PCR-SSP结合测序分析快速筛选HLA-A*0201亚等位基因  

Rapid Screening of HLA-A*0201 by PCR-SSP and Sequencing Analysis

作　　者：郭薇[1] 张建琼[1] 沈传来[1] 孟凡岩[1] 谢维[1] 

机构地区：[1]江苏省基因诊断与治疗重点实验室"HLA表达与肿瘤"国际参考实验室东南大学医学院遗传学研究中心,南京210009

出　　处：《上海免疫学杂志》2003年第4期240-243,共4页Shanghai Journal of Immunology

基　　金：教育部留学回国人员启动基金资金项目 (教外司留2 0 0 1/3 45 ) ;国家自然科学基金资助项目 (No 3 0 2 712 16)

摘　　要：建立相对简单经济的检测HLA A 0 2 0 1等位基因的方法 ,以便于从特定人群中快速筛选HLA A 0 2 0 1阳性的个体 ,满足科研的需要。根据第十二届HLA国际联合会提供的标准操作规程和一对HLA A位点特异性的引物 ,从外周血中提取基因组DNA ,扩增HLA A基因 ;再设计两对HLA A2组特异性引物 ,采用套式PCR分别扩增HLA A基因的 5’端和 3’端 ,如出现特异条带 ,则将后两对引物的PCR产物进行测序分析 ,对照HLA A2组等位基因的第 2和第 3外显子序列图 ,确定是否为HLA A 0 2 0 1 1～ 0 2 0 1 6的 6个等位基因。结果 :以HLA A 0 2 0 1阳性的T2细胞株为阳性对照进行检测 ,结果完全正确 ,并设立非HLA A 0 2 0 1的A2细胞株RML (HLA A 0 2 0 4 )及Raii细胞株 (非A2组 )为对照 ,同时随机检测 30份门诊病人血样 ,其中 7份为HLA A2阳性 ,经测序 ,2例为HLA A 0 2 0 1 1 ,1例为HLA A 0 2 1 1或 0 2 35 ,1例是HLA A 0 2 0 6或0 2 2 1或 0 2 4 1或 0 2 4 4或 0 2 5 1或 0 2 5 4 ,1例是HLA A 0 2 0 5或 0 2 1 4 ,1例是HLA A 0 2 34或 0 2 35 ,1例是 0 2 5 0。本方法利用位点特异性引物进行 2～ 3次PCR扩增 ,结合PCR产物测序便可相对简便经济地检测出HLA A 0 2 0 1的全部 6个亚等位基因 ,为快速检测其他特定HLA等位基因?To develop a simple and comprehensive method to detect the HLA A*0201 allele for the rapid screening of HLA A*0201 positive individuals, the genome DNA from PBMC was extracted according to the standard procedures of the 12th WHO HLA Union Committee with a pair of specific primers for HLA A loci; and then the HLA A alleles were amplified by nested PCR with two pairs of HLA A2 group specific primers to amplify the 5’ and 3’ termini of the HLA A genes As confirmed by DNA sequencing of the exon 2 and exon 3, this allele was identical to HLA A*02011～02016 By using this method, with HLA A*0201 positive T2 cell clone as positive controls and the non HLA A*0201 A2 clone RML(HLA A*0204) and Raji cell clone(non A2) as negative controls, 30 clinical blood samples were randomly selected for testing and the results showed that among these samples, 7 were HLA A2 positive, of which 2 were HLA A 02011; 1 was HLA A*0211 or A 0235, 1 was HLA A*0206, A*0221,A*0241, A*0244,A*0251 or A*0254; 1 was HLA A*0205 or A*0214; 1 was HLA A*0234 or A*0235 and the last was HLA A*0250 It concludes that this simple and cheap method can detect out all the 6 subtypes of HLA A alleles with implication for the selection of HLA A*02011～02016, and it is quite useful in basic and clinical researches.

关 键 词：HLA分型 PCR-SSP 测序 

分 类 号：R392.4[医药卫生—免疫学]