ABSTRACT
The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer.After designing specific gRNA sequences targeting HPV 16 E6,generating hCas9-EGFP and E6-gRNA-RFP plasmids,and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids,we determined the titer of the pseudotype virus using the TCID50 method.We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells.Experimental subjects were divided into control group,empty virus group,E6-gRNA transfected group,Cas9 transfected group and Cas9+E6-gRNA transfected group.The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis,and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time.RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups;the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups.Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6.The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells,and there were two cutting zones in the Cas9+E6-gRNA transfected group.However,the empty virus group,E6-gRNA transfected group and Cas9 transfected group had no corresponding zone.Compared with those in the control group,the empty virus group,E6-gRNA transfected group and Cas9 transfected group,the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9+E6-gRNA transfected group (P＜0.01).In addition,the proliferation and migration abilities of SiHa cells were significantly inhibited (P＜0.01).There were no significant differences among the other groups.In contrast to the control group,the HPV pseudotype virus carrying E6-gRNA and Cas9 plasmids could significantly delay the growth of tumor cells of the ectopic tumor transplantation model (P＜0.01).The CRISPR/Cas9 system mediated by the HPV pseudotype virus to knockout E6 gene expression exhibited a clear inhibitory effect on the biological function of SiHa cells,which indicated that knocking out the E6 gene using the CRISPR/Cas9 system mediated by the HPV pseudotype virus had a potential effect of eliminating HPV infection and inhibiting the growth of HPV-related tumors.Taken together,these findings provide insight into a new treatment strategy for the prevention and treatment of hr-HPV infected disease,particularly in HPV-related tumors.
ABSTRACT
The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer.After designing specific gRNA sequences targeting HPV 16 E6,generating hCas9-EGFP and E6-gRNA-RFP plasmids,and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids,we determined the titer of the pseudotype virus using the TCID50 method.We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells.Experimental subjects were divided into control group,empty virus group,E6-gRNA transfected group,Cas9 transfected group and Cas9+E6-gRNA transfected group.The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis,and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time.RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups;the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups.Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6.The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells,and there were two cutting zones in the Cas9+E6-gRNA transfected group.However,the empty virus group,E6-gRNA transfected group and Cas9 transfected group had no corresponding zone.Compared with those in the control group,the empty virus group,E6-gRNA transfected group and Cas9 transfected group,the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9+E6-gRNA transfected group (P＜0.01).In addition,the proliferation and migration abilities of SiHa cells were significantly inhibited (P＜0.01).There were no significant differences among the other groups.In contrast to the control group,the HPV pseudotype virus carrying E6-gRNA and Cas9 plasmids could significantly delay the growth of tumor cells of the ectopic tumor transplantation model (P＜0.01).The CRISPR/Cas9 system mediated by the HPV pseudotype virus to knockout E6 gene expression exhibited a clear inhibitory effect on the biological function of SiHa cells,which indicated that knocking out the E6 gene using the CRISPR/Cas9 system mediated by the HPV pseudotype virus had a potential effect of eliminating HPV infection and inhibiting the growth of HPV-related tumors.Taken together,these findings provide insight into a new treatment strategy for the prevention and treatment of hr-HPV infected disease,particularly in HPV-related tumors.
ABSTRACT
Objective To construct pseudotype retrovirus which integrates hemagglutinin(HA)of H5N1 avian influenza virus(AIV)isolated from human in Shenzhen.Methods AIV HA gene was amplified bv RT-PCR,then it was ligated with pGEM-T vector,and identified by restriction enzyme digestion and sequenced.HA gene was cloned into CMV/R vector at the site of Sal Ⅰ and BamH Ⅰ.pHR-Luc,pCMV&8.2 and CMV-HA were co-transfected into 293T cell by co-precipitation with calcium phosphate.The pseudotype virus supernatant was harvested 72 h post-transfection and ultracentrifugation,and the HA and P24 expression on the surface of pseudotype virus was analyzed by western blot.Meanwhile.the infection activity of HIV-HA pseudotype virus was identified in different kinds of cell lines,including MDCK,HeLa,CHO and 293T.Results A/Shenzhen/406H/06 belonged to subclade2.3 with open reading frame(ORF)of HA gene encoded 567 amino acides,whose accession number was EF137706 in GenBank.HA gene was cloned into CMV/R successfully.After co-transfection of above vectors,it revealed that HA protein could integrate pseudotype virus by western blot,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Finally.HIV-HA pseudotype virus could infect 4 kinds of cell lines,which indicated its property of infectivity and catholicity.Conclusion The pseudotype retrnvirns wassuccessfully constructed,which can integrate HA protein of A/Shenzhen/406H/06 and had property of infectivity.It call be used in the further research,including selection of neutralizing antibodies and epitope analysis.
ABSTRACT
The bacteriophage T7 RNAP gene was amplified via PCR from -lysogen DE3, and the gene was cloned into pBABEpuro retrovial vector, a recombinant plasmid named as pT7BABEpuro was constructed and sequenced. Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by liposomese, some pseudotype viruses were ingathered and transfected into IBRS-2 cell under polybrene. The IBRS-2 cell was propagated in DMEM with puromycin. The genome extraction from the cells transfected different times, the T7 RNAP gene was amplified from the genome by PCR, the mRNA of T7 RNAP protein expressed in IBRST7 cells was analyzed by RT-PCR, respectively, the results showed the T7 RNAP gene had been integrated into the chromosome of IBRS-2 cell and expressed stably at high level. To study whether T7 RNAP is of transcriptional activity in the established IBRST7 cell line, a plasmid pIERS-EGFP-ET with a reporter gene (EGFP) under control of the T7 promoter was constructed. IRES element from FMDV (for CAP-independent translation) was cloned into plasmid pET-43.1a-c(+) downstream of the T7 promoter sequence, then EGFP gene was cloned in frame downstream of the AUG codon of the FMDV IRES, resulting in the plasmid. IBRST7 cells were transfected with plasmid pIERS-EGFP-ET using lipfection, EGFP was expressed, the results showed the T7 RNAP in IBRST7 cells has transcriptional activity. IBRST7 cell line was directly transfected with linearized full-length cDNA of swine vesicular disease virus (SVDV) HK/70, infectious SVDV was efficiently recovered from the cDNA. The reverse genetic procedure is simplified to a faster, one step protocol to recover RNA virus and will be useful to understand the mechanisms of molecular pathology of RNA virus and develop effective vaccines.