Identification Of Different Strants Of IBDV And Experission Of VP2 Gene In Escherichia Coli

Infectious bursal disease (IBD) is an acute and highly contagious disease caused by IBDV. IBDV mainly invades bursa, the central immune organ of chickens. It can cause infected chickens dead and the production performance drop. Birds which survive the disease are permanently immunosuppressed. Immunosuppresssion leads to vaccination failure and susceptibility to other infections and diseases, Newcastle disease, Marek's disease and IBV. The bird researchers have payed more attention to it. IBD has been one of the main infectious diseases so far. Especially for the vvIBDV appeared in the 1981s, it made mortality rate of chicken infected up to 60% and brought serious economic losses.In 1957, the disease first broke out in broilers in Gumboro, Telahua state, America. The initial isolated strain caused high morbidity, but only caused low mortality less than 5% in usual, 20%-30% occasionally. The isolated strain was defined as the standard IBDV strain (vIBDV). In the late 1980s, the variant IBDV strain different significantly with standard strain in antigenicity was first appeared in America. In Europe, it first reported that highly virulent infectious bursal disease virus (vvlBDV) was appeared, causing more than 90% specific mortality. In the early 1990s, vvlBDV spread to Japan and other Asia parts. Now, it has spread all over the world, and espicially in those countries advanced in poultry industry, it was more severe.IBDV belongs to the genus Avibirnavirus of the Birnaviridae family. IBDV are nonenveloped icosahedrons, approximately 60 nm in diameter. The genome consists of two segments of double-stranded RNA. The smaller dsRNA segment (B) approximately 218kb of the genome of IBDV encodes a single polypeptide of 90ku (VP1), which is a RNA polymerase dependent on RNA. The large RNA segment (A) has two has two overlapping open reading frames (ORF). The ORF1 encodes for a polyprotein which is cleaved following translation into structural proteins, VP2 (41 000), VP3 (35 000) and VP4 (28 000). VP2 protein is the major structural protein and protective antigen of the virus. VP4 is a serine proteinase. VP3 is one of the structural proteins.ORF2 encodes a 16ku (VP5) protein, which is relate to the pathogenicity of virus, but is not indispensable for virus replication. The VP2 protein is the major host-protective antigen and a region of 130 amino acids in length (between amino acid residues 206 and 305) has been identified in VP2 as hypervariable region that is the major region of antigenicity. Alterations of molecular structure in this region lead to antigenic variation and pathogenicity variation in IBDV, and immunorespond variation to vaccinations, which make the classical vaccine unable to control the spread of IBDV. The hypervariable region consists of a seven-peptide sequence and two hydrophilic regions. Alterations are always appeared in the hydrophilic regions, and it is very important to the antigenicity of vvIBDV.It has differentiated the IBDV very-virulent strain, virulent and vaccine strain and expresses the IBDV VP2 gene in E.coli in this reseach. The main research and the results in this study as follows:1. Quickly identification of the very-virulent strain, virulent strain and vaccine strain of the IBDV by RT-PCR The primers were designed according to the conservative sequence of IBDV VP2 in Genbank. A 802bp conservative sequence in IBDV VP2 gene was amplified by RT-PCR, the PCR productions were digested, two groups of restriction enzyme: Aha I, Stu I and BamH I, Nsp I was used to identify the very-virulent strain, virulent strain and vaccine strain. The results showed that a 327bp fragment of very-virulent strain was digested with Aha I and Stu I, whereas a 270bp fragment could be digested with BamH I and Nsp I in both virulent strain and vaccine strain. It indicated that there was a difference in restriction sites between virulent strain, vaccine strain and very-virulent strain. Using this method, we can detect very-virulent IBDV in pathogenic avian accurately and quickly, which has provided a new way for IBD diagnosis.2. The expression of IBDV VP2 gene in E.coliThe VP2 gene was amplified with corresponding primers from IBDV cDNA separated from Shaanxi by PCR. The target gene cloned into expression vector pET-32a, and the recombinant plasmids pET-VP2 was obtained. The insert position, the orientation and the ORF were verified by PCR, digestion and sequence analysis. Comparing with the published sequence of gene IBDV showed that they were homologous. The recombinant plasmids were transferred into JM109. The 60ku target proteins were induced by IPTG. It provides the basic material to produce the mAb of anti-VP2 protein.