鸭疫里默氏菌OmpA与鸭IL-2融合基因的克隆与原核表达

Cloning and Expression of Riemerella anatipestifer OmpA with Duck Interleukin-2 Fusion Gene

以鸭疫里默氏菌吉林分离株JL-RA1OmpA基因(登录号HQ707077)和鸭DuIL-2成熟片段基因(登录号AY193713)为模板,分别设计带有linker的特异性引物,PCR扩增出序列大小为1182bp的OmpA-linker基因和序列大小为381bp的linkerDuIL-2成熟蛋白基因。利用SOE-PCR技术,成功构建OmpA-DuIL-2融合基因,片段大小为1551bp。将其转入大肠杆菌表达载体pET-28a中,构建pET-OmpA-DuIL-2融合表达载体,转化至大肠杆菌Rosetta(DE3)中,IPTG诱导表达,经SDS-PAGE分析得到分子量大小约为61 kDa的融合蛋白,经Western-blot分析该融合基因同时具OmpA和DuIL-2的反应原性。

Using the OmpA gene of Riemerella anatipestifer（RA）,which was isolated from Jilin and named JL-RA1（accession number HQ707077） and duck DuIL-2 mature fragment gene as a template（accession number AY193713）,the OmpA gene of size 1 182 bp and linker-DuIL-2 mature protein gene sequence size of 381 bp were amplified by PCR.The amplification of 1551bp which named OmpA-DuIL-2 fusion gene was obtained by SOE-PCR. The fusion gene was transferred into E. coli expression vector pET-28 a to construct a pET-OmpA-DuIL-2 fusion expression vector,and the expression vector was then transformed into E.coli Rosetta（DE3）.The DE3 contained pET-OmpA-DuIL-2 fusion expression vector can express target proteins by IPTG induction. The SDS-PAGE results showed that the recombinant protein has a molecular weight of about 61 KD,and the Western-Blot analysis showed the fusion protein owning immunogenicity of OmpA and DulL-2.