RESUMEN
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.
RESUMEN
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.
RESUMEN
Objective To compare the humoral immune response of BALB/c mice immunized by recombinant plasmids PeDNA3.1-M-NS1 and pcDNA3.1-N-NS1.Methods Dengue type 2 virus(DENV2)NS1 gene were constructed two partial sequences(1-413 bp)of the pcDNA3.1 eukaryotic plasmids and pET28a(+)plasmid for prokaryotic expression,identification,purification and quantification.The BALB/c mice were immunized by pcDNA3.1-M-NS1,pcDNA3.1-N-NS1 recombinant plasmids with adjuvant.Each animal received a primary inoculation and two boosts at 1-week intervals.Then the blood samples of BALB/c mice were collected from different experiment groups at day 7,14,28 and 56,respectively after first immunization.The specific IgM/IgG antibodies for NS1 protein in serum were confirmed by indirect ELISA.And then the activities of the specific protective antibody were determined by cytopathic effect inhibition(CPEI).Results Construction of the pET28a(+)-NS1 m/pET28a(+)-NS1n prokaryotic expression plasmid,SDS-PAGE analysis showed that,NS1 gene partial sequence was expressed,both the relative molecular weight of about 22.3×103:Western blot showed that the protein can bind anti-His tag monoclonal antibody;byNi affinity chromatographywith apurity of 92% protein,on the C6/36 cell toxicity,and can be used ELASA detection.The results showed that the levels of specific IgM/IgG antibody and neutralizing antibody activities were increased in pcDNA3.1-M-NS1 booster immunization group than other groups.The result had been observed longer duration of antibody level in peDNA3.1-M-NS1 booster immunization group.Conclusion Humoral immune response were significantly different between pcDNA3.1-M-NS1 and pcDNA3.1-N-NS1 recombinant plasmid immunized mice groups.