Rapid Selection Of Recombinant Orf Virus NA1/11Δ132 And Constructing The Recombinant Orf Virus NA1/11-VP1 Expressing The VP1 Proteins Of The Enterovirus 71

Orf virus(ORFV) is the prototype member of the genus Parapoxvirus,belong to the subfamily Chordopoxvirinae of the Poxviridae. The orf virus includes linear double-stranded DNA along with 64% G+C content.. The total length is 134 to 139 kb containing the conserved central structures and inverted terminal repeats (ITR). Poxviral genes essential for viral replication are located in the central region of the genome and are highly conserved among the different poxvirus species. Genes located near the genomic termini usually encode viral functions nonessential for replication, but play a role in pathogenesis, host and tissue tropism, and virulence.The host range of the orf virus is very restricted, mainly affacts sheep and goat, though reindeer, musk oxen and humans can also be infected. Virus transmission occurs via direct contact with infected animals, and mostly invade through the skin around the mouth, rarely extend into the other organs like the stomach, intestine, or the respiratory tract. As its self limiting, the virus does not cause systemic virus dissemination, and the overall death rate is very low. However the mortality may increase to 80% in lambs, children and those with immunodeficiencies.Therefore, it is very necessary and effective to research and develop the orf virus vaccine, which is the focus of the current study in the laboratories of the world. The traditional orf virus vaccine is mainly the inactivated vaccine, but they do not have a complete capsule, often only provide temporary protection, and the inactivated vaccine can not effectively active the immune cells in vivo, greatly limit the use of vaccine. Using gene engineering technology to knock out the virulence related gene of orf virus to construct the attenuated live virus vaccines becomes a new hot spot in the research of vaccine research. The orf virus encodes the vascular endothelial growth factor VEGF, which is similar to the ovine and other mammalian VEGFs, bind with the VEGF receptor-2(KDR), stimulates endothelial cell proliferation, vascular permeability and angiogenesis. The VEGF is the virulence gene of the orf virus, the recombinant orf virus lacking the gene of VEGF is attenuated, may become the new type of the live attenuated vaccine in the research. Besides, orf virus could be an ideal cancer therapeutic candidate in the future. Orf virus has been documented as an efficious immunotherapy in the models of the lung cancer by the Rintoul.What’s more, the location of the VEGF gene is very suitable for expressing heterologous antigens. Based on the attenuated ORFV strain D1701-V, recombinants were produced as the vaccine to protect the mice against the herpes virus, rabbit hemorrhagic fever virus, rabies virus, H5N1 and H1N1 influenza A virus. Ideal vector vaccine properties are theshort-lived ORFV vector-specific immunity allowing repeated immunizations, and still not entirely understood immunomodu-lating properties, which lead to the induction of strong innate and daptive Thl-Th2 balanced immune responses. And the orf virus replicate in the cytoplasm and does not insert a host cell chromosome causing mutations in the gene.Therefore, it has been proposed as a promising candidate for novel vectored vaccines.By now, not only the New Zealand OV strain NZ2 has the detailed information about the genetic structure and transcriptional organization, but also the American strain IA82, German strain D1701 and Chinese strain NA1/11 are exclusively available for the full gene sequence comparative analysis. Chinese OV strain NA1/11 was isolated from tissue collected from a farm located in Nongan County of Jilin Province, and its gene 132 encodes the vascular endothelial growth factor VEGF. In this experiment, we construct the shuttle plasmid pSPV-132LF-EGFP-132RF. The plasmids were transfected into OFTu cells and the GFP was incorporated into the NAl/11 by homologous recombination. The recombinant NA1/11A132 was selected by the green fluorescent signal. The deletion gene was identified by PCR and sequencing. Besides, the effects of knocking out of the ORFV132 were demonstrated by the virus titration and the test of stimulating vascular endothelial cell proliferation in vitro.The recombinant orf virus NA1/11 A132 was obtained successfully and quickly, and the deletion of ORFV 132 did not affect the replication in vitro, but could reduce its virulence. The attenuated recombinant orf virus NA1/11 Δ132 is expected to be a new live attenuated vaccine and proposed as a promising candidate for novel vectored vaccines.In addition, in order to construct the candidate vaccine of hand, foot and mouth disease, we also use the EV71 VP1 gene to replace the gene 132 of orf virus NA1/11 through homologous recombination method, and successfully select the recombinant orf virus by green fluorescent signal, named as the NA1/11-VP1. There are two parts in our study as follows.Part I Rapid Selection.of recombinant Orf virus NA1/11A132The Green fluorescent protein (GFP) is firstly found in Aequoreavictoria jellyfish in 1962 by Shimomura et al. The GFP, which is of great significance in the study of protein expression, protein and cell fluorescence tracing, protein structure and function, is widely used in the study of bacteria, plants and even human as a reporter. The traditional recombinant virus was selected mainly by the neomycin resistance and beta glucuronidase (neomycinresistance and beta -glucuronidase, neo/gus) blue white screening. However, the protocol is labor intensive and time-consuming.In this study, the flanking regions of the ORFV132 were amplified by PCR from the extracted virus NA1/11 DNA to construct the shuttle plasmid pSPV-132LF-EGFP-132RF. The plasmids were transfected into OFTu cells and the GFP was incorporated into the NA1/11 by homologous recombination. After two rounds of 96-well plates limited dilutions and three rounds of 6-well plates plaque assays, the recombinant Orf virus was selected and identified by PCR and sequencing.The ORFV132 deletion virus was named as NA1/11Δ132. Besides, the effects of knocking out of the ORFV132 were demonstrated by the virus titration and the test of stimulating vascular endothelial cell proliferation in vitro.Part II Constructing the recombinant Orf virus NA1/11-VP1 expressing the VP1 proteins of the enterovirus 71At the foreign research, the location of the VEGF is suitable for insertion and expression of foreign genes. In this study,we use the EV71 VP1 gene to replace the ORFV132 of NA1/11 by the homologous recombination method, constructing the recombinant orf virus NA1/11-VP1 for the candidate vaccine of hand, foot and mouth disease. The VP1 cDNAs were amplified by RT-PCR from the extracted viral RNA and cloned into the plasmids pSPV-132LF-EGFP-132RF. The plasmids were transfected into OFTu cells and the VP1 was incorporated into the NA1/11 to replace the ORFV132 by homologous recombination. The recombinant NA1/11-VP1 was selected by the green fluorescent signal. The inserted VP1 gene was identified by PCR and sequencing. And the expression of EV71 VP1 were demonstrated by the indirect immunofluorescence and Western blot. To our amazation, the GFP gene was successfully expressed and the green fluorescent signal was detected with a Microscope, but in the same location,the EV71 VP1 cannot be detected by the EV71 VP1 monoclonal and polyclonal antibody. There may be the following reasons:(1) The EV71 protein expression was too low; (2) In the same gene position, double promoter can not set up two different protein express; (3) The GFP protein influences the detection of VP1 protein. What anyway, the specific reasons are needed further study.Conclusion:1、The recombinant NA1/11A132 was selected by the green fluorescent signal. The deletion gene was identified by PCR and sequencing.2、The advantages of GFP fluorescent selection compared to conventional strategies are as follows:(i) visual monitoring of infected cells by fluorescent microscopy to assess the level of rORFV infection; (iii) easy to determine the optimal time to harvest cells; (ii) streamlining the procedures and shortening the time needed to isolate and purify the plaques (less than 15 days), compared to 3 or 4 months using neo/gusA method; (iv)much cheaper thanby fluorescence activated cell sorting (FACS) (v) picking plaques directly under fluorescent microscope,no antibiotics and substrates needed.3、The deletion of ORFV132 did not affect the replication in vitro, but could reduce its virulence.4、In the same gene position, double promoters can not set up two different protein express;5、The GFP protein influences the detection of VP1 protein.6、The EV71 VP1 protein expressed in the orf virus NA1/11 of the of 132 location should be in secreted protein form, but also may be inclusion body which affect the detection of the protein.