Discussion On The Recombinant Strategies Of FMD Gene Engineering Vaccine

Foot-and-mouth disease (FMD) was caused by FMDV, is a highly contagious, acute vesicular disease of cloven-hoofed animals, and is responsible for large economic losses, both as a direct result of the disease, and because of the necessary restrictions imposed on animal products and trade for disease control. Many researchers in the world pay attention to prevent and control this disease.Although both of attenuated FMD vaccine and inactivated vaccine are effective, major problems were encountered such as unpredictable virulence in the field, high-containment vaccine production facilities, carried a slight risk of residual contamination of inactivated vaccines with live virus. Its use would most probably complicate the discrimination of naturally infected and vaccinated animals. Several novel approaches to vaccine development are emerging, and these may enjoy significant advantages over conventional vaccines. However, as yet they remain too costly for general veterinary use, and their immunogenicity is often limited. One way to remove this shortcoming is to improve the adjuvants used in the formulation of the vaccine. In general, there are some potent adjuvants, such as interferon(IFN-a,IFN-β,IFN-γ); lymph cytokines(IL-2,IL-3,IL-4,IL-5,IL-10); monocytines(IL-1,IL-6,IL-8,IL-12) and other cytokines.In this study, the antigen of FMDV and some adjuvants (HSP70, PoIL-2, PoIFN-a) were co-expressed in methylotrophic yeast expression system and adenovirus vector expression system respectively. The affection of adjuvants on FMDV recombinant gene-engineering vaccine in mice was detected. This will provide novel ways for study of foot-and-mouth newly vaccine. All studies were listed as below:1. VP1-2A gene of FMDV type O was synthesized and cloned into pMD18-T, named pMD18-VP1-2A (It had been done by Shanghai Invitrogen company) . VP1 gene was amplified from pMD18-VP1-2A and subcloned to pET-32a(+), named pET-VP1, recombinant VP1 was expressed in E.coli induced with IPTG. Recombinant protein was testified by SDS-PAGE, and which was expressed successfully and relative molecular mass was about 44kD. Western-blot result indicated that the recombinant protein had specific immunogenic characteristic against antibody of FDMV. ELISA test indicated that 10.56μg/ml of purified recombinant VP1 protein was coated as antigen can distinguish positive serum and negative serum of FMDV. This study was the basic for further study.2. Multi-epitopes genes of 0 type foot-and-mouth diseases virus was synthesized and cloned into pMD18-T and named pMD18-EG (It had been done by Shanghai Invitrogen company). M.tuberculosis HSP70 gene was amplified from pMD18-HSP70. VP1 gene, EG gene and HSP70 gene were cloned into yeast expression vector pPICZaA. Four recombinant vectors were constructed and named pPICZaA-EG,pPICZaA-VP1,pPICZaA-EG-HSP70,pPICZaA-VP1-HSP70 respectively. Recombinant vector were transformed into methylotrophic yeast Pichia pastoris X-33 by electrophoration. The recombinant transformants were selected by Zeocin and induced by the addition of methanol every 24 h. The result of SDS-PAGE indicated that the recombinant proteins were successfully expressed in methylotrophic yeast Pichia pastoris and relative molecular mass were about 23kD(rVP1), 20kD(rEG), 89kD(rEG-HSP70), 92kD(rVP1-HSP70). Western blotting indicated that four recombinant proteins had specific antigenicity of FDMV. Mice were inoculated transcutaneous three times at a two-week interval with fusion protein, PBS and conventional inactivated vaccines. Level of IgG,IgG1,IgG2a,IL-4,IFN-γand proliferation T lymphocyte test were detected to evaluate humoral immune and cellular immune responses. The results indicated that fusion protein could elicit specific humoral immune and cellular immune responses. Compared with conventional inactivated vaccines, fusion protein elicited close to FMDV antibody level. Cellular immune responses in turn is rEG-HSP70, rVP1-HSP70, inactivated vaccine, rEG, rVP1, which suggest that HSP70 as molecular adjuvant can improve the immunogenicity of FMDV recombinant vaccine, thus provide valuable support for further development of FMDV genetic engineering vaccines.3. VP1 gene and multi-epitopes gene were synthesized and cloned into pAdeno Vator-CMV5-IRES-GFP, named pA5-EGS and pA5-VP1 respectively. pA5-EGS and pA5-VP1 were Linearized by Pme I, and then linearized vector and pAdeno VatorΔE1/E3 co-transformed into E.coli BJ5183 competent cells by electrophoration. The homologous recombinant plasmids pAd5-EGS and pAd5-VP1 were selected and Linearized by Pac I to expose the encapsidation signal. Linearized virus plasmids were transfected into 293A cells by Lipofectamine ^TM2000, and recombinant viruses were named rAd5-EGS and rAd5-VP1. Infection titer and rate were evaluated through monitoring green fluorescent protein (GFP) expression. The filter of rAd5-EGS and rAd5-VP1 are 10^10.62个TCID₅₀/ml and 10^11.87个TCID₅₀/ml respectively. The recombinant viruses were passaged 5 times in 293A and the titer is still stable.. Expression of VP1 and multi-epitopes of FMDV were testified by PCR and western-blot, wetern-blot result indicated that expression products had specific antigenicity of FDMV.4. VP1-2A gene of FMDV was and porcine Interleukin-2 gene was amplified from pMD18-VP1-2A and pMD18-poIL-2(pMD18-T contains porcine interleukin-2) respectively and cloned into pAdeno Vator-CMV5-IRES-GFP, named pA5VP1-2A-PoIL-2 and pA5-PoIL-2. Recombinant vectors were linearized by Pme I, and then linearized recombinant vectors and pAdeno VatorAEl/E3 co-transformed into E.coli BJ5183 competent cells by electrophoration. The homologous recombinant plasmids were selected and linearized by Pac I to expose the encapsidation signal. Linearized virus plasmids were transfected into 293A cells by Lipofectamine?2000, and recombinant viruses were named rAd5-VP1-2A-PoIL-2 and rAd5-PoIL-2. Western-blot indicated that rAd5-VP1-2A-PoIL-2 had specific antigenicity of FDMV and VP1-2A-PoIL-2 was cleaved by 2A with VP1-2A and PoIL-2. The lymphocyte proliferation test indicated that rPoIL-2 had biological activity.5. Porcine IFN-a (PoIFN-a) gene was amplified from pMD18-poIFN-a, and cloned into pAdenoVator-CMV5-IRES-GFP vector containing VP1-2A gene, named pAd5VP1-2A-PoIFN-a, which was linearized by Pme I, and then linearized pAd5VP1-2A-PoIFN-a and pAdeno VatorAEl/E3 co-transformed into E.coli BJ5183 competent cells by electrophoration. The homologous recombinant plasmids pAd5VP1-2A- PoIFN-a was selected and Linearized by Pac I to expose the encapsidation signal. Linearized virus plasmids were transfected into 293A cells by Lipofectamine?2000, and recombinant viruses were named rAd5VP1-2A-PoIFN-a. Western-blot indicated that rAd5-VP1-2A-poIFN-a had specific antigenicity of FDMV and VP1-2A-PoIFN-a was cleaved by 2A with VP1-2A and PoIFN-a.. Recombinant virus was verified to be of high PoIFN-a activity by inhibiting the cyto-pathogenic effect, 10⁵U/ml. 6. 6 weeks old female BALB/c mice were randomly separated into 14 groups, 12 mice per group, mice were inoculated intraperitoneal with recombinant viruses(rAd5VP1,rAd5EGS,rAd5poIL-2,rAd5poIFN-a,rAd5VP1-2A-poIFN-a and rAd5VP1-2A-poIL-2) three times at 2 weeks intervals, and with PBS and conventional inactivated vaccines as control. Level of IgG,IgG1,IgG2a,IL-4,IFN-γand proliferation T lymphocyte test were detected to evaluate humoral immune and cellular immune responses of recombinant viruses. The results indicated that rAd5VP1-2A-poIFN-a, rAd5VP1-2A-poIL-2 induced more stronger responses than inactivated vaccine done. The results indicated that cytokines (IFN-a and IL-2) enhanced immunity of recombinant virus. FMDV 2A separated the polyprotein into its single proteins and thus provide a maximal degree of freedom for cytokines activity, recombinant viruses showed VP1-2A-PoIL-2 and VP1-2A-PoIFN-a at a size of 25 kDa in Western blot analyses using an anti-FDMV antibody, revealing a proper cleavage of the fusion protein. ELISA test of antibody sub-type results indicated that recombinant viruses induced secretion both of IgG1 and IgG2a, partial to IgG1.The seem result elicited from cytokines ELISA test, secretion of both of Th1(γ-IFN) and Th2(IL-4) were induced by recombinant viruses, partial to Th2. This study demonstrates the VP1-2A-PoIL-2 (VP1-2A-PoIFN-a) fusion construct to be an efficient tool encoding two functions in one open reading frame. This promising candidate can be for instance in viral vectors vaccine studies. Thus provide valuable support for further development of FMD genetic engineering vaccines.