Isolation And Identification Of Pesudorabies Virus Variants And Preparation Of Ul42 Polyclonal Antibodies

Pseudorabies virus(PRV)is a pathogen causing pseudorabies(PR),which can infect pigs of all ages and lead to different degrees of clinical symptoms,mainly manifested in central nervous system symptoms of piglets,reproductive difficulties in breeding pigs,and respiratory symptoms in adult pigs,and thus caused huge economic losses of swine industry in China.For a long time,gene-deficient vaccines were widely used in China and effectively controlled PRV.However,after 2011,new variants of PRV spreaded from the northern China to all over the nation,indicating that current vaccines cannot provide complete protection for the newly emerged PRV variants,which aggravated the epidemic of PRV in China and increased the difficulty of prevention and control.Therefore,understanding the difference between PRV variants and classical strains is helpful to predict the way of viral mutation and lays a foundation for developing new vaccines.In this study,two strains of mutant strains were isolated and identified from suspected pseudorabies pig samples,and UL42 polyclonal antibodies were prepared.1.Isolation and identification of pseudorabies virus variantsThe pathogenic materials of suspected pseudorabies cases were collected from a swine farm in Jiangsu province.The gE and gB were amplified by PCR using genomic DNA extracted from collected samples as templates.The results showed that both gE and gB were amplified in these samples,but only gB was amplified in Bartha-61,a gE-deficient strain.This suggested the presence of PRV field strains in pathologic materials.After 6 blind passages in Vero cells,obvious cytopathic effects were observed.Interestingly,different sizes of plaques were formed.A certain number of plaques with different sizes were selected and inoculated Vero cells.It showed that there were two different lesions in the infected Vero cells.Then gE and gC were amplified and sequenced,the results showed that the identity of gE sequences was 100%and that of gC sequences was 94.33%.The genetic differences and phylogenetic analysis of gE showed that these two strains were closer to the PRV variants isolated from China after 2011 than foreign strains,which is consistent with the general characteristics of PRV variants isolated from China in recent years.Meanwhile,by analyzing the transmembrane region,secondary structure,hydrophilicity,antigenic index,surface probability and flexibility,we found that both N-and C-terminal of the gE protein have the potential to become dominant epitopes.These two strains of PRV were named JSB1-7 and JSB1-13.The one-step growth curve assay showed that the replication ability of JSB1-7 and JSB1-13 was lower than that of the vaccine strain Bartha-K61.2.Preparation of pseudorabies virus UL42 polyclonal antibodiesUL42,an accessory subunit of PRV-encoded DNA polymerase,is one of the early proteins necessary for viral replication and plays an important role in the entire PRV life cycle.In order to study its specific mechanism,UL42 polyclonal antibodies was prepared.The recombinant plasmid pET-28a-UL42 was constructed and transformed into Rosetta competent cells.Samples were collected after IPTG induced at different time points and detected by SDS-PAGE,the results showed that after 5-hour IPTG induction,the protein expression reached the highest level.After that,the supernatant and pellet were detected by SDS-PAGE and showed that the UL42 protein was mainly expressed as a soluble form.Then,mice were immunized with purified UL42 according to the immunization program.The ELISA test showed that polyclonal antibodies serum titer of mice reached 1:64000,and the polyclonal antibodies were suitable for WB and IFA.