KIR 3DL3基因cDNA分子克隆测序法鉴定1个常见型新等位基因

发布时间：2018-06-18 10:08

  本文选题：KIRDL基因 + cDNA　； 参考：《中国输血杂志》2017年10期

【摘要】：目的建立KIR3DL3基因c DNA分子克隆测序方法,鉴定在南方汉族人群中新发现的1个KIR3DL3等位基因。方法对1例KIR3DL3基因测序分型结果异常的标本,采集EDTA抗凝新鲜外周血样,提取mRNA,反转录成c DNA后,采用1对KIR3DL3基因特异性PCR引物对全部编码区序列做PCR扩增,扩增产物经切胶回收纯化后,做分子克隆和单体型测序。结果经分子克隆和测序,检出1个正常的KIR3DL3*01002等位基因和1个新变异的KIR3DL3等位基因,该新等位基因的序列与KIR3DL3*048最相近,但存在编码区(CDS)nt 1074 AG同义突变,位于第8外显子的第337密码子由CAA变成CAG,其序列提交国际Gen Bank(序列号:KU529269)和IPD-KIR Database(IWS40002178),已被世界卫生组织(WHO)HLA因子命名委员会KIR分委会正式命名为KIR3DL3*04802,该新等位基因在306名南方汉族无关个体中共检出12次,检出频率为3.92%。结论成功建立KIR3DL3基因c DNA分子克隆测序方法,在等位基因水平的KIR研究中具有良好的应用前景。
[Abstract]:Objective to establish a molecular cloning and sequencing method of KIR3DL3 gene and identify a new KIR3DL3 allele in southern Han population. Methods A sample with abnormal sequencing results of KIR3DL3 gene was collected, mRNAs were extracted from fresh peripheral blood samples of EDTA anticoagulant, then reverse transcribed into c DNA. All coding region sequences were amplified by PCR with a pair of KIR3DL3 gene specific PCR primers. The amplified products were recovered and purified by gel, then cloned and sequenced by haplotype. Results by molecular cloning and sequencing, a normal KIR3DL3p01002 allele and a new variant KIR3DL3 allele were detected. The sequence of the new allele was the most similar to that of KIR3DL3p048, but there was a synonymous mutation in the coding region of CDSNT 1074 AG. Codon 337, located at exon 8, changed from CAA to CAG.The sequence was submitted to Gen Bankinternational (serial number: KU529269) and IPD-KIR Database / IWS400021780.It has been officially named KIR subcommittee of WHO WHO HLA naming Committee for HLA factors KIR3DL3C04802. The new allele is located in 306 South China. A total of 12 cases were detected in unrelated individuals of the Fang Han nationality. The detection frequency was 3.92%. Conclusion the method of cDNA cloning and sequencing of KIR3DL3 gene has been successfully established and has a good prospect in allelic KIR studies.
【作者单位】： 深圳市血液中心;大连医科大学;
【基金】：国家自然科学基金(81373158)
【分类号】：R440
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本文编号：2035109