TALENs介导的CCR5△32/△32突变赋予CD4^+U87细胞抵抗HIV-1感染的能力  

TALEN-Mediated CCR5△32/△32 Mutations Endow CD4^+ U87 Cells with the Ability to Resist HIV-1 Infection

作　　者：贺朝勇 HE Chao-Yong(Department of cardiovascular medicine,Taihe Hospital,Habei University of Medicine,Shiyan 442000,Hubei,China)

机构地区：[1]湖北医药学院附属十堰市太和医院心血管内科,湖北十堰442000

出　　处：《中国生物化学与分子生物学报》2018年第10期1073-1079,共7页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：CCR5Δ32/Δ32(CC chemokine receptor type 5,CCR5)基因型的骨髓干细胞移植,可以治愈感染人免疫缺陷病毒-1(HIV-1)的患者。本研究运用TALENs结合同源重组技术产生纯合子的CCR5△32/△32突变,并赋予CD4^+ U87细胞抵抗HIV-1感染的能力。首先,采用重叠延伸PCR合成CCR5△32 donor DNA。构建CCR5-TALENs和CCR5△1-TALENs质粒,将CCR5-TALENs及CCR5△1-TALENs质粒分别结合CCR5△32 donor DNA,共转染野生型CD4^+ U87细胞。其次,用T7E1酶切分析转染后的TALENs和CCR5△1-TALENs打靶效率。通过单克隆培养筛选CCR5△32/△32突变的单克隆细胞。最后,将CCR5△32/△32 CD4^+ U87细胞和野生型CD4^+ U87细胞进行Bal HIV-1病毒攻击实验,采用ELISA方法检测上清中P24抗原含量。测序结果表明,通过重叠延伸PCR成功获得1 602 bp CCR5△32 donor DNA;CCR5-TALENs质粒第1轮、第2轮和第3轮转染,打靶效率分别为14.80%, 38.20%和50.40%;从29个单个细胞培养的克隆中成功筛选出1个CCR5△32/△1基因型CD4^+ U87细胞,CCR5与CCR5△32 donor DNA之间同源重组效率为1.7%;CCR5△1-TALEN质粒第2轮转染,打靶效率为23.5%,从34个单细胞培养的克隆中筛选出3个CCR5△32/△32基因型CD4^+ U87细胞,CCR5与CCR5△32 donor DNA之间同源重组效率达到8.8%。Bal HIV-1攻击实验表明,野生型CD4^+ U87细胞培养上清第2、4、6、8、10、12 d,平均P24抗原含量分别为58.47±2.35、162.23±4.78、458.78±27.34、613.35±26.78、580.35±24.73、483.34±30.85 pg/mL。而CCR5△32/△32基因型CD4^+U87细胞的培养上清平均P24抗原含量分别为11.30±1.76、5.13±0.88、3.43±0.44、3.84±0.69、3.21±0.44、4.24±0.46 pg/mL。本研究表明,TALENs结合同源重组技术无缝隙地介导了CCR5△32/△32突变,并赋予了CD4^+ U87细胞抵抗HIV-1感染的能力。Transplantation of bone marrow stem cells with the CC chemokine receptor type 5 （CCR5） △32/△32 genotype can cure patients infected with human immunodeficiency virus 1 （ HIV-1 ）. In this study, transcription activator-like effector nucleases （TALENs） combined with the homologous recombination technique was used to generate homozygous CCR5 △32/△32 mutations and endowed CD4± U87 cells with the ability to resist H1V-1 infection. Firstly, CCR5 △32 donor DNA was synthesized by overlapping extension PCR. CCRS-TALENs and CCR5 △1- TALENs plasmids were constructed. CCRS-TALENs and CCR5 △1- TALENs plasmids were co-transfected. CCR5-TALENs and CCRS△1-TALENs plasmids combined with CCR5 △32 donor DNA were cotransfected into wild-type CD4± U87 cells, respectively. Secondly, after transfection, the targeting efficiency of CCR5-TALENs and CCR5 △1-TALENs were analyzed by the T7E1 endonuclease. Lastly, monoclonal cells with CCR5 △32/△32 mutations were screened by the monoclonal culture. Both the wild-type CD4± U87 ceils and those with CCR5 △32/△32 genotype were challenged with Bal HIV-1 for 4 hours. The mean levels of P24 antigens in the supernatants were measured by ELISA analyses. Sequencing results demonstrated that CCR5△32 donor DNA of 1602-bp length was successfully obtained by overlap extension PCR. The targeting efficiency of CCRS-TALENs after first, second and third round of transfection were 14. 80% , 38.20% and 50.40% , respectively. The one CD4± U87 cell out of 29 single-cell cultured clones from the transfectcd cells were screened out with CCR5 △32/△1 genotype. Targeting efficiency of CCR5 △1- TALENs after second transfection was 23.5%. The three CD4± U87 cells out of 34 single-cell cultured clones from the transfection cells were screened out with CCR5 △32/△32 genotype, while the homologous recombination rate between CCR5 and the CCR5 △32 donor DNA also occurred considerably high as 8.8%. Bal HIV-1 attack experiment shows that the mean P24 levels determined at

关 键 词：同源重组 基因编辑 单克隆培养 

分 类 号：R739.86[医药卫生—肿瘤]