猪圆环病毒Cap蛋白的分泌表达及其免疫原性研究

Secretory expression of Cap protein of procine circovirus 2 and its immunogenicity

探索利用杆状病毒表达系统(Bac-to-Bac expression system)分泌表达猪圆环病毒2型(PCV2)Cap蛋白及将其作为亚单位疫苗的应用价值。将PCV2 Cap基因克隆到已插入蜂毒信号肽(Melittin)的转移载体pFastBacⅠ中,将鉴定正确的重组质粒(pFastBAC-Cap)转化至DH10Bac大肠杆菌感受态细胞,经抗性及蓝白斑筛选得到含Cap基因的重组杆状病毒DNA(Bacmid-Cap),转染提取的Bacmid-Cap DNA转染至sf9昆虫细胞,获得重组杆状病毒(rBac-Cap),应用无血清培养的High Five细胞进行重组蛋白的表达及条件优化。重组蛋白通过聚丙烯酰胺凝胶电泳分析(SDS-PAGE)证明:在重组杆状病毒感染的High Five昆虫细胞中获得分泌表达,并可产生较高浓度的重组蛋白;Western blotting结果显示:重组蛋白可被猪PCV2的特异性抗体所识别,表明重组蛋白具有反应原性;应用重组蛋白免疫BALB/C小鼠试验结果显示:该蛋白可刺激小鼠产生较高水平的特异性抗体。该研究成功分泌表达了PCV2 Cap蛋白,该蛋白能够刺激机体产生免疫应答,为PCV2亚单位疫苗的研制打下基础。

The gene fragment encoding Cap protein of porcine circovirus 2 （PCV2） was cloned into the Baculovirus pFastBac I transfer vector with a honeybee melittin signal peptide. The constructed pFastBae-Cap was transformed into Escherichia coli DH10Bac, resulting the recombinant Baculovirus DNA （Bacmid-Cap） which was confirmed by blue-white plaque assay and antibiotic resistance selection. The Bac- mid-Cap was then transfected into Si9 insect cells by the Cellfectin transfection reagent. The recombinant Cap protein was expressed in High Five cells in the serum-free medium. The SDS-PAGE and Western blotting analysis of recombinant protein showed that the protein could be expressed in insect cells and secreted into the culture medium. For the immunogenicity study, the recombinant protein was then inoculated into BALB/c mice and the results showed that the recombinant protein was able to stimulate B cells to produce special antibodies. In conclu- sions, the recombinant Baculovirus expressing Cap protein of PCV2 were successfully constructed. This study applies a basis for the develop- ment of PCV2 subunit vaccine.