Plasmids were extracted from the clones using QIA prep spin miniprep kit (Qiagen, Germany) following manufacture’s protocol

Plasmids were extracted from the clones using QIA prep spin miniprep kit (Qiagen, Germany) following manufacture’s protocol. to SG 13009. The recombinant protein expression was optimized using various concentration of isopropylthiogalactoside (IPTG) induction, further the expression was confirmed by Western blot analysis using anti-His antibody. Recombinant protein was PETCM purified under denatured condition using Ni-NTA affinity chromatography. Antibody was generated against the recombinant protein using alum adjuvant in BALB/c mice and tested for cross reactivity with other serotypes of as well as closely related clostridia. An ELISA test was developed for the detection of botulinum neurotoxin and the minimum detection limit was also estimated. Results: The recombinant protein was expressed at maximum yield at 4.3 h of post-induction with 0.5 mM IPTG concentration. The recombinant protein was purified using Ni-NTA affinity chromatography up to the homogeneity level. The polyclonal antibodies were raised in mice with a titre of 1 1:2048000. The developed antibody was highly specific with a sensitivity of detecting approximately 15 ng/ml of recombinant protein and not showing any cross-reactivity with other serotypes. Interpretation & conclusions: There is no commercial immunodetection system available in India to detect botulism. The developed detection system is highly specific. It will be useful for growing food industry to detect botulinum neurotoxin in food samples as well as in clinical samples. type B from Indian tropical fish was reported in 199018. Occurrence of in fresh and cured fish in retail trade in Cochin was also reported with a prevalence around 19 per cent19. In fresh retail fish most of the samples were found positive for serotype A, B, and D. Genetic diversity among toxigenic clostridia has also been reported20. The spores of type E were isolated from soil of Gwalior21. Mouse bioassay is considered as the gold standard for detection of botulism22. However, there are several shortcomings associated with mouse bioassay, mice can die non specifically during the process, this test takes 4 days to get the final results and it is intensive, requires animal facility and highly experienced and immunized person to perform the study. Moreover, mouse bioassay is not suitable for routine detection, quantification of samples and cannot meet the extent of real biodefence deployment since a large number of animals is required to get statistically significant results. In addition, there are several ethical issues of using animals for such testing in large number of samples23. Several new methods have been evolved to detect BoNTs; among those ELISA has been considered as one of the sensitive, easy and amenable methods to develop a high throughput system. Since there is no indigenous detection system available in the country, there is a need to develop an in-house system to detect botulism in food and clinical samples. The present study was therefore, aimed to construct the synthetic gene of truncated fragment of type B toxin gene, clone and express the recombinant protein, raise antibody against the recombinant protein and to develop an immuno detection system for botulinum neurotoxin type B. Material & Methods All the work was carried out in the biotechnology division of Defence Research and Development Establishment (DRDE), Gwalior. SG 13009 (Qiagen, Germany) cells by heat shock method. The transformants were selected on Luria broth (LB) agar plates supplemented with kanamycin (30 g/ml) and ampicillin (100 g/ml). Plasmids were extracted from the clones using QIA prep spin miniprep kit (Qiagen, Germany) following manufacture’s protocol. Further, these plasmids were screened for the confirmation of presence of inserts using BoNT/B synthetic gene specific primers mentioned above and also checked inframe using the combination of BoNT/B specific and PQE 30 UA vector specific primer. (ELISA): To determine SNRNP65 immunogenicity of recombinant BoNT/B, the presence of serum immunoglobulins specific to recombinant BoNT/B was determined by indirect ELISA. The purified recombinant BoNT/B was diluted to 5 g/ml in carbonate buffer (0.05M, ATCC-11437, ATCC-13124, ATCC-9714 (from ATCC, USA), 49205 (from CRI, Kasauli), (from culture collection Biotechnology Division, DRDE, Gwalior). All the culture and toxins were PETCM PETCM handled with utmost safety in BSL-3 biosafety cabinet. SG13009 cells. The recombinant transformant was selected by ampicillin and kanamycin marker. From the selected transformant, plasmid was isolated and checked for the presence of insert using BoNT/B forward and reverse primers. The synthetic gene was sequenced and aligned with the nucleotides of light chain of standard strain of type B (CP 000940). There was no ambiguity observed in the synthetic gene, it was perfectly matching with the standard sequence. The recombinant protein expression conditions was optimized the maximum yield of recombinant protein was obtained at OD600 0.8 with 0.5 mM IPTG induction with minimum incubation of 4.30 h after induction. The cells were harvested and localized PETCM for the release of PETCM recombinant protein and the results reveal that recombinant protein was present as inclusion bodies. Purification was carried out under.

Related Posts