伪狂犬病病毒鄂A株囊膜蛋白gD基因在杆状病毒载体系统中的表达研究

Expression of the Envelope Glycoprotein D Gene of Pseudorabies Virus Ea Strain

克隆了伪狂犬病病毒鄂A株囊膜蛋白gD基因并进行了序列分析 ,与国际标准毒株Rice株相比 ,两者之间核苷酸序列同源性为 98% ,推导氨基酸序列同源性为 97% .利用杆状病毒GST融合载体系统对其进行了高效表达 ,SDS 聚丙烯酰胺凝胶电泳及蛋白质印迹均证明了表达产物为 71ku的GST gD融合蛋白 ,其产量占细胞不溶蛋白量的 2 0 %左右 .以GST gD融合蛋白作为抗原进行小鼠免疫保护实验 ,结果表明表达的GST gD融合蛋白具有较好的免疫原性 ,不仅能诱导小鼠产生血清抗体 (1∶12 8) 。

The envelope glycoprotein D gene of pseudorabies, virus Ea strain was cloned by PCR technique. Sequence analysis displayed 98% nucleotide sequence homology and 97% deduced amino acid sequence homology between the cloned gD gene and PRV Rice strain gD gene. Then gD gene was expressed highly in the baculovirus GST fusion vector system. Both SDS-PAGE and Western-blot verified that the expression product was GST-gD fusion protein with the. molecular mass of 71 ku. Expressed GST-gD fusion protein accounted for about 20% of total cellular protein. The protective immune assay was performed in mice by using GST-gD fusion protein as antigen. The result, showed that all mice immunized by GST-gD could produce a certain level of antibody against PRV gD (antibody titer: 1: 128), and the mice in immunized group could be partly protected against challenge infection of highly virulent PRV Su strain at 2 x 10(5) PFU per mice. A good foundation has been laid down for developing PRV genetically-engineered subunit vaccine.