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

Infectious bursal disease(IBD),an acute and high contactable contagious disease,is caused by Infectious bursal disease virus(IBDV).It was first reported by Cosgrove in 1957, then characterized by the world popularity,and outbroke in over thirty countries, especially in the developed area of poultry-producing,it broke out in BeiJing,GuangZhou,ShangHai of our country in 1979 and became popular all over the country after that,which brought great economic losis,Although the government spent much money and take many measures to control and eradicate this disease,it is still one of the first contagious diseases which threat poultry industry nowadays due to the existence of very virulent and mutant strains.IBDV mainly do damage to the young chicken about 3-12 weeks,destroying B lymphocytes in the bursal and leading to immunosuppression,Therefore,it is easy to infected with other disease and manifest subclinical symptoms which bring difficulty to diagnose.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'-CGGAATTCACAGGGCTAAT-3'); P₂(5'-GCGTCGACTGCTAGTTCAGGATTTGG-3').Attenuated Vaccines were diluted by PBS,and SPF embryonating eggs were inoculated by the diluent,then infected the fibroblast egg cell with the allantoic,total RNA of virus was extracted from allantoic and the deposit of cell culture by the method of Trizol reagent,RT-PCR were used to amplify the fragment,results showed that total RNA from both allantoic and deposit of cell culture can be used as the template of RT-PCR,and fragment of 987 bp was successfully amplified which is consistent with what is expected.Site of restriction endonuclease was found in the accessed sequence by use of DNAStar and amplified fragment was identified by Restriction Endonuclease Reaction,then it was inserted into the vector PET-32a,and transformed it into Ecoli DH5α,positive clone was chosen by bacterial 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, AY321509 are 99.8%,99.2%,while with the foreign strains of AJ621158,AJ577092,AF508177 are 97.3%,99.3%,94.8%respectively,all these indicated that the expected fragment was successfully amplified and it found the basis for RT-PCR method to diagnose IBDV.Secondly,according to the sequence measured and the polyclonal sites,the other prokaryotic expression primer P₃(5'-GCGTCGACCTGTGATGAGAATTGGT-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 504bp 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 two means,partial VP2 gene include antigenic determinant was cloned and part of it 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.