IMMUNO INFORMATIC APPROACHES IN DESIGNING VACCINE AGAINST PATHOGENIC LEPTOSPIRA THROUGH PAN GENOME REVERSE VACCINOLOGY
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Date
2016-12
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT:
Leptospirosis is a globally important zoonotic disease caused by pathogenic
Leptospira and it is a disease of livestock, pet animals, wildlife and humans
throughout the world. The losses are due to reproductive problems in livestock and
mortality in case of humans. Current existing leptospiral vaccines are unsuccessful
due to their limitations .Hence there is a need to develop a novel and efficacious
vaccine to control the disease. To overcome this, reverse vaccinology is the right
choice. In recent trends, it is possible to target the common vaccine candidates with
genomic information of the single organism. Based on this concept the present work
was planned to study the Bioinformatics approaches to identify the vaccine candidates
in designing a vaccine against pathogenic Leptopsira in Bovines
In the present study complete proteomes of L.borgpetersenii hardjo bovis JB
197 and L550 were screened to identify common surface exposed proteins through Rxvi
language scripts and codes. Later these common surface exposed proteins were
subjected to DEG analysis. 49 essential proteins were identified .Further essential
proteins were subjected to non-homology analysis against both host and gut
microbiota to avoid autoimmunity using NCBI-BLAST.T-helper cell epitopes were
predicted from non-homologous proteins through MetaMHCII and ProPred homology
search against BoLA-DRB3, and evaluated using Vaxijen server.
Three dimensional structures were built for T-cell epitopes and BoLA-DRB3
using Modeller9v.15. A total of twenty five models were generated. The model with
high negative DOPE score was selected and validated using PROCHECK, ProQ, and
ProSA in determining protein quality. The structures of T-cell epitopes and BoLA
DRB3 were prepared before docking using protein preparation wizard of Schrodinger
2015-3. Docking and free energy calculations were performed with BioLuminate
Module v 2.0 of Schrödinger software suite 2015-3. The changes in structural
confirmation were monitored in terms of energy plot, RMSD and RMSF during 50 ns
MD simulations run time using Desmond v4.3.
In Silico analysis of L.borgpetersenii JB 197 and L550 retrieved three proteins
namely Ton B dependent receptor containing single epitope, ABC permease protein
with three epitopes and UVr ABC protein B with single epitope. L.ballum was
selected instead of L.borgpetersenii due to its non-availability of the culture during
the period of the study and 99% identity on BLASTp.
The nucleotide sequence corresponding to ABC permease gene containing
three epitopes was retrieved, primers were designed and PCR was standardized for
the amplification of ABC permease gene. PCR purified product on sequencing
analysis confirmed the presence of ABC permease gene. Then, the PCR purified
product was cloned in to PRSET vector using E.coli DH5α cells .The recombinant
plasmid was transformed in to E.coli BL21 (DE3) cells and expression was induced
by addition of 1mM IPTG. Finally recombinant protein was extracted from lysate of
E.coli BL21 (DE3) cells. Recombinant protein was analysed on SDS-PAGE for
characterization. The SDS-PAGE analysis yielded 20KD of expected recombinant
protein on staining with commassie brilliant blue
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