Cloning, Expression and characterization of β-haemolysin of a local isolate of staphylococcus aureus
| dc.contributor.advisor | Archana Sharma | |
| dc.contributor.author | Mahavir Singh | |
| dc.date.accessioned | 2017-08-01T10:45:18Z | |
| dc.date.available | 2017-08-01T10:45:18Z | |
| dc.date.issued | 2011 | |
| dc.description.abstract | Staphylococcus aureus is a Gram-positive bacterium that causes several clinically important infections in man and animals, including mastitis in cattle and buffaloes. More than 30 different exotoxins are secreted by different strains of S. aureus and are involved in pathogenesis of various clinical conditions. The role of S. aureus beta-haemolysin has not yet been clearly understood. Recombinant techniques have allowed large scale and cost-effective production of biologicals for diverse applications. The present study was undertaken to clone, sequence, express and characterize beta-haemolysin of a local Indian strain of S. aureus. Pathogenic coagulase+ beta-haemolysin+ S. aureus was isolated from milk samples of 10 bovines suffering from clinical mastitis in Haryana. The bacteria were identified using various tests such as Gram’s staining, catalase production, mannitol fermentation, ‘hot-cold’ lysis on blood agar and titration of beta-haemolytic activity in culture supernatant, SA-hlb and SA-coa gene-specific multiplex PCR and antibiotic sensitivity pattern. PCR product of SA-hlb gene from one of the above isolates was cloned into pGEM-Teasy vector for nucleotide sequencing. The nucleotide sequence (n=891 nt) of SAhlb MV1 clone was deposited at GenBank NCBI website and assigned GenBank accession no. JN580071. BLASTN and BLASTP searches retrieved several orthologs with >95% homology to its nucleotide and deduced amino acid sequences. The protein sequence possessed ‘nSMase’ conserved domain and was a member of EEP superfamily. Most interestingly, its 3D structure had remarkable similarities to 3i41B (PDB) hlb from S. aureus strain RN4220 of USA origin and all the residues involved in catalytic, metal binding and phosphate binding sites were 100% conserved. PCR product of SA-hlb gene was also directionally cloned into SacI-XhoI sites of pQE-Tri System vector to allow expression of ~37 kDa recombinant SA-hlb.8xHis in JM107 transformants. The r-SA-hlb MV3 clone showing the highest level of expression as revealed by SDS-PAGE and verified by immunoblotting, was produced in large amounts and purified by Ni-chelate chromatography. The purified r-SA-hlb possessed hot-cold lytic activity against sheep and buffalo erythrocytes. The hot-cold activity was thermostable upto <90ºC for 30 min. and could be neutralized by antibodies in convalescent bovine sera. The purified r-SA-hlb also exhibited cytocidal effect on buffalo mononuclear cells, maximum being at 125 ng/ml concentration. It was useful as coated antigen at 2.5 μg/ml concentration for optimally measuring antibody levels in bovine sera by indirect ELISA. Various other applications of this purified recombinant S. aureus beta-haemolysin have been speculated | en_US |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/5810027002 | |
| dc.keywords | Cloning, Characterization, Staphylococcus aureus,β- haemolysin | en_US |
| dc.language.iso | en | en_US |
| dc.publisher | LUVAS | en_US |
| dc.sub | Veterinary Microbiology | en_US |
| dc.theme | Cloning, Expression and characterization of β-haemolysin of a local isolate of staphylococcus aureus | en_US |
| dc.these.type | M.V.Sc. | en_US |
| dc.title | Cloning, Expression and characterization of β-haemolysin of a local isolate of staphylococcus aureus | en_US |
| dc.type | Thesis | en_US |