Expression And Immunogenic Analysis For Epitope Structural Domains Of The Newcastle Disease Virus Fusion Protein

Newcastle Disease(ND) which is also called chicken pest of Asia,caused by Newcastle disease virus(NDV),is a kind of highly contagious disease.NDV can cause many kinds of poultry such as chickens,turkeys,ostriches,pigeons,peacocks and other wild birds ill.The vaccination against ND does not eradicate periodic outbreaks of the disease in immuunized poultry,and enormously make economic losses.Though the reason of the outbreaks of the disease in immunized poultry have not been known,the varation of NDV may play a major role.It is well known that F fusion protein located on the membrane of NDV plays an important role in the course of NDV infection,such as its penetration into host cells,fusion of viral and cellular membranes and the transference of viral genetic material in the cells.F protein is also a kind of viral protective antigen and crucially virulent structure.Therefore,it is considered as the most predominant antigen in the vaccine including genetic engineering vaccine and DNA vaccine.In order to research the function and mechanism of F protein in immune protective response,we cloned, expressed and identified the antigenity of epitope configuration region of Fgene.Firstly,according to F gene sequence of F48E9 registered in GenBank and multi-cloning restriction enzyme sites of vector pGEX-4T-1 and pET-32-a,five pairs of specific primers for the section fragment encoding chicken Newcastle disease virus F gene were designed and synthesized respectively.Using total RNA from allantoic of SPF chicken embryonating eggs,five fragment about 509bp,81bp,108bp,105bp,102bp were amplified by RT-PCR respectively.The tandem-arranged multiple epitope gene of the F gene was obtained by appending complementary endonudease of genetic engineering means.Then the genes were inserted into vector pGEX-4T-1 and pET-32-a respectively, and the recombinant plasmids were transformed into E.coli BL21.The recombinant plasmid pGEX-4T-1/F509was identified by EcoRI+SalI digestion and the recombinant plasmid pET-32-a /F306was identified by EcoRI+Xhol digestion.Then,the positive recombinant clone was sequenced and analyzed.The results suggest that the epitope configuration region of Fgene and the tandem-arranged multiple epitope gene of the F gene have been successfully cloned from allantoic of SPF chicken embryonating eggs.Secondly,the constructed recombinant plasmids pGEX-4T-1/ F509,pET-32-a/F₁,pET-32-a/F₂,pET-32-a/F₃,pET-32-a/F₄ and pET-32-a/F306 were transformed into E.coli BL21 and then induced to express GST-F509,His-F₁,His-F₂,His-F₃,His-F_a and His-F306 fusion protein by IPTG after cultivating for 4 hours.After optimizing prokaryotic expression conditions,4h was determined as the optimum induction time and 0.5mmol/L was determined as the optimum concentration of IPTG..The solubility of fusion proteins was identificated and the result indicated that expressed fusion proteins were located in inclusion bodies.Under the optimal condition,the recombinant plasmids were induced to express large-scale fusion proteins and the induced recombinant bacteria were lysed by freeze-thaw and sonication.The obtained GST-F509,His-F₁,His-F₂,His-F₃,His-F_a and His-F306 inclusion body proteins were solubilized by sonication with the adding of the detergent lauroylsarcosine,and the solubilized fusion proteins were obtained at last.Finally,the fusion proteins were reacted with the obtained antiserum against the NDV, and the results showed that GST-F509,His-F₁,His-F₂,His-F₃,and His-F306 fusion protein had strong specific reaction.Then mice were immunizated with the purified GST-F509,His-F₁, His-F₂,His-F₃,His-F_a and His-F306 fusion proteins and the polyclonal antiserum against the epitope configuration region of F gene were collected.A specific reaction appeared between the antibodies and GST-F509,His-F₁,His-F₂,His-F₃,and His-F306 fusion protein in agar diffusion assay and in ELISA,these results indicated that fusion proteins,GST-F509, His-F₁,His-F₂,His-F₃and His-F306 had strong antigenity.In conclusion,in this study the genes of epitope configuration region of Fgene were cloned successfully,the recombinant of plasmids pGEX-4T-1/F509,pET-32-a/F₁,pET-32-a/ F₂,pET-32-a/F₃,pET-32-a/F₄ and pET-32-a/F306 were constructed and the fusion proteins GST-F509,His-F₁,His-F₂ His-F₃,His-F_a and His-F306 were expressed in prokaryotic cells and purified.The antigenity of the fusion proteins were identified,and the specific polyclonal antiserum against the epitope configuration region of Fgene were prepared.All these would provide some experimental materials for the further studies on the epitope vaccine of NDV F gene.