Development of Biofilm of Escherichia Coli with Subunits of Aeromonas Hydrophila
KrishiKosh
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Title |
Development of Biofilm of Escherichia Coli with Subunits of Aeromonas Hydrophila
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Creator |
Naveen Kumar, B.T.
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Contributor |
Shankar, K.M.
Pradhan, P.K. Shamsundar, B.A. Iqlas Ahmed Krishna Bhatt, C.A. |
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Subject |
Aeromonas hydrophila, Escherichia coli, Biofilm, Recombinant protein, Fish
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Description |
Ph.D. Thesis
The study was carried out on development, kinetics and A. hydrophila OMP gene expression in biofilm of E.coli for use in development of oral vaccine. Relative expression of OMP (48 and G) genes from biofilm and free cell of A. hydrophila was determined using real-time PCR. The OMP 48 and G genes in the biofilm cells upregulated compared to that in the free cell of A. hydrophila. Further, OMP 48 gene from A. hydrophila was amplified by PCR, constructed into vector pEXP-5-NT/TOPO® TA (Invitrogen) and propagated in TOPO 10 E.coli cells. Purified plasmid constructed with OMP 48 was transferred into BL21 (DE3) cells for protein expression and expression levels observed at different concentrations of IPTG. Development of biofilm of transformed E.coli with OMP 48 was studied at various nutrient concentrations and incubation time. Highest CFU of BF cells were obtained with 1.5% LB supplemented with 100ug/ml ampicillin and 0.3% chitin. Growth kinetics of BF cells of recombinant E. coli with OMP48 with different IPTG levels was studied for 6 days. Among the induced groups, 0.5 mM IPTG induced cells showed higher CFU g-1 of chitin than 1 mM IPTG. Recombinant protein expression in biofilm cells of E.coli with OMP 48 was determined by Western blot using rabbit anti OMP 48 serum. Evaluation of immune response against OMP 48 purified using Ni- NTA affinity chromatography from BF of E. coli (OMP 48) in fish was studied by ELISA. In Western blot, antibody has reacted to recombinant E. coli (induced) and A. hydrophila starins Ah1, Ah2, Ah3. Hence, recombinant protein expression in BF mode can be used for the development of oral vaccine and could probably be used for the protection of cultivated Indian major carps. |
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Date |
2016-06-30T16:15:38Z
2016-06-30T16:15:38Z 2014-01-03 |
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Type |
Thesis
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Identifier |
http://krishikosh.egranth.ac.in/handle/1/68339
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Language |
en
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Format |
application/pdf
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Publisher |
Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar
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