鸡BCL10基因的克隆与表达

Cloning and expression of chicken's BCL10 gene

通过电子克隆手段,克隆到大小为959bp的cDNA序列。然后从鸡的脾脏提取RNA,用RT-PCR方法扩增出目的片段。将该片段连接到原核表达载体pET-32a和真核表达载体pEGFP-C1中,采用SDS-PAGE检测原核表达载体pET-BCL10在大肠杆菌中的表达,脂质体转染法将真核表达载体pEGFP-BCL10转入vero细胞,Western blotting检测其在细胞中的表达。结果显示,PCR扩增到735bp的DNA片段,原核表达载体和真核表达载体经双酶切和核酸测序表明载体构建成功。SDS-PAGE电泳检测到pET-BCL10重组质粒在大肠杆菌中表达。Western blotting检测到pEGFP-BCL10重组质粒在vero细胞中表达。结论表明,BCL10基因在鸡体内存在,该基因的重组质粒能在大肠杆菌和vero细胞中表达。

To detect the existence of BCL10 gene in chickens and research how it expressed in E.coli and vero cells.A full-length cDNA of 959 bp was acquired by silico cloning and the ORF of 735 bp contained in the cDNA was cloned by RT-PCR.The chickens BCL10 gene was cloned to the prokaryotic expression vector pET-32a and eukaryotic expression vector pEGFP-C1.The expression product of prokaryotic expression vector pET-BCL10 was detected by SDS-PAGE.Eukaryotic expression vector pEGFP-BCL10 was transferred to vero cell line by lipofectin 2000 and GFP was detected by Western blotting.The results showed that the sequence obtained by silico cloning was the same as that obtained by sequencing.Prokaryotic expression vector and eukaryotic expression vector were both successfully constructed by sequencing.pET-BCL10 was expressed in E.coli confirmed by SDS-PAGE.BCL10 gene was successfully expressed in vero cells by GFP marker and Western blotting,The results indicated that the BCL10 gene definitely exists in chickens,which could be successfully expressed in E.coli and vero cells.