H3N2亚型猪流感病毒反向遗传学操作平台的建立  

Establishment of reverse genetic manipulation platform for swine influenza virus of H3N2 subtype

作　　者：崔平 CUI Ping(Binzhou Animal Husbandry and Veterinary Management Service Center,Binzhou 256600,China)

机构地区：[1]滨州市畜牧兽医管理服务中心,山东滨州256600

出　　处：《黑龙江畜牧兽医》2021年第4期78-82,157,共6页Heilongjiang Animal Science And veterinary Medicine

摘　　要：为了建立猪流感病毒反向遗传操作平台,试验利用RT-PCR技术扩增A/Swine/Shandong/JN1/2016(H3N2)流感病毒表面抗原基因HA和NA,连接至双向表达载体pHW2000,构建其感染性克隆。将A/PuertoRico/8/34(H1N1,PR8)毒株的6个内部基因片段重组质粒与H3N2的表面抗原基因HA和NA片段重组质粒毒共转染293Ta细胞,收获转染液接种鸡胚,收获尿囊液,测定其效价。对重组H3N2毒株进行血清学、形态、免疫荧光鉴定及传代稳定性、在犬肾传代细胞系(MDCK)上的病变效应。结果表明:反向遗传技术拯救获得重组猪流感病毒,命名为rH3N2。病毒液接种鸡胚后收获尿囊液,测定其效价为1∶256。经电镜观察,重组毒株rH3N2有囊膜,具备流感病毒一般特性;重组病毒rH3N2仅与猪流感病毒H3亚型特异性抗血清发生阳性反应;连续传代后,效价可稳定在1∶512;免疫荧光试验结果表明,rH3N2可以感染MDCK细胞,且可以连续传代,效价达1∶256。说明成功建立H3N2亚型猪流感病毒反向遗传学技术平台,获得重组毒株rH3N2,为猪流感病毒的致病机理、跨物种传播机制、基因结构与功能关系的研究以及猪流感流行疫苗候选株的快速筛选提供基础。In order to establish a swine influenza virus reverse genetic manipulation platform, RT-PCR technology was used in this experiment to amplify the A/Swine/Shandong/JN1/2016(H3 N2) Influenza A virus surface antigen genes HA and NA,which were cloned into the bidirectional expression vector pHW2000 to construct the infectious clone. The 6 recombinant plasmids of the internal gene fragments of A/PuertoRico/8/34(H1 N1,PR8) strain and the recombinant plasmids of the surface antigen gene HA and NA fragments of H3 N2 were co-transfected into 293 Ta cells. The harvested liquid was inoculated with chicken embryos and the allantoic fluid was harvested;the titer was determined. Serological, morphological, immunofluorescence identification and passage stability of recombinant H3 N2 strains, pathological effect on dog kidney cells(MDCK) were carried out. The results showed that reverse genetic technology rescued the recombinant swine influenza virus, named rH3 N2. The allantoic fluid was harvested after inoculating the chicken embryos with the virus fluid, and its titer was determined to be 1∶256. Observed by electron microscopy, the recombinant strain rH3 N2 had an envelope and the general characteristics of Influenza A virus. The recombinant virus rH3 N2 only reacted positively with Swine influenza virus H3 subtype specific antiserum;after serial passage, the titer can be stabilized at 1∶512. Immunofluorescence test results showed that rH3 N2 can infect MDCK cells and can be serially passaged, with a titer of 1∶256. The results suggested that the reverse genetics technology platform of H3 N2 subtype swine Influenza virus had been successfully established, and the recombinant strain rH3 N2 was obtained, which provided a basis for the study of the pathogenic mechanism of Swine influenza virus, the mechanism of cross-species transmission, the relationship between gene structure and function, and the rapid screening of swine influenza vaccine candidate strains.

关 键 词：猪流感 H3N2亚型 反向遗传学 病毒形态 增殖特性 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]