摘要
目的构建稳定表达人ABO~*A1.01等位基因的K562细胞株。方法在载体GV492(Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin)基础上构建ABO~*A1.01等位基因蛋白质编码区(coding sequence,CDS)全长重组质粒GV492-A,将慢病毒重组载体、2种慢病毒包装载体pHelper1.0和pHelper2.0共转染293T细胞做病毒包装;将重组慢病毒感染人慢性髓细胞白血病K562细胞,用嘌呤霉素筛选得到K562-A稳定株,荧光显微镜下观察K562-A稳定株;qPCR检测K562-A稳定株中ABO等位基因mRNA表达水平;对稳定株中的ABO等位基因测序;用流式细胞术检测稳定株细胞表面A抗原的表达情况。结果重组载体测序结果证实成功构建了ABO~*A1.01等位基因重组表达载体;包装慢病毒的滴度适中:ABO~*A1.01等位基因重组慢病毒为1.5E+9 TU/mL,载体GV492对照慢病毒为5.0E+8 TU/mL,说明重组慢病毒包装成功。重组慢病毒载体感染K562细胞后,得到稳定感染细胞株。K562-A稳定株的qPCR检测显示ABO等位基因的mRNA表达水平明显升高(P<0.01),GFP荧光蛋白阳性率>98%,ABO等位基因测序证实ABO~*A1.01等位基因在细胞内成功转录,流式检测显示细胞表面表达A抗原。结论成功构建表达ABO~*A1.01等位基因的K562-A细胞稳定株,细胞表面表达A抗原。
Objective To establish a K562 cell line with stable ABO~*A1.01 allele expression. Methods The full length of coding sequence of ABO~*A1.01 allele was cloned into the lentivirus vector GV492.The recombinant lentivirus vector GV492-A was cotransfected with pHelper1.0 and pHelper2.0 vectors into 293 T cells for packaging the virus, and packaged virus was transfected into human chronic myelogenous leukemia K562 cells. The stable cell lines(K562-A) was acquired by selecting with puromycin, and cell images was captured using a fluorescence microscope. The cellular expression of ABO gene mRNA was assessed by quantitative PCR. Sanger sequencing was used to ascertain the sequence of ABO allele in the K562-A stable cell line. Flow cytometry was used to detect the blood group A antigen on the cell surface. Results The sequencing results confirmed that the recombinant lentivirus vector of ABO~*A1.01 allele was successfully constructed. The titer of the ABO~*A1.01 allele recombinant lentivirus was 1.5 E+9 TU/mL and the titer of the GV492 lentivirus was 5 E+8 TU/mL, which manifested that the viruses were successfully packaged. After transfecting with recombinant lentivirus vector, a stable K562-A cell line was acquired. The rates of green fluorescent of K562-A achieved 98%.Quantity PCR results showed that the expression levels of ABO allele mRNA was significantly increased in K562-A(P<0.01). Sequencing results confirmed that the ABO~*A1.01 allele was expressed in K562-A cells. Flow cytometry results showed that the blood group A antigen was detectable on the surface of K562-A cells. Conclusion A K562 cell line(K562-A) with stable expression of ABO~*A1.01 allele and blood group A antigen expression on the surface has been established successfully.
作者
邹敏
陈鸿天
刘紫葳
赵晖
孔文兵
吕飘
刘持翔
周华友
ZOU Min;CHEN Hongtian;LIU Zi-wei;ZHAO Hui;KONG Wenbing;LV Piao;LIU Chixiang;ZHOU Huayou(Department of Blood transfusion,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China)
出处
《中国输血杂志》
CAS
2018年第12期1362-1367,共6页
Chinese Journal of Blood Transfusion
基金
南方医科大学南方医院人才引进科研启动经费(R10101009)
