| Zika virus(ZIKV)cause microcephaly in infants and is related to neurological diseases.Currently,there is no safe and effective vaccine.Studies have shown that pre-existing dengue virus antibodies can cross-intensify ZIKV infection,suggesting that traditional whole-attenuated live vaccine and inactivated vaccine strategies may be ineffective against ZIKV.ZIKV-specific protein-free domain III of the E protein(EDⅢ)that does not cross dengue virus(DENV).Therefore,we use ZIKV EDⅢprotein as the immunogenic gene displayed on the surface of the T7 phage to construct a new Zika vaccine that does not react with DENV and can provide neutral protection against ZIKV.T7 bacteriophage is an engineering carrier widely used in the field of life sciences.It is easy to cultivate,has a small genome,and is convenient for genetic engineering operations.Phage vectors can effectively display foreign genes without affecting the bacteriophage’s infection characteristics,while retaining the structure and biological functions of the foreign gene products,which is an ideal vector for new vaccines.Compared with traditional vaccines,phage carrier vaccine has the advantages of low cost,stable physicochemical properties,safety and reliability,and good immune effect.Objectives:In this experiment,the phage display system is used to study the biological characteristics of the recombinant phage T7-zEDⅢ Zika vaccine strain,and to study its immunogenicity.Methods:In this study,zEDⅢ gene and T7 phage 10B capsid protein gene were fusion expression.The expression correction of foreign protein EDⅢ on T7 phage were identification by PCR and Western blot.The successfully identified recombinant phage monoclones were added to e.coli BLT5403 in the logarithmic phase for extended culture.AfterPEG8000 concentration,the recombinant phage is precipitated and resuspended to obtain a high concentration of purified phage,and the phage titer is determined by the double agar sandwich method.The morphology of recombinant phage was observed by electron microscopy,and the activity of recombinant phage was studied at different pH and temperature.The genetic stability of phages was studied by continuous passage,and the one-step phage growth curve was studied by phage amplification in the host bacteria.The purified phage was inactivated by formaldehyde and mixed with an aluminium-hydroxide adjuvant to make a vaccine.The 6-week-old BABL/c mice were intraperitoneally injected with 1012pfu/ml and immunized with a booster shot at the 2th week afterthe first immunization.At the 2th,4th,6th,8th and 10th week after immunization,mouse serum were collected to detect the specific antibody by ELISA.Meanwhile,the neutralization titer of mouse antiserum against Zika virus was determined by serum neutralization test.The secretion of IL-2 and IFN-γ in mouse spleen cells induced by T7-zEDⅢ vaccine was detected by ELISPOT to determine the level of vaccine-induced cellular immunityResults:In this study,the foreign protein zEDⅢ can be correctly expressed in recombinant phage,and the recombinant phage vaccine strain was named T7-zEDⅢ.After purification,the titer of T7-zEDⅢ can reach above 7×1012pfu/ml.After exploring the growth characteristics of the recombinant bacteriophage,it was found that the recombinant bacteriophage T7-zEDⅢ has a wide pH range,and the optimal pH is 7.Studying its thermal stability,it was found to be stable at 40℃ and 50℃.The optimal multiplicity of infection was 0.001～1,while the 10th,20th,30th generation recombinant phage can be detected on the exogenous genes and proteins.By studying the one-step growth curve,the outbreak period of recombinant bacteriophage T7-zEDⅢ was 90 minutes,and the outbreak amount was 23.The mice were immunized twice with the recombinant T7-zEDⅢ vaccine,then blood samples were collected to detect specific anti-EDⅢ IgG titers.The antibody titers can reach 1:8000,1:128000,1:128000,1:64000,1:32000,1:32000 at the 1st,4th,6th,8th,10th,23rd week respectively,indicating that the vaccine can induced well and longer humoral immunization response than control groups in mouse model.Conclusion:We successfully constructed recombinant phage T7-zEDⅢ vaccine which showed stably inherited biological characteristics and good immunogenicity. |
