摘要
目的研制人类白细胞抗原(human leukocyte antigen,HLA)组织配型基因分型国家参考品。方法采集45份志愿者的新鲜外周血样本,经EB病毒转化,培养建立永生化细胞系。提取细胞系基因组DNA,分光光度计测定A_(260/280);细胞系基因组DNA反复冻融3次后,进行琼脂糖凝胶电泳分析;采用HLA-A、HLA-B、HLA-C、HLA-DRB1、HLADQB1位点基因高分辨分型试剂盒(测序法)进行全外显子基因分型,并与建系前外周血基因组DNA的分型信息进行比较。将检测结果符合要求的样本组成国家参考品,采用高分辨基因测序分型法(sequence-based typing,PCR-SBT)进行稳定性和均匀性验证。结果共获得38株永生化HLA细胞系,基因组DNA的A_(260/280)在1.80-2.15之间;琼脂糖凝胶电泳分析均未见拖带或弥散带,条带完整清晰;分型结果与建系前外周血基因组DNA的分型信息一致。以上述38份基因组DNA样本制备的HLA组织配型基因分型国家参考品,具有良好的均匀性和稳定性。结论成功建立了HLA组织配型基因分型国家参考品,可用于基因分型类检测试剂盒的质量评价。
Objective To prepare the national reference materials for typing of human leukocyte antigen(HLA) tissue matching genes.Methods Fresh peripheral blood samples were collected from volunteers and transformed by EB virus to establish immortalized cell lines.The genomic DNA was extracted from the cells and determined for A_(260/280) by spectrophotometer,then subjected to three cycles of freeze-thawing,and analyzed by agarose gel electrophoresis.Whole exon genotyping was carried out by using high resolution typing kits for HLA-A,HLA-B,HLA-C,HLA-DRB1 and HLA-DQB1 locus genes,and the results were compared with those of typing data of peripheral blood genomic DNA before establishment of the cell line.The national reference material was composed of the samples with qualified test results,and verified for stability and homo-geneity by high resolution gene sequence-based typing(PCR-SBT).Results A total of 38 immortalized HLA cell lines were obtained,of which the A_(260/280) of genomic DNA were 1.80 - 2.15.No tailing or diffuse bands were observed on agarose gel electrophoretic profile,and all the bands were intact and clear.The typing results were consistent with those of typing data of peripheral blood genomic DNA before establishment of the cell line.The national reference materials composed of the 38 samples showed good homogeneity and stability.Conclusion The national reference materials for typing of HLA tissue matching genes was successfully established,which might be used for the quality evaluation of genotyping kits.
作者
胡泽斌
高飞
刘湘
兰更欣
李丽莉
黄杰
HU Ze-bin;GAO Fei;LIU Xiang;LAN Geng-xin;LI Li-li;HUANG Jie(National Institutes for Food and Drug Control,Beijing 100050,China;不详)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2021年第10期1212-1215,共4页
Chinese Journal of Biologicals
基金
国家科技重大专项(2018ZX10732-401-003-009)。
关键词
人类白细胞抗原
组织配型
基因分型
高分辨基因测序分型法
国家参考品
Human leukocyte antigen(HLA)
Tissue matching
Genotyping
High solution gene sequence-based typing
National reference materials
