绵羊CD58基因的克隆与原核表达

Cloning and prokaryotic expression of sheep CD58 gene

通过对猪、绵羊CD58mRNA序列比对,设计出绵羊CD58的特异性引物,应用反转录PCR技术从绵羊淋巴细胞mRNA中扩增出绵羊CD58基因,将该基因克隆到原核表达载体pGEX4-1中进行原核表达.结果表明:克隆出的绵羊CD58cDNA长807bp,其中包含了开放式阅读框;该基因在大肠杆菌中进行表达,SDS-PAGE电泳在53ku处能观察到特异表达的融合蛋白,产物通过Western-blot检测证实,可以被CD58单克隆抗体识别.

CD58 plays an important role in mammal immune system.In this study,according to the porcine and sheep CD58 sequences,reference to the porcine CD58 mRNA primers,apair of primers was designed and used to clone sheep CD58 gene in lymphocyte mRNA by RT-PCR method.Then,this cDNA was ligated into pGEX-4T-1 and transformed to E.coli BL21cells.The results showed that an 807bp fragment of sheep CD58 cDNA with an ORF of 762bp was obtained.The recombinant transformant was induced with IPTG,SDS-PAGE showed the specific expression of the fusion protein was observed in 53ku and the expressed product could be recognized by CD58 monoclonal antibody in Western-blot.This research will help us to develop new adjuvant and immune-regulated drugs,and laid a solid foundation for further study on the structure of CD58 molecule and the mechanism of CD2-CD58 complex triggering immune system.