猪圆环病毒衣壳蛋白在昆虫细胞中表达形成病毒样颗粒的研究

本研究应用Bac-to-Bac杆状病毒/昆虫细胞表达系统,将猪圆环病毒（Porcine circovirus,PCV）PCV2b的ORF2基因插入供体质粒pFastBacTMⅠPPH启动子控制下的多克隆位点,构建的质粒转化DH10BAC感受态细胞,经抗性和蓝白斑筛选,获得重组穿梭质粒rBacmid-cap。将rBacmid-cap转染对数生长期的Sf9昆虫细胞,间接免疫荧光试验证明目的蛋白在重组杆状病毒感染的Sf9昆虫细胞中获得了表达。将该重组杆状病毒感染HighFiveTM细胞后,用Western blot在细胞培养上清中检测到了目的蛋白,并于d 5达到表达高峰。电镜观察结果显示上清中的目的蛋白可以装配成直径约为17 nm的病毒样颗粒。病毒样颗粒在培养上清中的大量存在,为目的蛋白进一步的分离和纯化提供了便利,也为PCV2基因工程疫苗的产业化奠定了基础。

EXPRESSION OF PORCINE CIRCOVIRUS CAPSID PROTEIN IN INSECT CELLS AND SELF-ASSEMBLED INTO VLPs

The capsid gene of PCV2b was cloned into the multiple cloning site of the pFastBacTMⅠvector of the Bac-to-Bac expression system under the control of PPH promotor.The recombinant plasmid rBacmid-cap was transformed into DH10Bac for bule/white selection and antibotics selection.The positive recombinant bacmid was transfected to Sf9 in logarithmic growth phase for harvesting recombinant Baculovirus.The expression protein in Sf9 which was infected by recombinant Baculovirus rvBac-cap was identified by IFA.HighFiveTM was infected by the rvBac-cap,and the culture supernatant was collected to detect the expression protein by Western blot and electron microscopy.The results showed that the recombinant protein was released into culture medium and self-assembled into PCV2-like particles（PCV2-VLPs） with a diameter of 17nm.The peak of protein expression was on the 5th day post infection.The efficient expression of PCV2-VLPs in cell culture could be a convenience for perotein purification and a basis for developing PCV2 genetically engineered vaccine.