表达牛病毒性腹泻病毒E2蛋白的嵌合狂犬病病毒生物学特性

Biological characteristics of chimeric rabies virus expressing bovine viral diarrhea virus E2 protein

牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)引起牛发热、腹泻、黏膜糜烂溃疡等症状,给养牛业带来严重经济损失,新型疫苗的研发意义重大。狂犬病病毒(rabies virus,RV)作为病毒载体可稳定表达外源蛋白,反过来外源蛋白也不会阻碍病毒的复制。本研究目的是利用反向遗传操作技术构建和拯救表达BVDV结构蛋白的重组RV,以期获得一种能表达高水平BVDV E2蛋白的重组病毒。选择Ⅰ型BVDV主要优势抗原E2蛋白基因序列进行优化后合成E2基因片段,利用Overlap PCR的方法将E2基因改造为DE2片段,以RV疫苗毒株LBNSE为病毒载体,利用BsiWⅠ和NheⅠ酶切位点,将DE2基因插入LBNSE基因组中,成功构建pRV-DE2重组质粒。脂质体法将全长重组质粒转染BSR细胞后,拯救重组病毒RV-DE2,并用直接免疫荧光和RT-PCR的方法对其鉴定,对构建成功的病毒进行生长特性、致病性、重组蛋白表达特性等生物学特性和小鼠体内免疫效力的研究。结果表明,RV-DE2病毒滴度在F3代之后趋于稳定,在相同的培养条件下比亲本LBNSE病毒滴度高。重组病毒对小鼠无致死性,E2蛋白表达量高于BVDV。将灭活的重组病毒免疫小鼠后成功产生了抗BVDV的中和抗体。表达BVDV E2的重组RV的成功拯救为BVDV新型疫苗的研发提供候选毒株,对BVD的防控提供了一种新的思路。

Bovine viral diarrhea virus(BVDV)causes symptoms such as fever,diarrhea,mucosal erosion,and ulcers in cattle,causing serious economic losses to the cattle industry.The development of new vaccines is of great significance.Rabies virus as a viral vector can stably express foreign proteins,which in turn will not hinder replication of virus.The purpose of this study is to construct and rescue the recombinant rabies virus expressing BVDV structural protein by reverse genetic technique,to obtain a recombinant virus that can express high level of BVDV E2 protein.The E2gene fragment was synthesized by optimizing the gene sequence of the main dominant antigen E2protein of type I BVDV.The E2 gene was transformed into DE2 fragment by overlap PCR.The rabies virus vaccine strain LBNSE was used as the viral vector.The BsiWⅠ and NheⅠ restriction sites were used to insert the DE2 gene into the LBNSE genome,and the pRV-DE2 recombinant plasmid was successfully constructed.After the full-length recombinant plasmid was transfected into BSR cells by liposome method,the recombinant virus RV-DE2 was rescued and identified by direct immunofluorescence and RT-PCR.The biological characteristics of the constructed virus,such as growth characteristics,pathogenicity,recombinant protein expression characteristics and immune efficacy in mice were studied.The results showed that the RV-DE2 virus titer tended to be stable after the F3 generation and was higher than the parental LBNSE virus titer under the same culture conditions.The recombinant virus was not lethal to mice,and the expression of E2 protein was higher than that of BVDV.The neutralizing antibody against BVDV was successfully produced by immunizing mice with the inactivated recombinant virus.The successful rescue of recombinant rabies virus expressing BVDV E2 provides a candidate strain for the development of new BVDV vaccines and a new idea for the prevention and control of BVD.