CX43基因慢病毒载体的构建与鉴定  

Construction and identification of lentiviral vectors carrying the connexin43 gene

作　　者：王亚萍 司英健 封志纯 邢国胜 王艳 章晟 

机构地区：[1]解放军陆军总医院附属八一儿童医院血液肿瘤科,北京100700

出　　处：《中国医师进修杂志》2017年第2期101-106,共6页Chinese Journal of Postgraduates of Medicine

基　　金：国家自然科学基金（31300474）

摘　　要：目的：构建间隙连接蛋白43（CX43）过表达载体，并对其进行鉴定。方法用内切酶EcoRI、XbaI双酶切含目的基因的质粒和慢病毒载体，将目的基因与酶切线性化的慢病毒载体进行连接，构建重组慢病毒表达载体pHBLV-CMVIE-IRES-ZsGreen-CX43；将构建的载体先进行酶切鉴定再对其测序分析，对测序正确的重组载体和包装质粒共同转染293FT细胞，进行病毒包装和生产，收集浓缩病毒液后在293FT细胞中测定病毒滴度，并采用实时定量聚合酶链反应（PCR）对CX43基因表达进行鉴定。结果酶切鉴定和测序分析均证明CX43慢病毒载体构建成功，包装慢病毒，收集浓缩后滴度为1×108/ml，实时定量PCR鉴定CX43基因在293FT细胞的表达明显增加。结论成功构建了稳定过表达CX43的慢病毒载体。Objective To construct and identify lentiviral vectors carrying the connexin43 （CX43） gene. Methods Plasmids containing the CX43 gene and lentiviral vectors were digested using EcoRI/XbaI restriction enzymes, and the target gene fragments were cloned into the lentival vectors to result in CX43 recombinant lentiviral vectors （pHBLV-CMVIE-IRES-ZsGreen-CX43）. CX43 recombinant lentiviral vectors were identified by restriction enzyme digestion and electrophoresis, and sequencing was carried on only for correct vectors after identification. The successfully-constructed CX43 recombinant lentiviral vectors and packaging plasmids were mixed and contransfected into 293FT cell for packagingnbsp;and producting virus. Then the virus was collecting, concentrated and titrated in 293FT cells. Finally, the expression of CX43 gene was assessed by real-time polymerase chain reaction（PCR）. Results The restriction enzyme digestion and electrophoresis and sequencing results proved that CX43 recombinant lentiviral vectors were constructed correctly. Lentiviral concentrated virus suspension titer was 1× 108/ml. The up-regulation expression of CX43 was detected correctly by real-time PCR in 293FT cells. Conclusions Stable lentiviral vectors expressing CX43 gene is constructed successfully.

关 键 词：间隙连接蛋白43 慢病毒载体 骨髓 

分 类 号：R457.7[医药卫生—治疗学]