摘要
根据GeneBank上的猫IFN-β基因序列,设计了1对特异性引物,对猫肝组织中提取的DNA进行PCR扩增,然后将扩增产物插入pUCm-T克隆载体进行了克隆。测序分析表明,克隆的IFN-β基因序列与GeneBank中的基因(NM_001009297)存在一个碱基差异,第446位碱基由T变为C,二者同源性高达99.82%,同时这个碱基的差异没有引起氨基酸的改变。将其双酶切后插入原核表达载体pGEX-KG,构建成功重组表达载体pGEX-IFNB,将其转化BL21(DE3)后成功进行了诱导表达。SDS-PAGE和Western-Blot分析表明,在35KDa处出现蛋白表达条带,表达量占菌体蛋白的12%。在Western-Blot中其能和GST抗体发生特异性反应,说明为GST-IFN-β融合蛋白。
A pair of specific primers were designed according to the sequences of feline IFN-β gene on NCBI website. The genome DNA was abstracted from the liver of cat and the interferon beta gene was amplified by PCR. The amplified product reclaimed was connected with the cloning vector pUCm-T. After identification by restriction enzyme and PCR, the gene was sequenced, The results of sequence analysis showed that the cloned feline IFN-β gene was at the length of 561bp and there was a different base（446th T→C） compared with the sequence NM_001009297 in the GeneBank. The homology between the both sequence reached 99,82% and the sequence of anomic acid sequence didn＇t change. Then the gene was connected with expressing vector pGEX-KG to form recombinant plasmid pGEX-IFNB. The E.coli BL21 （DE3） containing recombinant plasmid pGEX-IFNB was inducted for fusion protein expression. The results of SDS-PAGE and Western-blot showed that there is a protein band on 35KDa which can react with the GST antibody in Western-blot and the quantity of fusion protein is up to 12% of bacterial protein.
出处
《沈阳农业大学学报》
CAS
CSCD
北大核心
2008年第2期205-208,共4页
Journal of Shenyang Agricultural University
基金
沈阳农业大学青年教师科研基金资助项目(2006119)
关键词
Β干扰素
猫
克隆
表达
IFN-β
cat
cloning
expression
