新孢子虫表面抗原P40基因的克隆及原核表达

Cloning and prokaryotic expression of P40 gene of Neospora caninum surface antigen

目的克隆并原核表达新孢子虫表面抗原P40基因。方法 PCR扩增新孢子虫表面抗原P40基因,克隆至原核表达载体pET-28a中,构建重组原核表达质粒pET-28a-P40,转化入大肠埃希菌Rosseta(DE3),IPTG诱导表达,SDS-PAGE和Western blot鉴定表达产物。结果重组原核表达质粒pET-28a-P40经PCR及双酶切鉴定构建正确;表达的重组P40蛋白相对分子质量约为40 000,有效识别鼠抗新孢子虫阳性血清。结论成功在Rosseta(DE3)中表达了新孢子虫表面抗原P40基因,为新孢子虫病疫苗的研制及血清学检测方法的建立奠定了基础。

Objective To clone the P40 gene of Neospora caninttm surface antigen and express in prokaryotic cells. Methods P40 gene of the N. caninum surface antigen was amplified by PCR and cloned into prokaryotic expression vector pET-28a. The constructed recombinant plasmid pET-28a-P40 was transformed to E. coli Rosseta （DE3） for ex- pression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results The prokaryotic expression vector pET-28a-P40 was constructed correctly as proved by PCR and restriction analysis. The expressed recombinant P40 protein, with a relative molecular mass of about 40 000, recognized the positive mouse serum against N. caninum effectively. Conclusion P40 gene of the N. caninum surface antigen was expressed successfully in E. coli Rosseta （DE3）, which laid a foundation of preparation of N. caninum vaccine and development of methods for se- rologic diagnosis of neosporosis.