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Indian J Exp Biol ; 2015 July; 53(7): 429-439
Article in English | IMSEAR | ID: sea-178542


Unsatisfactory performance of the existing BCG vaccines, especially against the adult pulmonary disease, has urged the need for an effective vaccine against tuberculosis (TB). In this study, we employed differential proteomics to obtain a list of antigens as potential vaccine candidates. Bacterial epitopes being presented at early stages on MHC class I and class II molecules of macrophages infected with Mycobacterium tuberculosis (M. tb) were identified using iTRAQ labelling and reverse phase LC-MS/MS. The putative vaccine candidates, thus identified, were tested as plasmid DNA vaccines in mice to ascertain their protective efficacy against the aerosolized M. tb challenge, based on their ability to reduce the bacterial load in the lungs of infected mice. Here, we observed that 4 out of the 17 selected antigens imparted significant protection against the challenge of M. tb. The four shortlisted antigens were further assessed in a more stringent guinea pig model, where too, they demonstrated significant protection. It concludes that combining a proteomics approach with the in vivo assessment of vaccine candidates in animal models can be valuable in identifying new potential candidates to expand the antigenic repertoire for novel vaccines against TB.

Indian J Exp Biol ; 2014 Oct; 52(10): 935-942
Article in English | IMSEAR | ID: sea-153780


Development and study of dog mammary tumour xenograft in immunosuppressed Swiss Albino Mice adds a new dimension in cancer research as dog tumors have many similarities with human tumors regarding progression, histopathology, molecular mechanism, immune response and therapy. Failure of the immune system to recognize and eliminate cancer cells leads to cancer progression and the fight between immune cells and cancer cells has a great role in understanding the mechanism of cancer progression and elimination. Rejection and acceptance of tumour xenograft depends on efficiency of CD4+, CD8+ and NK cell populations. In the present investigation, dog mammary tumor xenograft in cyclosporine-A and γ-irradiated, immunosuppressed Swiss Albino mice was developed and the immune cell status of graft accepted and rejected mice was assessed. It was observed that all the major immune cells (CD4+, CD8+ and NK cells) play an equal role in tumour rejection.

Animals , CD4-Positive T-Lymphocytes/immunology , Dogs , Female , Graft Rejection/immunology , Immunocompromised Host , Killer Cells, Natural/immunology , Mammary Neoplasms, Experimental/immunology , Mammary Neoplasms, Experimental/pathology , Mice , Neoplasm Transplantation/methods , Transplantation, Heterologous/methods
Indian J Exp Biol ; 2012 Sept; 50(9): 618-624
Article in English | IMSEAR | ID: sea-145295


The canine Parvovirus 2, non-structural 1(NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1(rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS–PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 × His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.

Indian J Exp Biol ; 2012 May; 50(5): 325-331
Article in English | IMSEAR | ID: sea-145257


In the present study recombinant VP3 (rVP3) was expressed in E.coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS–PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37ºC. The 6×His-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.

Indian J Exp Biol ; 2011 Sept; 49(9): 654-659
Article in English | IMSEAR | ID: sea-145174


Parvoviruses are small, 260-Å-diameter, icosahedral, non-enveloped, single-stranded DNA viruses with a genome of approximately 5 kb. Non structural protein, (NS-1) is especially relevant, being both essential for virus replication and the main factor responsible for virus pathogenicity and cytotoxicity. This protein has also been reported to possess the property of killing of transformed cells. The present study was carried out to clone, characterize and express the NS-1 gene of canine parvovirus. NS-1 complete CDS 2020bp was amplified, cloned into eukaryotic expression vector pcDNA 3.1(+), sequenced and characterized by in vitro expression analysis. Functional activity of recombinant construct, pcDNA.cpv.NS-1, was evaluated by RT-PCR and flow cytometry for the expression of NS-1 specific mRNA and NS-1 protein, respectively, in transfected HeLa cells. This recombinant plasmid may serve as an important tool to evaluate the apoptotic potential of NS-1 protein of canine parvovirus in cultured HeLa cells.