Cloning And Expressing Of Partial VP2 Gene Of Infectious Bursal Disease Virus And Their Identification

Infectious bursal disease is an acute,highly contagious viral disease mainly imperiling young chickens.Infectious bursal disease virus can not only cause high mortality in 3-6week susceptible chickens,but more seriously induce immunosuppression in young chickens,and subsequent vaccination failures for important chicken infectious diseases,such as New Castle's disease,Marek's disease,infectious bronchitis etc.In order to construct a quick and accurate diagnostic technique,to analyze if the antigenic gene of vaccine mutated in our country,in this research,a RT-PCR technique was primarily established to detect IBDV,the partial gene of VP2 which include the epitope was obtained,prokaryotic expression recombinant plasmid was constructed,and the partial VP2 gene was successfully expressed.Firstly,according to the VP2 gene sequence accessed in GenBank and the research by eldership,a pair of primers which embrace the restriction endonuclease site and protective Base pair were designed as follows:P₁(5'-CGGAATTCCCAAAAA TGGTAGCCA-3');P2(5'-GCGTCGACTGCCACTCTTTCGTAGG-3').Attenuated Vaccines were diluted by PBS,and SPF embryonating eggs were inoculated by the diluent,total RNA of virus was extracted from allantoic by the method of Trizol rea-gent,RT-PCR were used to amplify the fragment,results showed that total RNA from allantoic can be used as the template of RT-PCR,and fragment of 504 bp was successsfully amplified which is consistent with what is expected,then it was inserted into the vector PGEX-4T-1,and transformed it into Ecoli BL21,positive clone was chosen by bact-erial culture PCR and sequenced after it was identified by plasmid PCR and Restriction Endonuclease Reaction,sequence analysis showed that the homology with Chinese strains of DQ202329 are 99.8%,while with the foreign strains of AY134874,AY598356,AF109154 are 93.5%,93.5%,94.8%.Respectively,all these indicated that the expected fragment was uccssfully amplified and it found the basis for RT-PCR method to rapid diagnose IBDV.Secondly,according to the sequence measured and the polyclonal sites,the other two prokaryotic expression primer P₃(5'-CGGAATTCATGCCATTCAATCTTGTG-3') P₄:(5'-GCGTCGACTGCTAGTTCAGGAATTGG-3') was designed.We added endonuclease site and protective Base pair in the 5' terminal of primer in order to express.Prokaryotic expression fragment of 273bp was amplified and then inserted into the vector PGEX-4T-1,transformed it into the Ecoli BL₂₁,recombinant clone was chosen by bacterial PCR and identified by restriction endonuclease reaction, recombinant bacteria was induced with IPTG,the expressive protein was detected by SDS-PAGE electrophoresis,the result showed that the molecular weight of expressive protein is in accordance with that expected.In a word,this research proliferate IBDV by SPF embryonating eggs,variable region in VP2 gene include antigenic determinant was cloned and part of it which include one antigenic determinant was successfully expressed,all these has offered experimental foundations for the construction of RT-PCR method to detect IBDV and the preparation of genetic engineering vaccine.