E. coli expressed proteins as diagnostic reagents for typing of foot-and-mouth disease virus.

Truncated proteins corresponding to the C-terminal half of VP1 of four vaccine strains and two field variants of foot-and-mouth disease virus (FMDV) were expressed in E. coli. The expressed proteins were affinity purified and their type specific reactivity was confirmed by immunoprecipitation with anti-virus antibodies. Antibodies were raised against the purified proteins in guinea pigs and the type specificity of the anti peptide antibodies was confirmed by antigen capture reverse transcription polymerase chain reaction (Ag-RT/PCR) where the sera against a particular type captured the homologous virus. Antibodies were purified by immuno-affinity chromatography and tested for specificity by various serological tests. Using the purified proteins and the antibodies raised against them, tests like ELISA, Ag-RT/PCR, and latex agglutination test (LAT) were standardized. Application of the reagents in various tests was studied by screening a few field samples and by nucleotide sequencing. Specific reactivity of antibodies raised against expressed protein was seen with both vaccine virus and field samples. Thus E. coli expressed proteins and antibodies to them may form an alternative and cheap source of diagnostic reagents. The studies showed that antibodies against peptides were mono-specific and therefore may be used in LAT for rapid typing of FMDV and Ag-RT/PCR for typing ELISA negative field samples.

Comparative studies on immunoreactivity of truncated recombinant proteins of foot and mouth disease virus (FMDV) produced in E.coli and insect cells.

For effective FMD control programme, India needs large quantities of cheaper diagnostics in addition to vaccine. Diagnostic reagents produced through conventional methods may not be able to meet such requirements. Alternatively, rDNA technology using suitable heterologous systems that permit production of recombinant antigens to the most native form may be exploited. Studies conducted in our laboratory have led us to select carboxy terminal part of VP1 for expression and evaluation. The protein, which was purified from E.coli under denaturing conditions, was renatured and its reactivity was compared with the protein expressed in insect cells through recombinant baculovirus. The expressed protein in the insect cell whole lysate reacted more efficiently with antibodies raised against whole virus than the purified and renatured protein produced in E.coli. But for its lower reactivity, protein produced from E.coli was found to be suitable in type detection. In addition, the size of the protein is small (16 kD) and production and purification of it from E.coli may be cost effective. Hence, it may be exploited for FMDV typing.

C-terminal region of VP1 of selected foot-and-mouth disease virus serotypes: expression in E. coli and affinity purification.

Foot-and-mouth disease (FMD), one of the most contagious and economically important diseases of farm animals, is caused by a FMD virus (FMDV) which belongs to the family of Picornaviridae. The virus occurs as seven serotypes of which four (A, O, C and Asia 1) are prevalent in India. Immunoprophylaxis supported by precise diagnosis is the prime requirement for achieving the success in controlling the disease. Recently, recombinant DNA technology is gaining importance for the production of cost-effective and safer diagnostics and immunogens. Based on this approach, cDNA of a part of gene for major variable immunogenic region, VP1, of FMDV of four serotypes (A22, O, C and Asia 1) was amplified by PCR and cloned into expression vector. The expression of the 16 K protein gene from the clones was induced with IPTG and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and [35S]-methionine labeling. The immunoreactivity of the labeled proteins was assayed by immunoprecipitation with anti-FMDV type-specific sera. Since the proteins contain 6 His residues at the N-terminal end, their affinity purification was carried out using nickel nitrilo-tri-acetic acid (Ni-NTA) agarose matrix. The proteins were found to be immunoreactive and the useful in the FMD diagnosis.