靶向人c-Cbl基因重组干扰慢病毒与过表达腺病毒载体的构建、鉴定以及病毒功效研究  

The Study of Recombinant Interfering Lentiviruses and Overexpressed Adenovirus Vectors Targeting Human c-Cbl Gene:Construction,Identification and Efficacy

作　　者：孙启鑫[1] 吴秉毅 姚倩倩[3] 黄志伟 朱志刚[1] SUN Qi-Xin;WU Bing-Yi;YAO Qian-Qian;HUANG Zhi-Wei;ZHU Zhi-Gang(Deparment of Geriatric Hematology and Oncology,Guangzhou First People's Hospital(The Second ffiliated Hospial of South China University of Technology),Guangzhou 510180,Guangdong Province,China;Deparment of Hematology and Oncology,Cancer Center of Sun Yal-sen University,Guangzhou 510060,Guangdong Province,China;Deparment of Hematology,Zhujiang Hospital of Southem Medical Universiy,Guangzhou 510260,Guangdong Province,China)

机构地区：[1]广州市第一人民医院(华南理工大学第二附属医院)老年血液肿瘤科,广东广州510180 [2]中山大学肿瘤研究中心血液肿瘤科,广东广州510060 [3]南方医科大学珠江医院血液科,广东广州510260

出　　处：《中国实验血液学杂志》2024年第1期274-281,共8页Journal of Experimental Hematology

基　　金：广东省医学科研基金(A2019508);广东省高水平大学建设项目(LC2016ZD026);广州市医学重点学科项目(ZDXK202103)。

摘　　要：目的:构建可调控c-Cbl基因表达的重组慢病毒与腺病毒并评估其功效。方法:应用基因重组技术,分别构建靶向人c-Cbl基因的干扰慢病毒和过表达腺病毒。采用定量PCR和免疫印迹法检测病毒感染后白血病细胞(HL60、THP1)c-Cbl基因表达与转录本的变化。结果:3个靶向人c-Cbl基因的重组干扰慢病毒载体经测序验证构建成功,包装的病毒滴度均大于1×10^(8)TU/ml,其中shRNA-2号慢病毒干扰效率最高,白血病细胞感染后c-Cbl基因的表达约下调95%,CBL蛋白的表达约下调60%;同时,靶向人c-Cbl基因的重组过表达腺病毒载体也经测序验证构建成功,包装的病毒滴度大于1×10^(9)TU/ml,细胞感染腺病毒后,c-Cbl基因表达可瞬时上调约10倍,CBL蛋白表达约上调1.5倍。结论:重组干扰慢病毒和过表达腺病毒均可高效感染白血病细胞,并能分别下调和上调c-Cbl基因与CBL蛋白的表达,为后续研究肿瘤细胞内c-Cbl基因功能打下前期基础。Objective:To construct recombinant lentivirus and adenovirus which regulate the expression of c-Cbl gene and evaluate their efficacy.Methods:The interference lentivirus and overexpressed adenovirus targeting human c-Cbl gene were constructed by gene recombination technology.Quantitative PCR and western blotting were used to detect the expression changes in c-Cbl gene and its transcription after leukemia cells(HL60,THP1)were infected by virus.Results:Three recombinant interfering lentiviral vectors targeting human c-Cbl genes to successfully constructed and were identified by DNA sequencing,and the titers of the packaged viruses were all greater than 1×10^(8) TU/ml.Among them,shRNA-2 lentivirus had the highest interference efficiency,and the expression of c-Cbl gene and CBL protein were decreased about 95%and 60%respectively after leukemia cells were infected with shRNA-2;In addition,the recombinant overexpression adenovirus targeting human c-Cbl gene was packaged successfully with the virus titer greater than 1×10^(9) TU/ml.When leukemia cells were infected with adenovirus,the expression of c-Cbl gene and CBL protein were up-regulated about 10 times and 1.5 times respectively.Conclusion:Both recombinant interfering lentivirus and overexpression adenovirus can efficiently infect leukemia cells and affect the expressions of c-Cbl gene and CBL protein.It will lay a preliminary foundation for the subsequent study on the function of c-Cbl gene in tumor cells.

关 键 词：c-Cbl基因 慢病毒载体 腺病毒载体 

分 类 号：R373[医药卫生—病原生物学]