一种具有G418抗性筛选标记的慢病毒RNA干扰表达载体的构建及应用  

Construction of a Lentivirus shRNA Vector with G418 Resistance and its Potential Application

作　　者：赵思捷 朱文琦 孙杰 侯俊 王友亮 李永利 李波 鲍春梅 张浩 李伯安 ZHAO Si-Jie;ZHU Wen-Qi;SUN Jie;HOU Jun;WANG You-Liang;LI Yong-Li;LI Bo;BAO Chun-Mei;ZHANG Hao;LI Bo-An(Center for Clinical Laboratory,Fifth Medical Centre,Chinese PLA General Hospital,Beijing 100039,China;Beijing Institute of Biotechnology,Beijing 100071,China)

机构地区：[1]解放军总医院第五医学中心临床检验医学中心,北京100039 [2]军事医学研究院生物工程研究所,北京100071

出　　处：《生物技术通讯》2019年第4期528-532,共5页Letters in Biotechnology

摘　　要：目的:构建具有新霉素抗性筛选标记的RNA干扰慢病毒表达载体,并检测它对乙型肝炎病毒x基因(HBx)mRNA表达的抑制作用。方法:从pcDNA3.0载体中扩增新霉素抗性基因并插入删除嘌呤霉素编码序列的pLKO载体中;将改构的RNA干扰载体包装成慢病毒后感染肝癌细胞HepG2,并检测感染细胞对G418的抵抗作用;为验证改构RNA干扰载体的有效性,设计了2条针对HBx的RNA干扰序列以及针对编码萤光素酶cDNA的干扰序列并插入该载体,将含新霉素筛选标记的HBx的RNA干扰载体与辅助质粒在293T细胞中包装成慢病毒并感染过表达HBx的HepG2(嘌呤霉素抗性)细胞,运用G418筛选出稳定混合克隆,提取细胞总RNA,运用RT-qPCR检测其在HepG2细胞中对HBx RNA表达的抑制作用。结果:构建的载体与辅助质粒包装出的慢病毒感染肝癌细胞后,细胞获得G418抗性;HBx RNA干扰序列克隆入该载体可有效抑制肝癌细胞中过量表达的HBx mRNA。结论:构建了具有新霉素抗性筛选标记的RNA干扰慢病毒表达载体,运用该载体可有效筛选出抑制目的基因表达的G418抗性的稳定细胞株。Objective:To construct a lentiviral short hairpin RNA(shRNA)vector with neomycin resistance and investigate its effect on inhibition of hepatitis B virus x gene(HBx)mRNA expression.Methods:The neomycin-resistant coding region was cloned into the lentivirus pLKO shRNA vector deleting the puromycin-resistant coding fragment,then the recombinant vector was packaged with three accessary packaging plasmids into lentivirus which was subjected to infect the liver cancer HepG2 cell lines to observe their response to G418,an inhibitor of protein synthesis.Further,two shRNA sequences targeting HBx cDNA were inserted into the construct and HepG2-X cells overexpressing HBx were infected with the lentivirus containing HBx shRNA vector and their pooled clones were screened with G418 treatment for one week.Lastly,RT-qPCR method was applied to detect the HBx mRNA expression level changes using total RNA extracted from the above pooled clones.Results:The liver cancer HepG2 cell lines gained G418 resistance after infected with lentivirus packaged with the recombinant G418-resistant shRNA vector and HBx mRNA level was significantly reduced with the new construct coding HBx shRNA.Conclusion:The lentivirus shRNA vector with G418 resistant screening marker and the stable cell lines both inhibiting target gene mRNA expression and resisting G418 cell-killing effect may be efficiently screened with the recombinant vector.

关 键 词：G418抗性 慢病毒载体 RNA干扰 乙型肝炎病毒X基因 

分 类 号：Q78[生物学—分子生物学]