DF-1细胞MHC单倍型的分子鉴定

Molecular identification of MHC haplotype in DF-1 cells

为了鉴定鸡胚成纤维细胞(DF-1细胞)主要组织相容性复合体(major histocompatibility complex,MHC)单倍型,试验选取生长状态良好的DF-1细胞,提取其总RNA并将总RNA反转录成cDNA;以DF-1细胞cDNA为模板,采用PCR方法扩增鸡MHC B基因座的微卫星LEI0258和MCW0371及BF2、BLB2基因并测序,同时采用“PCR+1”方法扩增BF2基因并测序,以根据上述序列及结构特征判定DF-1细胞的MHC单倍型。结果表明:微卫星LEI0258和MCW0371序列长度分别为357,205 bp,其中微卫星LEI0258含有1个CTATGTCTTCTTT重复单元和16个CTTTCCTTCTTT重复单元,与MHC B基因座B130、B131、B201、B5.1、B6.1、B21、B75单倍型的微卫星长度、重复单位的结构和数量完全相同;BF21基因序列长度为1117 bp,与GenBank收录3条鸡MHC B21单倍型的BF2基因序列(登录号分别为AF013493.1、S78682.1、NM-001031338.2)完全一致。说明DF-1细胞来源于MHC B21单倍型纯合鸡胚。

In order to identify MHC haplotype of chicken embryo fibroblasts(DF-1 cell),DF-1 cells with good growth status were selected in the experiment to extract total RNA,and the total RNA was reverse transcribed into cDNA.Using DF-1 cell cDNA as a template,the microsatellites LEI0258 and MCW0371 of chicken MHCB locus and BF2、BLB2 genes were amplified and sequenced using PCR method;BF2 gene was amplified and sequenced using the“PCR+1”method simultaneoustly,and determine the MHC haplotype of DF-1 cells based on above sequences and their structural characteristics.The results showed that the length of microsatellite LEI0258 and MCW0371 sequences were 357 bp and 205 bp,respectively,and microsatellite LEI0258 contained one CTATGTCTTCTTT repeating Unit and 16 CTTTCCTTCTTT repeating Units,which were exactly the same as the microsatellite length,repeating unit structure,and quantity of MHC-B locus B130,B131,B201,B5.1,B6.1,B21,and B75 haplotypes.The length of the BF21 gene sequence was 1117 bp,which was completely consistent with the BF2 sequences(registration numbers AF013493.1,S78682.1,NM-001031338.2)from all three B21 haplotype sources included in GenBank.The results indicated that DF-1 cells were derived from B21 haplotype homozygous chicken embryos.