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Purification of recombinant VP3, Western blot and ELISA (a)-SDS PAGE gel of purified protein of pET32a+.VP3, M-protein ladder and lane 1-purified protein approximately 34 kDa; (b)-Western blot of pET32a+.VP3 protein by using anti-His antibody, M-protein ladder, C-control and lane 1-the rVP3 at approximately 34 kDa; (c)-Western blot of rVP3 by using CAV specific antibody; (d)-Western blot of VP3 from CAV infected cell lysate using antibodies against rVP3, M-marker 11 kDa and lane 1 with VP3 at 13.4 kDa; (e)-ROC analysis of indirect ELISA.
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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 37 degrees C. The 6xHis-tagged fusion protein was purified on Ni-NTA and confirmed by Wes...
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... tubes and a time and dose dependent study was carried out by inducing cultures with different concentrations of IPTG and collecting samples at regular intervals after induction, respectively. SDS- PAGE analysis revealed maximum expression in E.coli induced with 1.5mM IPTG for 6 h at 37 ºC. The expressed protein was approximately 34 kDa in size (Fig. 2a) which included 13.4 kDa VP3 and 20.4 kDa fusion peptide having the 6×His-tag. The protein was finally expressed from 500 mL induced E.coli culture, extracted and purified. The purified protein was confirmed by Western blot using anti-His monoclonal antibody conjugated to HRP and CAV infected antiserum (Fig. 2b & 2c). The purified ...
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... was approximately 34 kDa in size (Fig. 2a) which included 13.4 kDa VP3 and 20.4 kDa fusion peptide having the 6×His-tag. The protein was finally expressed from 500 mL induced E.coli culture, extracted and purified. The purified protein was confirmed by Western blot using anti-His monoclonal antibody conjugated to HRP and CAV infected antiserum (Fig. 2b & 2c). The purified recombinant VP3 (rVP3) after dialysis, was used to raise polyclonal antibodies in albino rabbit. The serum collected was tested for its VP3 antibody ...
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... of Polyclonal antibodies against VP3- Polyclonal serum raised against CAV-VP3 was tested for specificity by Western blot and indirect ELISA using cell lysate infected with CAV. Antisera against rVP3 reacted with CAV infected cell lysate as shown by western blot (Fig. 2d) and indirect ELISA (Fig. 2e). The area under curve (AUC) indicated that the anti rVP3-ELISA test was on an average 97.2% accurate. The 95% confidence interval of the AUC for this ELISA ranged from 85.5% to 99.9%. The ROC analysis indicated that the optimal cut-off point was 0.141; this resulted in a sensitivity of 100% and specificity ...
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... of Polyclonal antibodies against VP3- Polyclonal serum raised against CAV-VP3 was tested for specificity by Western blot and indirect ELISA using cell lysate infected with CAV. Antisera against rVP3 reacted with CAV infected cell lysate as shown by western blot (Fig. 2d) and indirect ELISA (Fig. 2e). The area under curve (AUC) indicated that the anti rVP3-ELISA test was on an average 97.2% accurate. The 95% confidence interval of the AUC for this ELISA ranged from 85.5% to 99.9%. The ROC analysis indicated that the optimal cut-off point was 0.141; this resulted in a sensitivity of 100% and specificity of ...
Citations
... IFAT by Confocal microscope-For IFAT using confocal microscopy, after 48 h p.t., monolayer of the transfected cells on the glass cover slip were washed twice with cold PBS, fixed with 4% paraformaldehyde (PFA) for 20 min at RT, washed twice with ice-cold PBS for 5 min, permeabilized by treating with 0.2% Triton-X 100 in PBS for 5 min and washed again twice with cold PBS and blocked in 2% BSA (in PBS) for 1 h at 37 C 15 . After washing for 5 min with cold PBS, cells were incubated with polyclonal antisera against VP3 16 ...
... The recombinant clones containing the gene construct with VP3 and NS1 were confirmed by PCR for both the genes with their respective primers and by restriction enzyme digestion, BamHI and NheI for release of VP3 insert; and BspHI and AvrII for the release of NS1 insert (Fig. 1b & c). Confocal microscopy was performed to check the expression of NS1 and VP3 in HeLa cells using individual polyclonal antibodies of VP3 and NS1 16,17 . Fluorescence in the HeLa cells transfected with pVIVO.VP3.NS1 indicated the expression of NS1 and VP3 20 (Fig. 2a). ...
Viral gene oncotherapy is emerging as a non-infectious therapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. The use of chemo and radiotherapy, for treatment of cancer is limited due to genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.
... IFAT by Confocal microscope-For IFAT using confocal microscopy, after 48 h p.t., monolayer of the transfected cells on the glass cover slip were washed twice with cold PBS, fixed with 4% paraformaldehyde (PFA) for 20 min at RT, washed twice with ice-cold PBS for 5 min, permeabilized by treating with 0.2% Triton-X 100 in PBS for 5 min and washed again twice with cold PBS and blocked in 2% BSA (in PBS) for 1 h at 37 C 15 . After washing for 5 min with cold PBS, cells were incubated with polyclonal antisera against VP3 16 ...
... The recombinant clones containing the gene construct with VP3 and NS1 were confirmed by PCR for both the genes with their respective primers and by restriction enzyme digestion, BamHI and NheI for release of VP3 insert; and BspHI and AvrII for the release of NS1 insert (Fig. 1b & c). Confocal microscopy was performed to check the expression of NS1 and VP3 in HeLa cells using individual polyclonal antibodies of VP3 and NS1 16,17 . Fluorescence in the HeLa cells transfected with pVIVO.VP3.NS1 indicated the expression of NS1 and VP3 20 (Fig. 2a). ...
Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells. Cancer, the leading cause of death worldwide, is a challenge to mankind. Use of chemo and radiotherapy for treatment of cancer is limited due to the genotoxic side effects on healthy cells and involvement of anti-apoptotic signal transduction pathways that prevent cell death. Apoptosis is a physiological process that plays an important role in embryonic development and tissue homoeostasis
... The insert of VP3 gene was subcloned in pcDNA 3.1(+) eukaryotic expression vector (Invitrogen, USA) at BamHI and XbaI restriction sites [16] (Fig. 1a-d). The recombinant plasmid pcDNA.cav.vp3 was found to express VP3 in HeLa cells [17]. ...
The use of viruses for treatment of cancer overcomes the bottlenecks of chemotherapy and radiotherapy. Several viruses and their proteins have been evaluated for oncolytic effect. The VP3 protein (apoptin) of chicken anemia virus is one such protein with an inherent ability to lyse cancer and transformed cells while leaving normal cells unharmed. In the present study, the apoptosis inducing potential of VP3 protein of CAV was evaluated in human cervical cancer cell line (HeLa). It was found that in VP3-induced apoptosis, caspase-dependent intrinsic pathway plays an important role with the cleavage of poly (ADP-ribose) polymerase (PARP) and there was no evidence of involvement of death receptor-mediated extrinsic pathway. The results of this study provide intuitive information and strengthen the candidacy of apoptin as a viral oncotherapeutic agent.
... Expression of cloned genes at protein level was confirmed by immune-fluorescence assay (IFAT and FAT) as described in previous studies (11,20,22,23) using NDV infected polyclonal sera of chicken and human anti-TNFa FITC conjugated monoclonal antibody, respectively. Results of these assays demonstrated bright fluorescence in pVIVO.nd.hn.hu.tnf transfected HeLa cells for HN and TNF-a; whereas, dim or no fluorescence was observed in pVIVO2 empty vector transfected HeLa cells (Fig. 2a), suggesting expression of the cloned genes in pVIVO.nd. ...
The viral gene oncotherapy in combination with cytokines emerges as an exciting strategy for cancer therapy due to its minimal side effects and tumor specificity. HN is the surface protein of NDV which is involved in virus infectivity and is known to kill many cancerous cell types. TNF-a, a multifactorial cytokine has direct anti-tumor activity by activating the extrinsic pathways of apoptosis. In the present study, HN gene of NDV and TNF-a of human were cloned at multiple cloning sites (MCS) 1 and 2 of bicistronic expression vector pVIVO2. Expression pattern of recombinant clone was checked on transcriptional and translational level by RT-PCR, Immunofluorescence assay and flow cytometry. On flow cytometric analysis HN gene expression was found to be 28.30 1.21; 5.22 0.60%, and TNF-a gene expression was found to be 15.44 0.42; 6.51 0.757%, in HeLa cells transfected with pVIVO.nd.hn.hu.tnf and pVIVO2 empty vector control, respectively. These assays confirm that HN and TNF-a act synergistically in the induction of apoptosis in HeLa cells.
Outcome of seven years long (2007-2014) extramural research in frontier areas of agricultural sciences, in consortium mode, financed under the National Agricultural Innovation Project (NAIP) by the World Bank and Government of India, and implemented by the Indian Council of Agricultural Research (ICAR) is presented in this report. The research fetched 85 patent applications, 723 research publications, and over 2.81 million genomic resources deposited in the GenBanks besides commercialization of few technologies through licensing, and cues for furthering the research in various areas/fields.
