Expression And Immunogenicity Of Truncate F Protein Of NDV

Newcastle disease （ND） is a highly contagious disease, caused severe economic loss in global poultryindustry. Newcastle disease virus （NDV） mainly infects chickens, turkeys, ostriches and other birds. Thefusion protein （F） and hemagglutinin neuraminidase （HN） of NDV are the major protective antigens, whichplayed important role in the pathogenic process, so that the F protein and HN protein have been the betterchoice for a subunit vaccine antigen.Referred to gene sequences of the LaSota strains published in GenBank, two pairs of specific primers havedesigned according to the F protein epitopes. The chorioallantoic fluid was obtained from chicken embryosinoculated with attenuated LaSota strain.The two cDNA fragments of the F₇₈₀and F₇₆₀were amplified fromfresh chorioallantoic fluid by RT-PCR, respectively, and then were inserted into the pMD18-T vector. After theidentification of positive recombinants, those gene fragments were cloned into the pET30expression vectorbetween BamH Ⅰa ndHind Ⅲ. The plasmids called pET30-F₇₈₀and pET30-F₇₆₀were transformed into E. coliBL21（DE3）. After IPTG induction, the target proteins which sizes were approximately27.9kDa and31.1kDahad been expressed by SDS-PAGE screening. Westem-blotting showed that the recombinant F₇₈₀and F₇₆₀proteins were of reactiongenicity.Intramuscular immunization of chicken was performed with the recombinant protein of F₇₈₀or F₇₆₀orboth purified from E. coli BL21after induced by IPTG. The chickens were randomly divided into three dosagegroups （I, II, III;50ug/bird、100ug/bird、150ug/bird） which were further divided to three antigen parts (F₇₈₀,F₇₆₀, both) per goup. The goup IV and V intramuscularly inoculated with attenuated LaSota vaccine strainand PBS as control. The specific antibody titers were detected by HI. The results showed that significant HItiters in goup IV were higher then goup I, II and III with significant difference from PBS control （P<0.01）. TheHI titers of different antigen in goup I were lower then that in goup II and goup III，but there was notdifference between goup II and goup III. The results of HI titers in same antigen parts indicated that immuneeffect of both F₇₈₀ and F₇₆₀ was not better than that of F₇₈₀ or F₇₆₀, but there were significant differentfrom PBS control. Specific IgG antibody in serum were detected by indirect ELISA.The results showed IgGtiters in recombinant protein groups and in attenuated LaSota group were significant higher then the control ondays14after immunization. The challenge was performed with10⁵EID₅₀F48E8virulent strain on days14after the second immunization.25%，12.5%，25%, in group I,50%,25%,50%in group II,50%、37.5%、50%in group III and100%in LaSota goup survived after challenged with F48E8virulent strain, all dead inthe control group. All those data demonstrated that the recombinant proteins have some immunoprotectionagainst the virulent Newcastle Disease Virus.