KIR2DL1框架基因cDNA的分子克隆测序及一个新等位基因的鉴定

Cloning and sequencing of KIR2DL1 framework gene cDNA and identification of a novel allele

目的建立K工R2DLI框架基因cDNA的分子克隆测序方法，并鉴定南方汉族人群一个新的KIR2DL1等位基因。方法对-K豫2DL1框架基因测序分型结果异常的外周血样，提取mRNA，反转录为cDNA，用1对KIR2DLl框架基因特异性PCR引物进行扩增，特异性扩增产物（1．2kb）经切胶回收纯化后，进行分子克隆和单倍型测序。结果KIR2DL1特异性PCR扩增产物，经分子克隆和单倍型测序，检出1个正常的K／R2DL1＊00302等位基因和1个新变异的KIR2DL1等位基因。该新等位基因的序列与KIR2DL1＊00302最相近，但存在编码区nt 188A〉G点突变、位于第4外显子的第42密码子由GAG变成GGG，导致第42位氨基酸残基发生GIu42Gly改变；其序列提交GenBank（序列号：KP025960）和IPD-KIR数据库（IWS40001982），已被世界卫生组织HLA因子命名委员会KIR分委会正式命名为KIR2DL1＊031。结论我们建立了KIR2DL1框架基因cDNA分子克隆测序方法，在等位基因水平的KIR研究中具有广泛的应用前景。

Objective To develop an assay for cDNA cloning and haplotype sequencing of KIR2DL1 framework gene and determine the genotype of an ethnic Han from southern China. Methods Total RNA was isolated from peripheral blood sample, and complementary DNA （cDNA） transcript was synthesized by RT-PCR. The entire coding sequence of the KIR2DLI framework gene was amplified with a pair of KIR2DL1 -specific PCR primers. The PCR products with a length of approximately 1. 2 kb were then subjected to cloning and haplotype sequencing. Results A specific target fragment of the KIR2DL3 framework gene was obtained. Following allele separation, a wild-type KIR2DL1 ＊00302 allele and a novel variant allele, KIR2DL1 ＊ 031, were identified. Sequence alignment with KIR2DL1 alleles from the IPD- KIR Database showed that the novel allele KIR2DL1 ＊ 031 has differed from the closest allele KIR2DL1 00302 by a non-synonymous mutation at CDS nt 188A〉G （codon 42 GAG〉GGG） in exon 4, which has caused an amino acid change Glu42Gly. The sequence of the novel allele KIR2DL1 ＊ 031 was submitted to GenBank under the accession number KP025960 and to the IPD-KIR Database under the submission number IWS40001982. A name KIR2DL1 ＊ 031 has been officially assigned by the World Health Organization （WHO） Nomenclature Committee. Conclusion An assay for cDNA cloning and haplotype sequencing of KIR2DL1 has been established, which has a broad applications in KIR studies at allelic level.