构建表达猪瘟病毒E0基因的重组杆状病毒

Construction of recombinant baculovirus expressing CSFV E0 gene

为了得到可溶性重组猪瘟病毒E0蛋白并便于观察重组蛋白的表达情况，将增强型水母绿色荧光蛋白基因（EGFP）与E0基因相连插入昆虫杆状病毒转移载体中，与线性杆状病毒DNA共转染Sf9细胞后通过噬斑纯化得到纯的重组杆状病毒，将其感染Sf9细胞制备P1种子液，同时用荧光显微镜观察绿色荧光蛋白的表达情况，剔除表达效果差的重组杆状病毒。再用P1种子液感染Sf9细胞制备高效价的P2种子液。通过病毒液的梯度稀释和噬斑测定，确定P2种子液的病毒滴度达1．14×10^7pfu／mL。将P2种子液以MOI5～10接种指数期SD细胞．3d后呈现出最强的荧光。重组杆状病毒在Sf9细胞中的初步成功表达为进一步制各重组E0糖蛋白．研究E0蛋白的结构与功能、免疫诊断新方法和猪瘟基因工程疫苗奠定基础。

In order to get the soluble recombinant classical swine fever virus E0 protein and inspect the protein expression situation conveniently, the EGFP and E0 gene are ligated and inserted into baculovlrus transfer vector in tandem. After co-transfecting Sf9 cells of baculovirus recombinant transfer vector and linearized viral DNA, and plaque purification in the posttransfection procedure, the pure recombinant baculovirus is harvested, which further infect the Sf9 cells for amplifying to generate a P1 stock. In the meantime, the fluorescence microscopy detection indicates expressed EGFP protein to get rid of poorly expressed recombinant baculovirus. The P1 stock from a pure plaque is used to generate a high titer P2 stock, which is determined in titer as 1.114 × 10^7 pfu/mL by performing a plaque assay. When Sf9 cells at log phase are infected by P2 stock with MOI 5-10 for expression, the strong fluorescence is observed 3 days later. The initially successful expression of recombinant baculovirus in Sf9 cells allows us to prepare recombinant E0 protein, study E0 protein structure and function, establish new methods for immunological diagnosis and develop the qenetic engineering vaccine for CSFV.