HLA-Cw基因全长序列分子克隆及测序方法的建立

Establishment of an assay for cloning and sequencing the full-length HLA-Cw gene

目的建立可靠的HLA-Cw基因全长序列的分子克隆和测序技术。方法设计合成HLA-Cw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，扩增HLA-Cw基因非翻译区（untranslatedregion，5′-UTR）区、8个外显子、7个内含子和3′-UTR区，全长约4．5kb。PCR产物纯化后进行分子克隆，筛选阳性克隆，提取质粒DNA，采用自行设计的测序引物进行全长双向测序。12份已经AlleleSEQRHI。A-Cw测序分型试剂盒进行PCR产物直接测序、基因型已知的样本，分别用TaKaRaLATaq酶和StratagenePfuTaqDNA聚合酶进行HI，A-Cw基因全长扩增，以及PCR产物分子克隆和序列测定，克隆测序结果分别与PCR产物直接测序结果进行对比分析。结果PCR扩增获得了特异性目的片段，测序获得了HLA-Cw基因-962～3576位碱基全长序列。克隆测序结果的对比表明，Pfu酶保真性高于LATaq酶。比较本文测定的Cw＊010201与Cw＊07020101等位基因序列，在5′上游-962～-284位碱基区域存在11个单核苷酸多态（single nucleotide polymorphisms，SNPs）和2个插入（或）缺失多态性位点；3′-UTR下游3067～3576位碱基区域存在11个SNPs和1个插入（或）缺失。结论建立了HLA—Cw基因全长序列分子克隆及测序方法，在HLA—Cw基因全长序列分子多态性及表达调控等研究领域，具有广泛应用前景。

Objective To establish a reliable assay for cloning and sequencing the full-length HLA- Cw gene. Methods In this study, a fragment of 4.5 kb full-length HLA-Cw gene was amplified using the self-designed PCR primer pair by long template PCR, purified PCR products was cloned into the pGEM- Teasy plasmid vector and the plasmid DNA isolated from positive clones was subjected to haplotype sequencing by both directions. A total of 12 samples having been previously-genotyped by PCR sequence- based-typing （PCR SBT） were amplified by using the TaKaRa LA Taq and Stratagene Pfu polymerase, respectively. PCR products of full length HLA-Cw gene were subjected to cloning and sequencing and the obtained haplotype sequence were compared with the PCR-SBT results. Results The specific target fragment of HLA-Cw gene could be amplified and the full-length HLA-Cw allele sequence covering from nucleotide position 962 in 5′untranslated region （5′-UTR） to nucleotide position 3576 in downstream area of 3′ UTR region could be obtained using our method. The results of cloning and sequencing analysis indicated that the Stratagene Pfu polymerase had better fidelity than the TaKaRa LA Taq polymerase in this experiment. By comparing the sequences of Cw＊ 07020101 with Cw＊ 010201, 11 SNPs as well as 2 insertions/ deletions in nt-962--284 of 5′-UTR, and 11 SNPs as well as 1 insertion/deletion in nt3067-3576 downstream of 3′-UTR were identified. Conclusion Our results indicate that the technique for cloning and sequencing full-length HLA-Cw gene has been established, it has a broad application in full-length HI.A-Cw gene polymorphism study and the regulation and expression of HLA-Cw gene.