一种新的突变型人血小板内皮细胞黏附因子1的基因克隆与序列分析

Gene cloning and sequence analysis of a new mutant type human platelet-endothelial cell adhesion molecule-1

目的克隆一种新的突变型人血小板内皮细胞黏附因子1(PECAM1)基因,并应用生物信息学方法进行序列分析。方法以提取的X射线辐射诱导的人鼻咽癌耐药细胞CNE1/R中的总RNA为模板,RT-PCR扩增获得PECAM1基因片段,克隆至pMD19-TSimple载体并测序鉴定。应用生物信息学方法分析PECAM1基因的序列同源性与结构特征。结果成功克隆了突变型人PECAM1基因,但与GenBank中的人PECAM1基因参考序列相比有三点不一致,分别位于1452、1688和2008nt处。与马、猪、牛、大鼠、小鼠的PECAM1基因具有高度同源性,同源性分别为82%、82%、79%、73%、73%。突变序列与参考序列相比,1688和2008nt翻译对应的氨基酸发生变化,即563、670aa分别由丝氨酸、精氨酸改变为天冬酰胺、甘氨酸。人PECAM1蛋白39～127aa、235～322aa、327～404aa、408～496aa和502～594aa均为Ig-2免疫球蛋白超家族结构域,而突变型人PECAM1基因编码的563aa突变刚好位于502～594aa的Ig-2免疫球蛋白超家族结构域中。结论突变型人PECAM1基因在基因水平相较人PECAM1基因参考序列有3个位点改变,应用生物信息学方法进行序列分析,发现在蛋白水平改变导致的563aa突变位于Ig-2免疫球蛋白超家族结构域,这为进一步研究其生物学功能及其在X线照射后人鼻咽癌细胞CNE1/R产生多药耐药中的作用提供了依据和线索。

Objective To clone a new mutant type of human PECAM1 gene,and apply bioinformatics methods for sequence analysis.Methods The DNA fragment of PECAM1 gene was amplified by reverse transcription polymerase chain reaction（RT-PCR）with mRNA from CNE1/R cells as the template,and then was cloned into pMD19-T simple vector.After sequencing,the correct DNA fragment was identified.Bioinformatics technique was used to analyze the gene sequence homology and structural feature of PECAM1.Results The mutant type of human PECAM1 gene was cloned successfully.But comparing the cloned PECAM1 gene with the reference sequence in NCBI GeneBank,there were three inconsistencies,which were located at No.1452,1688 and 2008 nt,respectively.The cloned PECAM1 gene was highly homologous to that of horse（NM_001101655）,pig（NM_213907）,cattle（NM_174571）,rat（NM_031591）and mouse（NM_008816）in GenBank with the homology of 82%,82%,79%,73% and 73%,respectively.Comparing with the reference sequence,No.1688 and 2008 nt of the mutant type changed,correspondingly the post-translational amino acid changed,namely No.563 aa and 670 aa changed to asparagine and glycine by serine and arginine,respectively.Human PECAM1 gene reference protein sequence 39-127 aa,235-322 aa,327-404 aa,408-496 aa and 502-594 aa are all Ig-2 immunoglobulin superfamily domains,and the No.563 aa of the mutant type of human PECAM1 gene protein sequence is just located in the 502-594 aa of Ig-2 immunoglobulin superfamily domains.Conclusions Compared to human PECAM1 gene reference sequence,three nucleotides of the mutant type of human PECAM1 gene changed at the genetic level.Sequence analysis with bioinformatics technique showed that the changes of nucleotide induced No.563 aa mutation,which was just located in Ig-2 immunoglo-bulin superfamily domains.The changes may affect its function,and it provides some evidences and clues for the further study on biological functions of human PECAM1 gene,and on its possible roles in the multi-drug resistance effect of CNE1/R cells.