Construction, Identification And Experimental Immunization Of Goatpox Virus Gene Deleted Mutant And DNA Vaccine

Goat pox, caused by goatpox virus (GTPV), is an acute feverish and contagious in goats characterized by fever, generalized papules or nodules, vesicles, internal lesions, and death. As the most important of all pox disease of domestic animals, goat pox causes high mortality and significant economic losses, poses a major obstacle in international trade. Accordingly, goatpox is categorized as notifiable diseases and the first group disease by the World Organization for Animal Health (OIE), and the Ministry of Agriculture of China, respectively.Both live attenuated and inactivated vaccines are useful in the prevention and control of goat pox. Inactivated vaccines give only short-term immunity. Although highly immunogenic and longer term of immunoprotection do live attenuated vaccines possess, nowadays, they still have some problem need to solve, including the latent dangers of persistence infection and virulence reversion, and the difficulty in distinguish vaccinated animals from infected ones. AV41, the live GTPV vaccine strain widely used in China, has good immunogenicity and potential to be developed as a live viral vector. However, recently increasing reports showed that it may cause generalized skin nodules and misbirth in goats of native breed in southern China, imported pure breed and crossing breed. Therefore, it is necessary to develop a more safe and effective gene deleted attenuated GTPV.In is study, we chose AV41 strain as parental virus. At first, an 830 bp genome fragment contain thymidine kinase gene (ORF66), and 430 bp E coli gpt gene were cloned, respectively. Then, a GTPV transfer vector pTK-Eg was constructed, which contains an EGFP reporter gene and gpt resistance gene. Co-transfected pTK-Eg together with AV41 in Vero cell, a recombinant GTPV, designated as vTK-Eg, was obtained after 3 rounds selections of MPA, plague purification and PCR identification. Then, vTK-Eg was examined by PCR and TCID50 during passaging to 10th in BT and Vero cells. The result showed that vTK-Eg was stable, and the impairment of TK gene had no obvious effect on CPE, the morphology and multiplication of virus in culture cells. Furthermore, experimental immunization in goats showed vTK-Eg had lowed virulence, causing less skin lesions and fever, however, still remained good immunogenicity of AV41. Moreover, it could also elicit antibody response towards foreign gene-EGFP.Basing the successful experience obtained by Paoletti during they constructed the VACV gene deleted attenuated mutant NYVAC, it is maybe a promising approach to attenuate GTPV by delete large noncaonservative genome fragment. Therefore, genome fragment of AV41 ORF7～ORF8, and ORF18～ORF19 were cloned as flanking sequences for homologous recombinant, respectively. Then, another transfer vector pCF-Eg was constructed by insert the expression cassettes of EGFP gene and gpt gene. Through homologous recombinant of pCF-Eg and AV41 in Vero cell, an ORF8～ORF18 deleted GTPV was obtained. And it is the first report to obtain a recombinant GTPV with 9 ORFs deleted.Serpin gene encoded by poxvirus was reported to be a virulence gene and non-essential gene of poxvirus. In this study, serpin gene (ORF149) of GTPV AV41 with length of 1041bp was cloned. Then, the gene fragment was sequenced and analysised. And Result shows that serpin gene of AV41 share highest sequence homology with those of GTPV (≥97%), LSDV and SPPV (93%～95%). Moreover, it has 36%～38% amino acid sequence homology with that of SPI-2 of VACV and a conservative VADC motif, suggesting they have same P1 amino acid- Asp (D) in RSL, and similar bio-activity. Then, an expression plasmid pcDNA-Serpin was constructed. And a HeLa cell stably expressing GTPV-serpin was obtained, after transfected pcDNA-Serpin into HeLa cell and selected using G418. Results of morphology observation, cell viability and caspase activity measure showed, GTPV serpin could hamper the activation of caspase-8, inhibit about 69% apoptosis of HeLa cell induced by TNF-αand cycloheximide. It is the first report for the research on the bio-function of GTPV serpin. Moreover, a transfer vector pSp-LgE was obtained by insert the expression cassettes of LacZ, EGFP and gpt gene. And finally, a recombinant GTPV with serpin gene disrupted, designated as vSp-LgE, was obtained after homologous recombinant and selections.Envelope protein genes A27L (ORF117), L1R (ORF122), A33R (ORF141) and B5R (ORF60) of GTPV AV41 were cloned, respectively, and linked side-by-side with 2A gene of FMDV, to generate two dual-cistron expression cassette AAL and BAA. Two suicide DNA vaccine plasmids pCS-AAL and pCS-BAA were constructed by insert AAL and BAA into a modified semliki forest virus RNA replicon basing expression vector pCSm. And the expressions of foreign genes were identified by IFA and Western blot. In the meantime, A27L gene, truncated A33R and B5R genes with secrete signal pipette and trans-membrane domain encoding sequences deleted were obtained by PCR, and inserted into prokaryotic expression vector pET32a, and expressed in E coli with Trx gene fusion. Via metal chelate chromatography, inclusion body washing and gel-cutting retrieval, recombinant proteins were prepared, with purity of 90%, and output of 20.8mg/L, 5.5mg/L and 12mg/L. At last, experimental immunization in mice demonstrated, these two suicide plasmids could elicit specific humoral and cellular responses against GTPV. And the strongest immune response was achieved by inoculated two DNA simultaneously.Also in this study, a duplex PCR was developed and optimized for simultaneous detection and differentiation of capripoxvirus (including goatpox virus and sheeppox virus) and orf virus. Detection limit of this duplex PCR assay was 1 PFU for both capripoxvirus and orf virus. At last, two suicide plasmids were injected into goats, followed by the boost of AV41 or two plasmids themselves. Results shows that these two DNA could elicit specific reaction antibodies, Neutralizing antibodies, lymphproliferation response towards GTPV, not only provide partly protection against viral challenge, but also significantly reduce the pathogenicity of AV41 on goat, but not affect latter's immunogenicity.In summary, these researches can provide a useful research platform for safer, high effective live GTPV attenuated vaccine and viral vector development, and basic data for the studies on the pathopoiesis and immunoregulation mechanism of poxviruses.