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Research
(Published
online: 19-10-2014)
18.
Polymerase chain reaction
amplification and cloning of immunogenic protein NAD-dependent
beta hydroxybutyryl CoA dehydrogenase gene of
Clostridium
chauvoei -
Saroj K. Dangi, Ajay
P. Singh, Satyaveer S. Dangi, Prasad Thomas, Santosh K. Gupta,
Rajesh K. Agarwal and K. N. Viswas
Veterinary World, 7(10): 848-851
doi:
10.14202/vetworld.2014.848-851
Saroj
K. Dangi:
Division of Bacteriology & Mycology, Indian Veterinary Research
Institute, Izatnagar, Bareilly, Uttar Pradesh, India;
sarojsvet@gmail.com
Ajay P.
Singh:
Division of Bacteriology & Mycology, Indian Veterinary Research
Institute, Izatnagar, Bareilly, Uttar Pradesh, India;
drajay_vet@yahoo.co.in
Satyaveer S. Dangi:
Division of Physiology & Climatology, Indian Veterinary Research
Institute, Izatnagar, Bareilly, Uttar Pradesh, India;
dangivet@gmail.com
Prasad
Thomas:
Division of Bacteriology & Mycology, Indian Veterinary Research
Institute, Izatnagar, Bareilly, Uttar Pradesh, India;
prasadthomas99@gmail.com
Santosh
K. Gupta:
Division of Bacteriology & Mycology, Indian Veterinary Research
Institute, Izatnagar, Bareilly, Uttar Pradesh, India;
skgupta15@rediffmail.com
Rajesh
K. Agarwal:
Division of Bacteriology & Mycology, Indian Veterinary Research
Institute, Izatnagar, Bareilly, Uttar Pradesh, India; grace_bly@yahoo.com
K. N.
Viswas:
Division of Bacteriology & Mycology, Indian Veterinary Research
Institute, Izatnagar, Bareilly, Uttar Pradesh, India;
vkn111@gmail.com
Received:
28-06-2014, Revised: 14-09-2014, Accepted: 20-09-2014, Published
online: 19-10-2014
Corresponding author:
K. N. Viswas, e-mail: vkn111@gmail.com
Abstract
Aim:
The present study was aimed at polymerase chain reaction (PCR)
amplification and cloning of NAD-dependent betahydroxybutyryl
coenzyme A dehydrogenase (BHBD) gene of Clostridium chauvoei.
Materials and Methods: C. chauvoei was cultured and
confirmed by 16-23S rDNA spacer region primers. The primers for
nad-bhbd gene of C. chauvoei were designed to aid in
cloning into pRham-N-His SUMO-Kan vector, and nad-bhbd gene
was amplified by PCR. The amplified nad-bhbd gene was
purified and cloned into pRham-N-His SUMO-Kan expression vector.
The recombinant plasmid was transformed into E. cloni 10 G
cells and the clone was confirmed by colony PCR using the
pRham-SUMO-NAD-For and pRham-SUMO-NAD-Rev primers and also by
sequencing.
Results: PCR amplification of nad-bhbd gene yielded a
product length of 844 base pairs which was cloned into pRham-NHis
SUMO-Kan vector followed by transformation into E. cloni
10G chemically competent cells. The recombinant clones were
characterized by colony PCR, sequencing, followed by basic local
alignment search tool (BLAST) analysis to confirm the insert.
Conclusions: Immunogenic protein NAD- dependent BHBD of C.
chauvoei was cloned and the recombinant clones were confirmed
by colony PCR and sequencing analysis.
Keywords: black quarter, Clostridium chauvoei,
NAD-beta-hydroxybutyryl coenzyme A dehydrogenase.
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