表达NDV HN和IBDV VP2融合蛋白的重组减毒沙门菌的构建及其生物学特性分析

Construction and biological characteristics analysis of recombinant attenuated Salmonella expressing IBDV VP2 and NDV HN fusion protein

为构建表达鸡新城疫病毒(NDV)HN和传染性法氏囊病病毒(IBDV)VP2融合蛋白的重组减毒沙门菌,并分析其生物学特性。本研究扩增了NDVHN和IBDVVP2主要抗原基因片段,通过重叠延伸PCR扩增获得HN-VP2融合基因,构建重组质粒pYA-HN-VP2,将该重组质粒电转化至减毒鼠伤寒沙门菌SL1344ΔcrpΔcyaΔasd中,获得表达HN-VP2融合蛋白的减毒重组菌SL1344ΔcrpΔcyaΔasd(pYA-HN-VP2),并分析其生化特性、遗传稳定性和毒力等生物学特性。Westernblot结果显示,重组减毒沙门菌可分泌表达HN-VP2融合蛋白,大小为84ku,且该融合蛋白分别能与相应特异性阳性血清发生反应;生物学特性结果显示,重组菌株失去了利用麦芽糖、山梨醇及乳糖等碳源的能力,其生长速度显著慢于野生菌株SL1344;毒力较野生菌株至少下降5个对数单位,且能够稳定遗传重组质粒pYA-HN-VP2。本研究为鸡新城疫-传染性法氏囊病-沙门菌病的三联口服疫苗研制奠定重要基础。

In order to construct the recombinant attenuated Salmonella which expresses Newcastle disease virus (NDV) HN and Infectious bursal disease virus (IBDV) VP2 fusion protein and analyze its biological characteristics, the recombinant vector pYA-HN-VP2 was constructed by inserting the fusion gene fragment HN-VP2 into prokaryotic expression vector pYA3493, and SL1344ΔcrpΔcyaΔasd (pYA-HN-VP2) was constructed by transform the recombinant vector pYA-HN-VP2 into attenuated Salmonella typhimurium SL1344ΔcrpΔcyaΔasd. The results of western blot showed that SL1344ΔcrpΔcyaΔasd (pYA-HN-VP2) could express and secrete 84 ku HN-VP2 fusion protein, which could react with specific positive sera against HN or VP2 protein, respectively. Biological characteristics results showed that recombination incapacitates the strain digesting ferment maltose, sorbitol and lactose, and the growth rate of the recombinant strain was slower than wt SL1344. LD50 of recombinant strain reduced at least 5 Logarithmic units compared with SL1344 and recombinant plasmid pYA-HN-VP2 could be stably inherited in the recombinant salmonella strain. These results provide new insights for triple oral vaccine candidate of ND, IBD and salmonellosis investigation.