中国小型猪FasL的cDNA克隆及序列分析

Molecular Cloning and Characterization of Chinese Miniature Pig Fas Ligand cDNA

以广西巴马小型猪活化的外周血淋巴细胞为材料,抽提总RNA,反转录成cDNA.参照人FasL基因保守序列设计引物,扩增得到包括全长阅读框序列的特异性DNA片段.将其克隆于T vector,以双脱氧法完成测序.在国际上首次完成中国小型猪FasL基因序列(GenBank登录号:AY033634).该序列全长846bp,编码282个氨基酸的CD95L分子,为Ⅱ型膜蛋白,其膜内部分富含脯氨酸残基.通过比较分析发现,小型猪与人FasL基因的核苷酸序列同源性为87%,氨基酸序列同源性为88%,比人的FasL基因多一个氨基酸.这些数据将为进一步用小型猪作为实验动物进行生物学和免疫应答研究提供必要的资料.

Fas ligand (FasL) gene of Guangxi Bama Miniature Pig was studied. Peripheral blood lymphocytes were activated in a culture medium containing ionomycin to increase the expression of the Fas ligand gene for RNA extraction and cDNA synthesis. The Fas ligand cDNA was used as a template for polymerase chain reaction (PCR). The PCR products were cloned into pGEMT vector and sequenced. The cDNA sequence has revealed that FasL gene of Guangxi Bama Miniature Pig is an 846 bp long type II transmembrane protein encoding 282 amino acids. The information of this sequence can be retrieved from the GenBank (accession number AY033634). The FasL of miniature pig has one amino acid more than human FasL. The cytoplasmic region is rich in proline residues. The sequence homologue of miniature pig FasL and human FasL is 87% at the nucleotide level and 88% at the amino acid level. These results will provide necessary infarmation for further immunoresponse and cellular apoptosis studies on the miniature pig, which is a very useful animal model for transplantation.