Molecular Characterisation Of Infectious Bursal Disease Virus And Expression Of Vp2 Protein For The Development Of Diagnostic Kit And Recombinant Vaccine
Mat Isa, Nurulfiza (2008) Molecular Characterisation Of Infectious Bursal Disease Virus And Expression Of Vp2 Protein For The Development Of Diagnostic Kit And Recombinant Vaccine. PhD thesis, Universiti Putra Malaysia.
Outbreak of infectious bursal disease (IBD) in chickens due to highly pathogenic strain of IBD virus (vvIBDV) was first reported in Europe in late 1980’s and in Malaysia in 1991. The disease caused significant economic losses, estimated more than RM72 million per year in Malaysia alone due to high mortality and immunosuppression. Treatment of IBD is of no value and the disease can only be controlled and prevented by proper vaccination programme and biosecurity. It was the objectives of the study to determine the molecular characteristic of Malaysian field isolates of IBDV and expression of the VP2 gene of the isolate for the development of diagnostic kit and recombinant vaccine. Three IBDV isolates identified as UPM04178, UPM04190 and UPM04238 were characterised. Based on their pathogenicity and sequence characteristic, the highest similarity (98%) concerning both nucleotide and amino acid sequences, the IBDV isolates were characterized as vvIBDV strains. Evolutionary relatedness of the isolates to vvIBDV strains was demonstrated by three phylogenetic methods: bootstrap values of 100%, 95% and 90% for nucleotide sequences and those of 58%, 86% and 96% for amino acid sequences were obtained by the distance, maximum parsimony and maximum likehood methods, respectively. Phylogenetic analysis revealed clustering of the isolates with vvIBDV strains of serotype 1, which originate from a common ancestor of IBDV strains present in Malaysia. Using informative characteristics of the isolate, both diagnostic kit and recombinant vaccine were successfully developed using a new wild-type field vvIBDV strain of UPM04190 isolate. A safe and effective recombinant IBD vaccine was developed base on the construction of recombinant VP2 gene of the isolate cloned into an Escherichia coli expression system. The VP2 gene was inserted into pRSET B vector as a fusion protein with histidine tag, which can be easily purified. The recombinant VP2 protein bands were expressed to their expected sizes of ~50 kDa from cell lysate. The pRSET vectors are pUC-derived expression vectors and expression of the gene of interest from pRSET is controlled by the strong phage T7 promoter that drives expression of gene 10 (Φ10) which provides protein stability and help to maintain the original structure of the protein. High-level production (3 mg/ml) of soluble product of VP2 recombinant protein was achieved with modified techniques of expression conditions and approaches. Efficacy test demonstrated that the recombinant vaccine of various fractions could provide protection ranging from 75% to 100% in highly susceptible chickens (specific pathogen free chickens) when challenged with vvIBDV (B00/81) at 104.25 EID50/ml per chicken following vaccination. One-step-immunostrip kit which is highly specific and sensitive was developed using whole virus as capture antigen and high-affinity polyclonal IBD antibodies coated with gold particles. Rapid detection of IBD antibody can be achieved as fast as two minutes in a clinical or field environment. The kit is highly sensitive as it can detect as low as 250 ELISA units compared to commercial ELISA kit that only goes to 391 ELISA units for positive samples. The specificity of the kit was evaluated against antibody of other chicken viruses. No signal of reactivity or cross react exists among the antibodies tested. Thus, it was highly specific to IBDV. It was concluded that the local IBDV isolates were proven to be vvIBDV strain, the constructed recombinant vaccine provide a safe and effective protection and, the developed one-step-immunostrip kit is rapid, specific, sensitive, safe and economic in detection of IBDV infection and monitoring immune status of chicken against IBD.
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