Development Of Recombinant Antigens For Diagnosis Of Avian Mycoplasmosis
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Date
2006
Authors
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Publisher
Tamil Nadu Veterinary and Animal Sciences University
Abstract
Avian Mycoplasmosis is widespread and is one of the most common
respiratory diseases of poultry causing significant economic losses in poultry
industry. The disease occurs when birds infected with Mycoplasma
gallisepticum (MG) or Mycoplasma synoviae (MS) are stressed. An attempt
has been made in this study to express the antigenic proteins of MG and MS
in E.coli and use them in diagnostic assay.
Primers were designed with built in restriction enzyme sites for
amplification of p75 and p29 (TM-1) genes of MG PG31, PCR amplification
of the genes was done, cloned and sequenced. The genes were cloned into
expression vector pPROEx HTb and expressed in E. coli DH5α. The full
length vlhA gene of local field isolate MS427 was amplified using published
primers and the 5’ end corresponding to MSPB protein sequences were
sequenced. Based on the sequence, primers were synthesized, amplified , a
smaller part of MSPB (MSPB-I) and a larger part of MSPB (MSPB-II) gene
was cloned in the expression vector pPROEx HTb and expressed in E. coli
DH5α.The recombinant proteins were purified using Ni-NTA affinity
chromatography.The four recombinant proteins obtained in this study were
characterized and evaluated as diagnostic antigen. The recombinant P29 of
MG and recombinant MSPB-II protein of MS showed a potential to be used
as diagnostic antigens. A highly sensitive indirect ELISA, a rapid simple easy
to perform latex agglutination test and flow-through enzyme immunoassay
were developed to diagnose MG and MS separately using their respective
recombinant proteins. The MG and MS indirect ELISA had perfect
agreement with commercial ELISA kit used in this study. The latex
agglutination tests and flow-through enzyme immunoassays had substantial
agreement with commercial kit.
Description
Keywords
Avian Mycoplasmosis, Mycoplasma gallisepticum, Mycoplasma synoviae, Cloning, Recombinant expression, ELISA, Latex agglutination test, Flow-through enzyme immunoassay