CAJ | 학술논문

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基因非翻泽区(untranslated region,5'-UTR)区、8个外显子、7个内含子和3'-UTR区,全长约4.5 kb.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AlleleSEQR HLA-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TaKaRa LATaq酶和Stratagene Pfu Taq DNA聚合酶进行HLA-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LA Taq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.
목적 건립가고적HLA-Cw기인전장서렬적분자극륭화측서기술.방법 설계합성HLA-Cw기인전장서렬PCR인물화탐색PCR반응체계,채용장거리PCR기술,확증HLA-Cw기인비번택구(untranslated region,5'-UTR)구、8개외현자、7개내함자화3'-UTR구,전장약4.5 kb.PCR산물순화후진행분자극륭,사선양성극륭,제취질립DNA,채용자행설계적측서인물진행전장쌍향측서.12빈이경AlleleSEQR HLA-Cw측서분형시제합진행PCR산물직접측서、기인형이지적양본,분별용TaKaRa LATaq매화Stratagene Pfu Taq DNA취합매진행HLA-Cw기인전장확증,이급PCR산물분자극륭화서렬측정,극륭측서결과분별여PCR산물직접측서결과진행대비분석.결과 PCR확증획득료특이성목적 편단,측서획득료HLA-Cw기인-962～3576위감기전장서렬.극륭측서결과적대비표명,Pfu매보진성고우LA Taq매.비교본문측정적Cw*010201여Cw*07020101등위기인서렬,재5'상유-962～-284위감기구역존재11개단핵감산다태(single nucleotide polymorphisms,SNPs)화2개삽입(혹)결실다태성위점;3'-UTR하유3067～3576위감기구역존재11개SNPs화1개삽입(혹)결실.결론 건립료HLA-Cw기인전장서렬분자극륭급측서방법,재HLA-Cw기인전장서렬분자다태성급표체조공등연구영역,구유엄범응용전경.
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 (5f-UTR) to nueleotide 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 HLA-Cw gene polymorphism study and the regulation and expression of HLA-Cw gene.