PCV-2和PCV-3 Cap蛋白在昆虫细胞-杆状病毒系统中的表达和鉴定

Expression and Identification of Cap Protein of PCV-2 and PCV-3 in Insect Cell-Baculovirus System

为了实现猪圆环病毒2型(PCV-2)和3型(PCV-3)Cap蛋白在昆虫细胞-杆状病毒系统中表达,本试验利用刚性连接肽连接PCV-2和PCV-3 Cap基因,构建杆状病毒转移载体pFastBac-PCV-2-3-Cap,双酶切验证正确后,将其转化至大肠杆菌DH10Bac感受态细胞,经蓝白斑筛选和PCR鉴定,获得重组杆粒。将重组杆粒转染SF9细胞(昆虫卵巢细胞),盲传3代,获得重组病毒。将重组病毒接种SF9细胞96 h后,通过细胞免疫荧光检测、Western blot和电镜观察对其进行验证,并利用噬斑法检测其效价。结果显示:成功构建重组质粒pFastBac-PCV-2-3-Cap,获得了重组杆粒rAcBacmid-PCV-2-3-Cap,并制备出重组杆状病毒rvAc-PCV-2-3-Cap。重组病毒感染SF9细胞后,目的蛋白成功表达,并可以形成病毒样颗粒,病毒效价为3.41×10^(6) PFU/mL。本试验成功在昆虫细胞-杆状病毒系统中共表达了PCV-2-3-Cap,为防治PCV-2和PCV-3混合感染的二联疫苗研究提供了数据支持。

To enable the expression of the Cap protein of porcine circovirus type 2(PCV-2) and porcine circovirus type 3(PCV-3) in the insect cell-baculovirus system,this study constructed a baculovirus transfer vector pFastBac-PCV-2-3-Cap by connecting the Cap gene of PCV-2 and PCV-3 with rigid junction peptide.Upon double digestion verification,the vector was transformed into E.coli DH10Bac competent cells.Following blue and white spot screening and PCR identification,the recombinant rod-shaped particle was transfected into SF9 cells and passaged for three generations to obtain the recombinant virus.After inoculation into SF9 cells for 96 h,the recombinant virus was validated through cell immunofluorescence detection,Western blot,and electron microscopy observation,and the virus titer was detected using plaque assay.The results showed that the recombinant plasmid pFastBac-PCV-2-3-Cap was successfully constructed,the recombinant rod-shaped particles rAcBacmid-PCV-2-3-Cap were obtained,and the recombinant baculovirus rvAc-PCV-2-3-Cap was prepared.After the recombinant virus infection of SF9 cells,the target protein was expressed and virus-like particles were formed,with a viral titer of 3.41×10^(6) PFU/mL.The successful co-expression of PCV-2-3-Cap in the insect cell-baculovirus system provides technical data for future development of a dual vaccine against PCV-2 and PCV-3 mixed infection.