应用新型假包膜糖蛋白提高慢病毒感染人T细胞效率的研究  

Preliminary study on application of novel pseudo-envelope glycoproteins to improve efficiency of lentivirus infection in human T cells

作　　者：张伟光 王露 尹昌林 ZHANG Weiguang;WANG Lu;YIN Changlin(Department of Critical Care Medicine,Southwest Hospital/the First Affiliated Hospital of the Army Military Medical University,Chongqing 400038,China)

机构地区：[1]陆军军医大学第一附属医院/西南医院重症医学科,重庆400038

出　　处：《重庆医学》2023年第8期1121-1127,共7页Chongqing medicine

基　　金：国家自然科学基金项目(81573110)。

摘　　要：目的利用基因工程方法构建一种新型假包膜糖蛋白表达载体来提高慢病毒感染人T细胞的效率。方法采用分子克隆技术构建CD5-scFv-VSV-G质粒;慢病毒包装载体共转染293T细胞,包装慢病毒并感染Jurkat细胞,流式细胞术检测转导率并计算其滴度;慢病毒包装载体共转染293T细胞,包装慢病毒感染健康人T细胞获得嵌合抗原受体T细胞。结果与野生型WT VSV-G慢病毒比较,CD5-scFv-VSV-G慢病毒能够提高感染率1.5倍(10%的CD5-scFv-VSV-G慢病毒对Jurkat细胞的感染率为63.1%),而在293T细胞中,不同比例CD5-scFv-VSV-G慢病毒的感染率都低于野生型WT VSV-G慢病毒。当感染复数(MOI)=5时,与野生型WT VSV-G慢病毒比较,CD5-scFv-VSV-G慢病毒能够提高感染率1.6倍(33%的CD5-scFv-VSV-G慢病毒对CD3+T细胞的感染率为44.1%);当MOI=10时,与野生型WT VSV-G慢病毒比较,CD5-scFv-VSV-G慢病毒也能够提高感染率1.5倍(33%的CD5-scFv-VSV-G慢病毒对CD3+T细胞的感染率为78.8%)。结论该研究成功构建了一种新型假包膜糖蛋白表达载体,与野生型慢病毒WT VSV-G比较,新型假包膜糖蛋白包装的慢病毒可提高其转导人T细胞效率。Objective To use the genetic engineering method to construct a new type of pseudo-envelope glycoprotein expression vector to improve the efficiency of lentivirus infection in human T cells.Methods The CD5-scFv-VSV-G plasmid was constructed by the molecular cloning technology;the lentiviral packaging vectors co-transfected 293T cells,packaged the lentivirus and infected the Jurkat cells,the flow cytometry was used to detect the transduction rate and calculate the titers;the lentiviral packaging vectors co-transfected with 293T cells to obtain the chimeric antigen receptor T cells from lentiviral infection in healthy person.Results Compared with the wild-type WT VSV-G lentivirus,CD5-scFv-VSV-G lentiviral particles could increase the infection rate by 1.5 times(the infection rate of 10%of CD5-scFv-VSV-G lentiviral particles on CD5+Jurkat cells was 63.1%),while in 293T cells,the infection rates of viral particles produced by different proportions of CD5-scFv-VSV-G all were lower than that of WT VSV-G lentivirus particles.When the multiplicity of infection(MOI)was 5,compared with the wild type WT VSV-G,the CD5-scFv-VSV-G lentivirus particles could increase the infection rate by 1.6 times(the infection rate of 33%of CD5-scFv-VSV-G lentivirus particles on the CD3+T cells was 44.1%);when MOI=10,the CD5-scFv-VSV-G lentivirus particles could increase the infection rate by 1.5 times(the infection rate of 33%of CD5-scFv-VSV-G lentivirus particles on CD3+T cells was 78.8%).Conclusion This study successfully constructs a new type pseudo-envelope glycoprotein expression vector.Compared with the wild-type WT VSV-G lentivirus,the new type pseudo-envelope glycoprotein packaged lentivirus could increase its transduction efficiency of human T cells.

关 键 词：慢病毒 基因治疗 假包膜糖蛋白 嵌合抗原受体T细胞 

分 类 号：R392-3[医药卫生—免疫学]