猪圆环病毒2型Cap蛋白在大肠杆菌中的可溶性表达及在小鼠体内免疫特性研究

The Soluble Expression of Porcine Circovirus Type 2 Cap Gene in Escherichia coli and Its Immunogenicity in Mice

猪圆环病毒2型(PCV2)Cap蛋白基因是该病毒基因工程疫苗的重要目的基因,但其在大肠杆菌表达系统中的表达产物通常以包涵体形式存在,影响其作为亚单位疫苗使用的免疫保护作用。将ORF2基因密码子改造为大肠杆菌偏爱的密码子,或构建MPG与ORF2基因融合,分别克隆至表达载体p ET28a,再转化至大肠杆菌BL21(DE3),经IPTG诱导表达。SDS-PAGE和Western blot结果证实改造后的PCV2 Cap蛋白基因实现了可溶性表达。将上述两种重组蛋白纯化后,分别与GEL01、ISA206或ISA15A三种佐剂混合配制疫苗,小鼠免疫保护试验结果证明,以GEL01为佐剂的两免疫组PCV2 ELISA抗体和中和抗体水平最高。攻毒试验结果显示,除ISA15A组外,其他各免疫组脾脏中PCV2含量都显著低于非免疫对照组,表明两种重组蛋白与佐剂GEL01和ISA206制成的疫苗可诱导产生一定水平的免疫保护作用,为PCV2亚单位疫苗的研制奠定了基础。

Porcine circovirus type 2（PCV2）Cap gene has been used as target gene for developing genetic engineering vaccine.However,it is usually expressed as the form of inclusion body in E.coli which having low protective efficacy against PCV2 challenge.The PCV2 ORF2 gene condons was optimized and fused with MPG gene.Then,they were cloned into expression vector p ET28 a and induced for expression in E.coli BL21（DE3）individually.SDS-PAGE and Western blot results confirmed that they could be expressed with the production of soluble proteins.After purification,the two kinds of proteins were mixed with GEL01,ISA206 and ISA15 A adjuvant respectively.Mice experiment results showed that the GEL01 groups induced the highest levels of antibodies against PCV2 with ELISA and neutralization antibody assay.Following challenge with PCV2,the PCV2 virus loads in spleen in immunized groups except the ISA15 A were significantly lower than challenge control group.It indicated that the vaccine prepared by the two recombinant proteins and GEL01 and ISA206 could induce immune protection in mice against PCV2 infection,which laid a foundation for the development of PCV2 subunit vaccine.