Segmental Expression And Immune Activity Analysis Of Latent Antigenic Epitopes For NDV HN Protein

On the basis of the amino acid sequences of NDV F48E9 and LaSota HN structure protein,this research took the methods of Chou-Fasman,Garnier-Robson,Emini, Kyte-Doolittle and Karplus-Schulz to predict the secondary structure,superficial possibility, hydrophilicity and flexibility of the HN protein,to analyze the structure and nature of the HN protein in the single parameter;then,by the Jameson-Wolf method,it evaluated comprehensively the antigenicity of the HN protein.The results indicate that there are thirteen different latent antigenic epitopes between F48E9 and LaSota HN structure protein, and the discrepancy in the 10th(212-218AA) and 21st(492-498AA) epitope is extremely remarkable,which suggests that the 10th and 21st epitope maybe the protective antigenic epitope of NDV.This research provides theoretical basis for further testing protective antigenic epitopes of NDV HN structure protein and developing the effective ND vaccine. HN protein is the main component of surface spike of NDV capsule,which can stimulate the body to produce antibodies,so it is the major protective antigen of NDV.To identify the position of protective epitopes of HN protein,this research cloned and expressed segmentally HN gene,and analyzed the immune activity of HN segmental recombinant protein.Firstly,according to NDV F48E9 HN gene sequence registered in Gen Bank and multi-cloning sites sequence of pGEX-4T-1 vector,one pair of specific primers were designed to amplify the whole HN gene from chicken embryo allantoic fluid.Then on the basis of discrepancy in latent antigenic epitopes of HN protein,four pairs of specific primers were designed to divide HN gene to four segments,pGEX-4T-1-P225, pGEX-4T-1-P435,pGEX-4T-1-P444 and pGEX-4T-1-P558 recombinant plasmids were constructed.The results of PCR and sequencing indicated that the recombinant plasmids have been constructed successfully.Secondly,GST-HN225,GST-HN435,GST-HN444 and GST-HN558 segmental recombinant proteins were induced to express in prokaryocyte by IPTG,expression forms were identified,expression conditions were optimized,then fusion proteins were expressed largly.The induced recombinant bacterias were lysed by freeze-thaw and sonication, inclusion body proteins were obtained,which could be solubilized by sonication after the detergent lauroylsarcosine was added.The results of SDS-PAGE indicated that four segments of HN gene were expressed successfully in E.coli,the MW of the fusion proteins were about 33.5KD,40.5KD,40.8KD and 44.6KD,all the expressed fusion proteins were located in inclusion bodies,and 0.75mmol/L 3h was the optimum expression condition.Finally,in order to examine the immune activity of HN segmental recombinant proteins,mice were immunizated with the purified GST-HN225,GST-HN435,GST-HN444 and GST-HN558 to develope multi-antibodies.The results of Western blotting indicated that all the HN segmental recombinant proteins had strong immune activity.In conclusion,in this research NDV HN gene was cloned and expressed segmentally, purified fusion proteins were obtained,corresponding multi-antibodies were generated,and the immune activity of HN segmental recombinant protein were proved.This research provides basis for further identifying protective antigenic epitopes of NDV HN protein.