| Porcine circovirus type 2(PCV2) is the causative agent of post-weaning multisystemic wasting syndrome(PMWS) and is prevalent in most swine herds, causing enormous economic losses to the swine industry. Mostly, there are only inactivated and genetic engineering subunit vaccines to control PCV2 infection. Recently, PCV2 always coninfects with pseudorabies virus(PRV) seriously and clinically, resulting in severe respiratory syndromes on piglets and reproductive failure on sows. PRV variants with high pathogenicity have occurred recently and brought new challenge and problem in terms of PRV control. This study aims to insert PCV2 Cap gene into the viral live vector of PRV to develop genetic engineering vaccines of PCV2 and PRV.TK gene is one of PRV genes which are closely related to PRV virulence. It has been reported that PRV strains with TK gene-deleted can weaken its virulence obviously without affecting its replication ability. So this study firstly chose TK gene as the target site to insert PCV2 Cap gene to construct TK gene-deleted recombinant PRV virus that could express PCV2 Cap. To begin with, this study constructed two transfer vectors, one of which was pMD18T-LR(TK)-EGFP that was consisted of left and right arms and EGFP expression cassette while other one was pMD18T-LR(TK)-Cap that was consisted of left and right arms and Cap expression cassette. Subsequcently, the transfer vector pMD18T-LR(TK)-EGFP was cotransfected with PRV-JF genome DNA into PK-15 cells, hoping that the TK gene could be replaced by EGFP expression cassette based on the theory of homologous recombination. By selecting the plaque with green fluorescence and PCR amplication, the TK gene-deleted stain rPRV-TK-/EGFP+ was purified. At last, the recombinant virus rPRV-TK-/Cap+ containing one Cap expression cassette was also generated because of the replacement of the EGFP expression cassette through plaque purification and PCR amplication. Unluckily, IPMA test could not detect the Cap protein in this recombinant virus.In order to construct recombinant PRV virus that could express PCV2 Cap successfully, this study chose gE gene as another news target site. First of all, this study constructed two transfer vectors, one of which was pMD18T-LR(gE)-EGFP that was consisted of left and right arms and EGFP expression cassette while other one was pMD18T-LR(gE)-Cap(CI) that was consisted of left and right arms and Cap expression cassette. Then the transfer vector pMD18T-LR(gE)-EGFP was cotransfected with rPRV-TK-/Cap+ genome DNA into PK-15 cells, hoping that the gE gene could be replaced by EGFP expression cassette based on the theory of homologous recombination. By selecting the plaque with green fluorescence and PCR amplication, the TK/gE double gene-deleted stain rPRV-TK-/Cap+/gE-/EGFP+ was purified. Then recombinant virus rPRV-TK-/Cap+/gE-/Cap+(CI) containing two Cap expression cassettes were also generated based on the recombinant virus rPRV-TK-/Cap+/gE-/EGFP+ as materials by plaque purification and PCR amplication. The detection of the Cap expression by IPMA and Western blot tests was positive. To further increase the amount of Cap protein of the recombinant virus, this assay changed the promoter of the Cap expression cassette and used the optimized codon sequence of Cap gene. Consequently, recombinant viruses rPRV-TK-/Cap+/gE-/Cap+(CA) whose Cap expression cassette possessed double promoters and rPRV-TK-/Cap+/gE-/Cap+opti(CA) whose Cap expression cassette had both double promoters and optimized codon sequence of Cap were also purified. Both of these recombinant viruses expressed Cap protein effectively through IPMA and Western blot analysis. But, in terms of the amount of the Cap protein among the three different viruses, there was no obvious difference. Growth properties of the three different viruses were also similar but weaker than parent virus. Anminals tests showed these viruses were safe to rabbits with reduced virulence.The pseudorabies virus(PRV) UL42 protein, known as the DNA polymerase processivity factor, is an essential protein required for viral replication. The in vitro function of UL42 has been characterized; however, there is little information concerning the linear B cell epitopes of UL42 that are recognized during humoral immune responses. This study analysed six UL42-reactive mAbs(2D5, 4E2, 2G11, 5F8, 7C11 and 8F9) which were generated in our lab before. Through Western blot analysis, two regions of UL42(amino acids 39~148 and 302~384) that reacted with these mAbs were identified. Then a panel of UL42-derived peptides spanning the two regions were synthesized and screened by the six mAbs. Finally, three linear epitopes(116SGGVLDALK124, 354KRPAAPR360 and 360RMYTPIAK367) were identified by enzyme-linked immunosorbent assays. The 116SGGVLDALK124 epitope was located at the amino-terminus, while the other two epitopes were at the carboxy-terminus. Amino acid sequence analysis showed that the linear epitopes of UL42 were highly conserved among PRV strains. Using these mAbs, IFA result showed that UL42 localized to the nucleus during viral replication, and could be immunoprecipitated from PRV-infected PK-15 cells by immunoprecipitation assay. A UL42 mAb-based immunoperoxidase monolayer assay for the determination of PRV titers was also established. Taken together, our results indicate that the six generated mAbs could be useful tools for investigating the structure and function of UL42 during viral replication. |
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