Characterization of Dichelobacter nodosus and Expression of its fimA gene
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Date
2020-01-27
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Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J&K)
Abstract
The study was conducted to determine the prevalence of ovine foot rot in Jammu and Kashmir (J&K) and to study the serological diversity of Dichelobacter nodosus (D. nodosus) in the area of study. Further, cloning and expression of the fimbrial subunit gene (fimA) ofthe most prevalent serogroup of D. nodosus in the area of study was undertaken. A total of 104 flocks with 2685 sheep from the six administrative districts of J&K (Jammu, Doda Samba, Anantnag, Pulwama and Shopian) were investigated for ovine footrot. Four hundred and sixty six (466) sheep were found to exhibit clinical footrot. The overall prevalence of ovine footrot was 17.35%, ranging from 14.20% to 19.74%. Of the 352 exudate samples (swabs) collected from sheep affected with severe foot lesions, 111(33.43%) were positive for D. nodosus by polymerase chain reaction (PCR), using species-specific 16S rRNA primers. All the D. nodosus positive samples were subjected to serogrouping by multiplex PCR, using nine (A-I) serogroup specific primers. Eighty four (75.60%) samples belonged to serogroup B of D. nodosus, twenty three (20.70%) samples to serogroup E, mixed infection with the presence of both B and E serogroups in same samples was shown by two samples and two of the D. nodosus positive samples were untypable.The present study revealed that serogroup B is the most prevalent serogroup in the area of study, followed by serogroup E. However, besides fimA gene of serogroup B, fimA gene of serogroup E was also undertaken for cloning and expression, due to occurance of serogroup E in significant proportion from the samples taken from Jammu division. Sequence analysis after cloning of fimA genes of D. nodosus serogroups B and E, revealed that the cloned genes had maximum homology with strains JKS-01B and JKS-O8E respectively. The coding sequence of fimA gene (cdsfimA), encoding fimbrial subunit protein was characterized and successfully expressed in E. coli BL21 (DE3), using pET32a(+) expression vector. However, the target protein was found to be toxic to BL21 cells, as the transformed cells could not grow on LB/ampicillin agar without the addition of 1% glucose in the media. This was substantiated by the sizeable decline in viability and division of recombinant BL21cells post induction of expression (IPTG), as judged by decline in turbidity of induced control as compared to the uninduced control. The recombinant D. nodosus fimbrial subunit proteins did not assemble to mature fimbriae in E. coli, after the observation that the expressed protein could not be released from the host without the use of detergent (SDS).Upon SDS- PAGE analysis, the estimated molecular weight of recombinant D. nodosus fimbrial subunit fusion protein (Thioredoxin- 6x Histidine tag) was approx. 35 kDa, which is very close to expected molecular weight. It was further elucidated that increasing the incubation time post induction of expression as well as repeated freeze thawing of cell lysate, increases the level of expression of recombinant protein.The exact identity of recombinant protein was confirmed by western blotting, using Ni-NTA conjugate antibody directed against polyhistidine tagged recombinant fimbrial protein. The fusion protein was purified using Ni-NTA affinity chromatography where relatively purified fraction of the target protein was obtained in elution fractions (2nd& 3rd). Work needs to be continued to assess the protective immune response to the recombinant protein in laboratory animals and eventually formulate a recombinant vaccine using recombinant fimA protein to assess the immune response in sheep.