利用Red/ET重组技术构建表达非洲猪瘟病毒CP530R和F317L基因的重组伪狂犬病毒

Construction of recombinant pseudorabies virus expressing CP530R and F317L genes of African classical swine fever virus by Red/ET recombinant technology

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)感染引起的一种高度接触型传染病,由于该病毒感染机制极为复杂,能够引起世界范围内的猪群发生致病性死亡。本研究参考Georgia 2007/1株(GenBank登录号:FR682468)的CP530R和F317L基因序列,人工合成PUC57-CMV-HA-CP530R-F317L-BGH质粒,利用Red/ET重组工程技术和ccdB反向筛选技术,将ASFV特定基因定向插入到伪狂犬病毒TK位点,构建重组毒株rBartha-K61-CP530R-F317L。结果表明,构建成功的感染性克隆在PK-15细胞上完成适应性传代并持续传至20代,PCR扩增得到正确基因条带,测序结果未发现缺失与插入。本研究将外源基因成功插入到pBeloBAC11-Bartha-K61载体上,重组伪狂犬病毒构建成功,为进一步研制新型ASF活载体疫苗奠定了基础。

African swine fever(ASF)is a highly contagious disease caused by African swine fever virus,due to the complexity of its infection mechanism,ASF can cause the death of pigs worldwide.In this study,referring to the CP530 R and F317 L gene sequences of Georgia 2007/1 strain(GenBank number:FR682468),PUC57-CMV-HA-CP530 R-F317 L-BGH plasmid was synthesized.The specific gene of African swine fever virus was inserted into the TK site of pseudorabies virus by Red/ET recombinant engineering technology and ccdB reverse screening technology,yielding the recombinant strain rBartha-K61-CP530 R-F317 L.The results showed that the infectious virus was successfully constructed and passaged on PK-15 cells for 20 generations.The correct gene band was obtained by PCR amplification,and the sequencing result showed that there was no deletion or insertion.In conclusion,the foreign gene was inserted into the vector pBeloBAC11-Bartha-K61,and the recombinant pseudorabies virus was successfully constructed,which lays a foundation for the further development of a new live vector vaccine against ASF.