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Nagaleekar, Viswas Konasagara
- Cloning and Sequence Analysis of Hyaluronoglucosaminidase (nagH) Gene of Clostridium chauvoei
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Materials and Methods: C. chauvoei strain ATCC 10092 was grown in ATCC 2107 media and confirmed by polymerase chain reaction (PCR) using the primers specific for 16-23S rDNA spacer region. nagH gene of C. chauvoei was amplified and cloned into pRham-SUMO vector and transformed into Escherichia cloni 10G cells. The construct was then transformed into E. cloni cells. Colony PCR was carried out to screen the colonies followed by sequencing of nagH gene in the construct.
Results: PCR amplification yielded nagH gene of 1143 bp product, which was cloned in prokaryotic expression system. Colony PCR, as well as sequencing of nagH gene, confirmed the presence of insert. Sequence was then subjected to BLAST analysis of NCBI, which confirmed that the sequence was indeed of nagH gene of C. chauvoei. Phylogenetic analysis of the sequence showed that it is closely related to Clostridium perfringens and Clostridium paraputrificum.
Conclusion: The gene for virulence factor nagH was cloned into a prokaryotic expression vector and confirmed by sequencing.
Authors
Affiliations
1 Division of Bacteriology and Mycology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122, Uttar Pradesh, IN
1 Division of Bacteriology and Mycology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly-243122, Uttar Pradesh, IN
Source
Veterinary World, Vol 10, No 9 (2017), Pagination: 1104-1107Abstract
Aim: Blackleg disease is caused by Clostridium chauvoei in ruminants. Although virulence factors such as C. chauvoei toxin A, sialidase, and flagellin are well characterized, hyaluronidases of C. chauvoei are not characterized. The present study was aimed at cloning and sequence analysis of hyaluronoglucosaminidase (nagH) gene of C. chauvoei.Materials and Methods: C. chauvoei strain ATCC 10092 was grown in ATCC 2107 media and confirmed by polymerase chain reaction (PCR) using the primers specific for 16-23S rDNA spacer region. nagH gene of C. chauvoei was amplified and cloned into pRham-SUMO vector and transformed into Escherichia cloni 10G cells. The construct was then transformed into E. cloni cells. Colony PCR was carried out to screen the colonies followed by sequencing of nagH gene in the construct.
Results: PCR amplification yielded nagH gene of 1143 bp product, which was cloned in prokaryotic expression system. Colony PCR, as well as sequencing of nagH gene, confirmed the presence of insert. Sequence was then subjected to BLAST analysis of NCBI, which confirmed that the sequence was indeed of nagH gene of C. chauvoei. Phylogenetic analysis of the sequence showed that it is closely related to Clostridium perfringens and Clostridium paraputrificum.
Conclusion: The gene for virulence factor nagH was cloned into a prokaryotic expression vector and confirmed by sequencing.
Keywords
Black Quarter, Clostridium chauvoei, Hyaluronoglucosaminidase.Full Text
- Cloning and Expression of P67 Protein of Mycoplasma leachii
Abstract Views :143 |
PDF Views:0
Materials and Methods: P67 gene was amplified from genomic DNA of M. leachii. The polymerase chain reaction (PCR) product was inserted in pRham N-His SUMO Kan vector and was used to transform competent Escherichia cloni 10G cells. Recombinant protein expression was done by inducing cells with 0.2% Rhamnose. Purification was done using nickel nitrilotriacetic acid affinity chromatography. Western blot and dot blot analysis were performed to assess the immunoreactivity of P67 protein.
Results: PCR amplicon size of P67 gene was found to be 1500 base pair. The size of the fusion protein with SUMO tag was 79 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The recombinant P67 fusion protein expressed in pRham N-His SUMO Kan vector was found to be immunogenic in both western blot and dot blot analysis.
Conclusion: Western blot and dot blot analysis of P67 protein of M. leachii revealed that the protein is immunogenic. Further work is needed to evaluate the role of P67 antigen of M. leachii as an immunodiagnostic agent.
Authors
Sabarinath Thankappan
1,
Rajneesh Rana
1,
Arun Thachappully Remesh
1,
Valsala Rekha
1,
Viswas Konasagara Nagaleekar
1,
Bhavani Puvvala
1
Affiliations
1 Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, IN
1 Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, IN
Source
Veterinary World, Vol 10, No 9 (2017), Pagination: 1108-1113Abstract
Aim: The present study was undertaken to clone, express and study the immunogenicity of P67 protein of Mycoplasma leachii.Materials and Methods: P67 gene was amplified from genomic DNA of M. leachii. The polymerase chain reaction (PCR) product was inserted in pRham N-His SUMO Kan vector and was used to transform competent Escherichia cloni 10G cells. Recombinant protein expression was done by inducing cells with 0.2% Rhamnose. Purification was done using nickel nitrilotriacetic acid affinity chromatography. Western blot and dot blot analysis were performed to assess the immunoreactivity of P67 protein.
Results: PCR amplicon size of P67 gene was found to be 1500 base pair. The size of the fusion protein with SUMO tag was 79 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The recombinant P67 fusion protein expressed in pRham N-His SUMO Kan vector was found to be immunogenic in both western blot and dot blot analysis.
Conclusion: Western blot and dot blot analysis of P67 protein of M. leachii revealed that the protein is immunogenic. Further work is needed to evaluate the role of P67 antigen of M. leachii as an immunodiagnostic agent.
Keywords
Cloning, Dot Blot, Expression, Mycoplasma leachii, P67 Protein, Western Blot.Full Text
- Overhauling the Communicational Inconsistencies in Indian Science
Abstract Views :249 |
PDF Views:77
Authors
S. Badrinarayan
1,
Bhavisha P. Sheth
2,
Kavita Pal
3,
Kshama Lakshman
4,
Palatty Allesh Sinu
5,
K. Sri Manjari
6,
Viswas Konasagara Nagaleekar
7,
Wairokpam Premi Devi
8
Affiliations
1 Electronics and Communication Engineer, Bengaluru - 560 085, IN
2 Entrepreneurship Development Institute of India, Gandhinagar - 382 428, IN
3 ACTREC, Tata Memorial Centre, Mumbai - 410 210, IN
4 Everwell Health Solutions, Bengaluru - 560 025,, IN
5 Central University of Kerala, Kasaragod - 671 316, IN
6 Osmania University, Hyderabad - 500 032, IN
7 ICAR-Indian Veterinary Research Institute, Izatnagar - 243 122, IN
8 Entrepreneurship Development Institute of India, Gandhinagar - 382 428, IN
1 Electronics and Communication Engineer, Bengaluru - 560 085, IN
2 Entrepreneurship Development Institute of India, Gandhinagar - 382 428, IN
3 ACTREC, Tata Memorial Centre, Mumbai - 410 210, IN
4 Everwell Health Solutions, Bengaluru - 560 025,, IN
5 Central University of Kerala, Kasaragod - 671 316, IN
6 Osmania University, Hyderabad - 500 032, IN
7 ICAR-Indian Veterinary Research Institute, Izatnagar - 243 122, IN
8 Entrepreneurship Development Institute of India, Gandhinagar - 382 428, IN
Source
Current Science, Vol 116, No 1 (2019), Pagination: 20-21Abstract
No Abstract.
Keywords
No Keywords.Full Text