人类白细胞抗原-G5过表达慢病毒载体的构建以及JAR-HLA-G5稳转细胞株的建立  

Construction of human leukocyte antigen-G5 overexpression lentiviral vector and establishment of JAR-HLA-G5 stable cell line

作　　者：曹维维 袁成良[2] CAO Wei-wei;YUAN Cheng-liang(College of Medical Technology,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,Sichuan Province,China;Department of Laboratory,Deyang People’s Hospital,Deyang 618000,Sichuan Province,China)

机构地区：[1]成都中医药大学医学技术学院,四川成都611137 [2]德阳市人民医院检验科,四川德阳618000

出　　处：《中国临床药理学杂志》2021年第18期2495-2498,共4页The Chinese Journal of Clinical Pharmacology

摘　　要：目的构建过表达人类白细胞抗原G5(HLA-G5)慢病毒载体,筛选建立稳定表达HLA-G5的JAR稳转细胞株。方法根据HLA-G5基因序列设计合成引物并使用聚合酶链反应(PCR)扩增目的基因片段。将目的基因定向接入经BamHI/AgeI酶切的载体质粒GV492中构建重组慢病毒质粒GV492-HLA-G5。筛选阳性克隆和测序鉴定后,使用三质粒包装系统将含有目的基因的重组质粒GV492-HLA-G5和辅助包装质粒共转染至293T细胞进行病毒包装及纯化并测定病毒滴度。设置过表达实验(OE)组和阴性对照(NC)组,在感染复数为20的条件下感染JAR细胞。用荧光显微镜观察各组绿色荧光蛋白的表达情况。用浓度为2μg·mL^(-1)嘌呤霉素筛选出稳定表达HLA-G5的JAR细胞株。用qRT-PCR和Western blot法检测JAR细胞中HLA-G5的mRNA和蛋白表达水平。结果经PCR及测序鉴定证明,序列与设计序列一致,成功构建重组慢病毒载体。病毒滴度结果显示,OE组的滴度为2×108TU·mL^(-1),NC组的滴度为4×108TU·mL^(-1)。qRT-PCR显示OE组JAR细胞中HLA-G5的mRNA(108.69±4.25)表达水平较NC组(1.00±0.76)明显增高(P<0.01)。Western blot显示OE组JAR细胞中HLA-G5的蛋白(65.84±14.11)表达水平较NC组(1.35±0.37)明显升高(P<0.01)。结论成功构建HLA-G5慢病毒载体并筛选建立JAR-HLA-G5稳转细胞株。ObjectiveTo construct a lentiviral vector of overexpressing human leukocyte antigen-G5(HLA-G5)and to screen and establish a stably JAR-HLA-G5 cell line expressing HLA-G5.Methods The primers were designed and synthesized according to the sequence of HLA-G5 gene and the target gene fragment was amplified by polymerase chain reaction(PCR).The recombinant lentiviral vector GV492-HLA-G5 was constructed by inserting the target gene into the vector GV492 digested by Bam HI/Age I.After the positive clones were screened by PCR and identified by sequencing,the recombinant plasmid GV492-HLA-G5 containing the target gene and the auxiliary packaging plasmid were co-transfected into 293T cells for virus packaging and purification and the virus titer was determined.The overexpression experimental(OE)group and negative control(NC)group were set up.JAR cells were infected under the condition of multiplicity of infection was 20.The expression of green fluorescent protein was observed by fluorescence microscope.Puromycin at a concentration of 2μg·m L-1was used to screen JAR cell lines stably expressing HLA-G5.The mRNA and protein expression of HLA-G5 in JAR cells were detected by qRT-PCR and Western blot.Results The recombinant lentiviral vector was successfully constructed.The virus titer of OE group was 2×108TU·m L-1.The titer of NC group was 4×108TU·m L-1.qRT-PCR showed that the mRNA level of HLA-G5 in JAR cells of OE group(108.69±4.25)was higher than that in NC group(1.00±0.76,P<0.01).Western blot showed that the expression of HLA-G5 protein in JAR cells of OE group(65.84±14.11)was higher than that in NC group(1.35±0.37,P<0.01).Conclusion The lentiviral vector of HLA-G5 was successfully constructed and the stable cell line of JAR-HLA-G5 was established.

关 键 词：人类白细胞抗原G5 慢病毒载体 人绒毛膜癌细胞 类风湿关节炎 

分 类 号：R972[医药卫生—药品]