采用PCR扩增T7 RNAP基因片段,将其克隆至慢病毒载体中,构建pCDH-CMV-T7 RNAP重组质粒。筛选阳性克隆及测序鉴定后,利用脂质体将包含重组质粒pCDH-CMV-T7 RNAP的包装系统和辅助包装质粒共转染至HEK-293T细胞,进行慢病毒的包装及纯化,获得高病毒滴度的重组慢病毒。将收集的病毒液分别以感染复数(multiplicity of infection,MOI)为1、5、10和20感染BHK-21细胞,72 h后观察荧光情况,筛选最佳MOI。将重组慢病毒在最佳MOI条件下感染BHK-21细胞,通过绿色荧光蛋白copGFP的表达观察感染细胞的阳性率。使用4 μg/mL嘌呤霉素作为抗性筛选物进行多轮筛选,最终筛选得到BHK-21-T7 RNAP细胞株。进一步对所筛选的细胞株设计3对检测引物进行RT-PCR检测,并通过检测试验组与空白对照组细胞中海肾荧光素酶(hRluc)报告基因的活性差异来反映T7 RNAP驱动T7启动子下游基因的能力。【结果】本研究成功扩增了大肠杆菌BL21(DE3)细胞基因组中T7 RNAP基因,成功构建重组慢病毒载体,并获得了滴度为1.0×10⁸ TU/mL的重组慢病毒。成功筛选到BHK-21-T7 RNAP细胞株,此重组细胞株经RT-PCR扩增能够检测到T7 RNAP的特异性DNA序列。将含有T7启动子驱动的hRluc的质粒(pT7-hRluc)转染此细胞株后发现,BHK-21-T7 RNAP细胞株中hRluc的活力与空白对照组BHK-21细胞相比极显著升高(P<0.01)。【结论】本研究获得了稳定表达T7 RNAP的BHK-21细胞株,且所筛选的BHK-21-T7 RNAP细胞株中的T7 RNAP能够驱动pT7启动子下游基因hRluc的转录,研究结果为RNA病毒的反向遗传学平台建立奠定了细胞基础。

关键词: T7 RNA聚合酶; 慢病毒载体; 细胞株; 稳定表达

Abstract: 【Objective】 BHK-21 cell line stably expressing T7 RNAP was constructed by establishing a lentivirus vector system overexpressing T7 RNA polymerase (T7 RNAP).【Method】 In this study,T7 RNAP gene was amplified using the primers designed according to Escherichia coli BL21 (DE3) genome sequence published in GenBank (accession No.:NZ_CP081489.1),and then cloned into lentiviral vector to obtain the recombinant plasmid pCDH-CMV-T7 RNAP.After screening positive clones and sequencing identification,the packaging system and auxiliary packaging plasmid containing recombinant plasmid pCDH-CMV-T7 RNAP were co-transfected into HEK-293T cells by liposome,and the lentivirus was packaged and purified to obtain a recombinant lentivirus with high virus titer.The collected virus solution was infected with BHK-21 cells with multiplicity of infection (MOI)=1,5,10 and 20 respectively.The fluorescence was observed after 72 h to screen the best MOI.The recombinant lentivirus was infected with BHK-21 cells under the optimal MOI conditions.The positive rate of infected cells was observed by the expression of green fluorescent protein copGFP.4 μg/mL puromycin was used as the resistance screening material for multiple rounds of screening,and finally BHK-21-T7 RNAP cell line was obtained.Further,three pairs of detection primers were designed for RT-PCR detection of the selected cell lines,and the ability of T7 RNAP to drive the downstream gene of T7 promoter was reflected by detecting the activity difference of sea kidney luciferase (hRluc) reporter gene between test and blank control groups.【Result】 T7 RNAP gene in the genome of Escherichia coli BL21 (DE3) cells was successfully amplified,the recombinant lentivirus vector was successfully constructed,and the titer of the recombinant lentivirus was 1.0×10⁸ TU/mL.BHK-21-T7 RNAP cell line was successfully screened.The specific DNA sequence of T7 RNAP could be detected by RT-PCR.After transfection of plasmid containing T7 promoter driven hRluc (pT7-hRluc) into this cell line,it was found that the activity of hRluc in BHK-21-T7 RNAP cell line was extremely significantly higher than that blank control BHK-21 cells (P<0.01).【Conclusion】 In this study,BHK-21 cell lines stably expressing T7 RNAP were obtained,and T7 RNAP in the selected BHK-21-T7 RNAP cell lines could drive the transcription of the downstream gene hRluc of pT7 promoter.The results laid a cellular foundation for the establishment of RNA virus reverse genetics platform.

Key words: T7 RNA polymerase; lentiviral vector; cell strain; stable expression

中图分类号:

Q786

陈宇婧, 张艺艺, 黄正阳, 石建州, 刘阳坤, 邱礽, 姚伦广, 李娜. 稳定表达T7 RNA聚合酶BHK-21细胞株的构建[J]. 中国畜牧兽医, 2022, 49(4): 1253-1261.

CHEN Yujing, ZHANG Yiyi, HUANG Zhengyang, SHI Jianzhou, LIU Yangkun, QIU Reng, YAO Lunguang, LI Na. Establishment of BHK-21 Cell Strain for Stably Expressing T7 RNA Polymerase[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1253-1261.

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