重组杆状病毒表达猪圆环病毒2型Cap蛋白的优化及蛋白免疫原性研究

为获得高蛋白含量和良好免疫原性的抗原,利用杆状病毒表达体系进行猪圆环病毒2型(PCV2)重组Cap蛋白表达,采取正交试验设计确定三因素(High five细胞浓度、病毒感染量、蛋白表达时间)的最佳组合,对重组病毒株vBac-SP-PCV2的表达条件进行优化,利用His Bind蛋白质纯化试剂盒对表达产物进行纯化,纯化蛋白作为标准蛋白用于Western blotting中蛋白质定量分析和蛋白免疫原性检测。结果显示:2.0×10^6/mLHigh five细胞浓度、1.5MOI病毒感染量、蛋白表达时间168h为重组PCV2-rCap蛋白表达的最佳条件,表达产物纯化良好,并能被PCV2多克隆抗体识别,免疫豚鼠可诱导产生高水平PCV2抗体。研究表明纯化PCV2-rCap蛋白可作为标准蛋白用于后续表达蛋白的定量分析和PCV2亚单位疫苗研发候选抗原。

The Optimization and Its Immunogenicity by the Recombinant Baculovirus Virus Expressed Capsid Protein of Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) recombinant Cap protein was expressed by baculovirus expression system in order to obtain antigen with high protein content and good immunogenicity.,Optimum conditions of three factors (cell concentration,virus infective dose,time) for protein expression has been confirmed by orthogonal test.The purified protein was obtained by use of His·Bind affinity chromatography.The purified protein was observed by SDS-PAGE electrophoresis and was further confirmed by Western blotting.The purified protein as standard protein applied to quantifying Protein expression in Western blotting analysis,to protein of immunogenicity determination.Results showed that 2.0×10^6 /mL High five cell concentration,1.5 MOI,infection time of 168 hours were the optimum condition for expression proteins.The results showed that the recombinant Cap had been well purified and could react with the polyclonal antibody against PCV-2.The results of Protein expression in Western blotting analysis were in agreement with the quantitative approach to computation of protein expression percentage.The purified protein could induce high titer of the specific PCV2 antibodies in guinea pig.The purified protein as standard protein applied to quantifying protein expression.It is an ideal antigen candidate of recombinant sub-unit vaccines.