KIR 3DL3基因cDNA分子克隆测序法鉴定1个常见型新等位基因

Identification of a common KIR3DL3 novel allele by molecular cDNA cloning and haplotype sequencing

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 A>G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。

Objective To identify a novel KIR3 DL3 allele in southern Chinese Han individuals by complementary DNA（ c DNA） cloning and haplotype sequencing. Methods One sample with inconclusive result was identified in sequencingbased typing on the KIR3 DL3 gene. We isolated the total RNA from the fresh peripheral blood of the individuals and c DNA transcripts were synthesized by RT-PCR. The KIR3 DL3-specific PCR products covering the entire coding sequence of the KIR3 DL3 gene was amplified by one pair of KIR3 DL3-specific PCR primers,the purified PCR products were then subjected to cloning and haplotype sequencing. Results After allele separation by molecular cloning, we identified a normal KIR3 DL3＊01002 allele and a novel variant allele,KIR3 DL3＊04802. Nucleotide sequence alignments with KIR3 DL3 alleles showed that the novel allele KIR3 DL3＊04802 differed from the closest allele KIR3 DL3＊048 by a synonymous mutation at coding sequence（ CDS） nt 1074 A〉G（ codon337 CAA〉CAG） in exon 8. The sequence of this novel allele KIR3 DL3＊04802 was submitted to Gen Bank under the accession number KU529269 and the IPD-KIR Database under the submission number IWS40002178. The name KIR3 DL3＊04802 has been officially assigned by the World Health Organization（ WHO） Nomenclature Committee for factors of HLA system KIR subcommittee. This novel allele was identified in 12 unrelated individuals from southern Han population with a observation ratio of 3. 92%. Conclusion Our results indicate that this particular assay approach utilizing c DNA cloning and the sequencing of KIR3 DL3 gene presents promising applicational significance in KIRassociated studies at an allelic level.