摘要
构建大片吸虫硫氧还蛋白过氧化物酶(Fg.TPx)原核表达质粒,经大肠埃希菌Rosetta表达融合蛋白(Fg.TPx/His)并对其进行纯化和初步鉴定。PCR扩增Fg.TPx的基因片段,亚克隆至pET32a(+)中构建重组表达载体pPET32a-Fg.TPx。IPTG诱导表达,经镍离子亲和层析分离纯化后,进行SDS-PAGE和Western blot分析。重组质粒经限制性内切酶双酶切和测序分析表明构建成功,表达产物以可溶性和包涵体形式存在,纯度为90%,Wsetern blot证实该蛋白可与抗His单克隆抗体发生特异性结合反应,分子质量为TPx和His分子质量之和,表明是融合蛋白。重组蛋白可以被大片吸虫感染水牛阳性血清特异性识别。免疫家兔产生的抗体效价最高可达1∶4 000。成功构建了p ET32a-Fg.TPx原核表达质粒,重组蛋白得到高浓度表达,具有抗原性,为进一步研究其在大片吸虫病诊断中的应用奠定了基础。
To construct a prokaryotic expression plasmid （Fg. TPx / His） transformed into E. coli Rosetta, the expressed fusion protein was identified and purified. Fg. TPx was amplified by PCR, and the gene fragment was subcloned into pET32a （＋）, recombinant expression vector pPET32a-Fg. TPx. was con- structed. Under induction of IPTG, the expressed products were purified by Ni2＋-NTA affinity chroma- tography and identified by SDS-PAGE and Western blot. The enzyme digestion analysis and sequencing proved that recombinant plasmid pET32a-Fg. TPx was constrcted successfully. The expressed products were in forms of inclusion body, thioredoxin peroxidase reached purities of 90% in soluble proteins. The purified recombinant protein could be specifically recognized by serum of buffalo infected with Fasciola gigantica by ELISA and Western blot. The titers of polyclonal antibodies prepared by immunizing the rab- bit with recombinant protein was more than 1 ： 4 000. The purified protein could be used for diagnosis of Fasciola gigantica infection.
出处
《动物医学进展》
CSCD
北大核心
2012年第6期11-14,共4页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(30560111
39660061)
