减毒沙门氏菌为载体在Vero细胞中表达新城疫病毒融合蛋白

RT PCR扩增了新城疫病毒 (NDV)F4 8E9株的融合蛋白 (F)基因并插入到 pcDNA3的CMV启动子下游 ,构建成真核表达质粒pcDNA3 F ,高压电转化dam和 phoP基因双突变株减毒鼠伤寒沙门氏菌 (ZJ111株 ) ,并直接感染Vero细胞 ,分别提取细胞总DNA和总RNA ,DIG标记探针均可检测到阳性杂交信号。FITC标记的羊抗鸡IgG进行间接免疫荧光试验 ,可检测到特异性的黄绿色荧光。ELISA检测F蛋白结果表明 ,转染后 4 8h开始表达 ,随后逐渐增多。SDS PAGE和Western印迹可检测到 5 5kD的蛋白质条带。上述试验结果证实减毒沙门氏菌不仅可将目的基因呈递给Vero细胞 ,而且还得到了转录和表达 ,表达的F蛋白具有免疫反应性 ,为研制减毒沙门氏菌为载体的口服NDVDNA疫苗创造了条件。

Expression of the Newcastle Disease Virus Fusion Glycoprotein in Vero Cells Using Attenuated Salmonella typhimurium as Transgenic Carrier

The full length cDNA of fusion protein (F) gene of newcastle disease virus (NDV) strain F48E9 was amplified by RT PCR and inserted into pcDNA3 under the control of human cytomegalovirus (hCMV) immediate early enhancer and promoter. The resulting recombinant plasmid pcDNA3 F was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 ( dam - and phoP - ), which was then used to transfect the Vero cells. The DNA and RNA dot blotting revealed that the F gene was transcribed into mRNA in the Vero cells. There was expression of the F protein as shown by indirect immunofluorescent assay. The expression began at 48 h post infection and increased thereafter, as indicated by ELISA. A 55 kD band of the F protein was identified by SDS PAGE and Western blotting. These results clearly show that the expressed fusion protein was immuno reactive with chicken anti NDV serum.