摘要
目的建立可靠的HLA-DRB1等位基因全长序列的分子克隆和测序方法。方法设计、合成HLA-DRB1基因全长序列PCR引物和探索PCR反应体系,采用长距离PCR技术,对10份经PCR产物直接测序、基因型已知的标本,扩增HLA-DRB1基因5'-UTR区、6个外显子、5个内含子和3'-UTR区,全长约11 kb。PCR产物纯化后作长片段分子克隆,筛选阳性克隆,提取质粒DNA,采用自行设计的测序引物测序。结果 PCR扩增获得了特异性目的片段,克隆测序后获得了全部10种等位基因的全长序列。将HLA-DRB1等位基因全长序列划分为9个亚区,群体遗传学分析发现第2外显子的平均核苷酸变异率(π)8.653%,高于其他外显子,并且非同义突变率(dn)9.029%大于同义突变率(ds)7.846%。第5内含子的平均核苷酸变异率高达13.690%,DRB1*09∶01∶02和DRB1*07∶01∶01∶02这2个等位基因第5内含子的序列与其他等位基因序列差别较大。结论采用HLA-DRB1基因全长序列分子克隆及测序方法获得了HLA-DRB1基因各亚区多态性分布特点等基础资料。
Objective To establish the reliable assay for cloning and sequencing full-length HLA-DRB1 al- leles. Methods The genomie full-length HIA-DRB1 gene covering 5 ＇-UTR through 3＇-UTR with 11 kb was amplified using the self-designed PCR primer pairs by long template PCR, purified PCR products was cloned into the pMD19-Tsimple plus- mid vector and the plasmid DNA isolated from positive clones was subjected to haplotype sequencing. A total of 10 samples having been previously-genotyped by PCR sequence based typing were subjected to full length HLA-DRB1 allele cloning and haplotype sequencing. Results The specific target fragment of HLA-DRB1 gene could be amplified and the full-length se- quences of 10 HLA-DRB1 alleles covering from 5 ＇-UTR to 3＇-UTR region could be obtained using our method. The further population genetic study indicated that the π value of exon 2 （8. 653% ） was much more than the other exonic sub-regions, moreover,the dn value （9. 029% ） of exon 2 was more than that of ds（7. 846% ）. The most polymorphic sub-region was in- tron 5 with π value of 13. 690% ,the sequences of intron 5 for DRB1 ＊ 09：01 ： 02 and DRB1 ＊ 07：01 ： 01 ： 02 were distinct from other alleles. Conduslon The technique of cloning and sequencing full-length HIA-DRB1 gene has been established and the polymorphisms of DRB1 sub-regions and its characterizations has been elucidated.
出处
《中国输血杂志》
CAS
CSCD
北大核心
2012年第6期538-542,共5页
Chinese Journal of Blood Transfusion
基金
广东省自然科学基金资助项目(9451803501004124)
