Establishment Of A Stable Cell Line Expressing The Non-structural Protein NS1 Of Human Bocavirus And Preliminary Study On The Transactivation Of NS1 Protein

Human Bocavirus 1(HBo V1)is a member of the genus Bocavirus of the subfamily Parvovirinae of the family Parvoviridae.The infection tissues of the HBo V1 are the human respiratory tract and can cause severe respiratory diseases.The most common symptom of HBo V1 infection is acute asthma.The infection rate is high among infants and young children from aged 6 months to 2 years.HBo V1 is extremely harmful to human health.The NS1 is the major non-structural protein of HBo V1.The NS1 gene encodes two isoforms of the NS1: the NS1 protein(NS1-100)with the size of approximately 100 k Da through the alternative splicing and polyadenylation mechanism,and the NS1 protein(NS1-70)with the size of approximately 70 k Da without an alternative splicing.As a multifunctional protein of the Parvoviridae family,NS1 plays an important role in the replication,transcriptional activation,apoptosis and cytopathic effects of parvovirus.As one of the most important functions of NS1 protein,transactivation can significantly increase the transcription level of viral genome,and accelerate the multiplication of the virus in cells.Therefore,studying the NS1 transactivation of HBo V1 is of great significance for the further in-depth study of the transactivation mechanism of HBo V1.In this study,the specific primers were designed with the gene sequence of HBo V1 WHL-1(Accession Number of NCBI: GU139423),the plasmid p WHL-1 constructed by our laboratory was used as a template.The target gene of NS1-100 and NS1-70 was amplified by PCR and cloned into the lentiviral expression vector p LVX-TRE3G-Zs Green1 to generate the lentiviral expression recombinant plasmids p LVX-NS1-100 and p LVX-NS1-70,respectively.Then these two plasmids were co-transfected into HEK293 T cells with the plasmid ps PAX2 and p MD2.G,then the cell supernatant was collected at 72 hours after transfection to obtain the virus solution.The virus solution was Purified and concentrated to obtain a concentrated virus solution with a titer of 1×105 TU/m L.Then 100?L of virus concentrated solution was used to infect the HEK293 T cells.The infected cells were observed under fluorescent microscope,the results indicated that all cells showed fluorescence.The stable cell line was selected by screening the cells with 4?g/m L puromycin selection medium for 10~14 days.Almost all viable cells expressed green fluorescent protein,suggesting that the stable cell lines expressing NS1-100 and NS1-70 protein were established.The lentiviral recombinant plasmid was transfected into HEK293 T cells,and the monoclonal cells expressing the target protein were selected by limiting dilution method.The same method described above was used for screening the cells and the cell linesstable expression of NS1-100 and NS1-70 were established as well.The stable cell lines were subjected to Western Blot detection,the expression of NS1-100 and NS1-70 protein was high,indicating that the stable cell lines expressing NS1-100 and NS1-70 was successfully constrcted.The target gene of NS1-100 and NS1-70 was cloned into the eukaryotic expression vector p CAGGS to obtain the eukaryotic expression recombinant plasmids p CAGGS-NS1-100 and p CAGGS-NS1-70,respectively,then these two constructs were co-transfected into HEK293 T cells with p GL3-HBo V1 or p GL3-CMV plasmid respectively.The activity of dual-luciferase was detected after 24 hours.The results showed that the HBo V1 promoter activity was 1.96 times higher than that of the CMV promoter and NS1-70 protein,the HBo V1 promoter activity was 1.64 times higher than that of the CMV promoter.The p GL3-Hbo V1 or p GL3-CMV plasmid was transfected into the the stable expression of NS1-100 protein or NS1-70 protein cell line.The activity of dual-luciferase was detected after 24 hours.The results showed that the transactivation activity of HBo V1 promoter by the stable expression of NS1-100 protein was 4.6 times higher than that of the CMV promoter and was 10 times higher than that of the CMV promoter.In the stable expression cell lines,the activity of HBo V1 promoter was higher than that of transient expression.Our results demonstrated that establishment of a regulatory and stable cell line for further exploring the mechanism of NS1 protein involved in transactivation provides an in vitro cell model and helps us to fully understand the function and mechanism of the non-structural protein NS1 of HBo V1 as well as the NS1 of other parvoviruses.