摘要
设计1对特异性引物对羊布鲁菌16M总DNA进行外膜蛋白omp10的PCR扩增,得到了一个大小为330bp的目的基因片段(去掉17个氨基酸编码的信号肽),测序证实它与国外报道的羊布鲁菌omp10基因完全一致。将其克隆到表达载体PET-30a中,经酶切、PCR扩增和测序分析,表明重组表达载体构建成功。将此重组质粒转化入大肠埃希菌BL21(DE3)中,IPTG诱导表达,该基因以包涵体的形式在大肠埃希菌中表达,经过包涵体的变性、复性和亲和层析纯化,成功获得大小为14.2ku的融合蛋白,与理论推测的蛋白分子质量一致;Westernblot和间接ELISA试验证明,纯化之后的OMP10重组蛋白可以被布鲁菌阳性血清识别。
An ompl0 gene, which was deleted signal peptide, was amplified and a 330 bp brand was obtained by PCR method from total DNA of Brucella melitensis with a pair of specific primers. The product was inserted into an expression vector PET-30a and was confirmed that the expression vector PET-30a/ omp10 was correctly constructed after restriction enzyme analysis and PCR. The PET-30a/omp10 was transformed into E. coli BL21（DE3）cell and induced by IPTG. The recombinant protein OMP10 of 14.2 ku in size was expressed in the form of inclusion body and purified by degeneration, renaturation and an affinity chromatography. Furthermore,the results from Western-blot and iELISA showed that recombinant protein can be recognized by positive serum against Brcuella.
出处
《动物医学进展》
CSCD
北大核心
2009年第12期25-29,共5页
Progress In Veterinary Medicine
基金
家畜疫病病原生物学国家重点实验室基金
