This study aimed to construct the dual-gene expression vector p Hsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The...This study aimed to construct the dual-gene expression vector p Hsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. Hep G2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in Hep G2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBs Ag and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection（P〈0.01 vs. control group）. RT-PCR showed that the level of HBV mRNA decreased by 80.2%（t=–99.22, P〈0.01）, the genomic HBV DNA by 92.8%（t=–73.06, P〈0.01）, and the supernatant of HBV DNA copy number by 89.8%（t=–47.13, P〈0.01） in pHsa-miR16-siRNA transfected group. It was suggested that the dual-gene expression vector pHsa-miR16-siRNA can inhibit the replication of HBV more efficiently than a single-gene expression vector.展开更多
Virus-based gene expression is a simple and powerful approach for functional genetic studies in plants.Here,sugarcane mosaic virus(SCMV,Potyvirus sacchari)was engineered as a dual gene expression vector for simultaneo...Virus-based gene expression is a simple and powerful approach for functional genetic studies in plants.Here,sugarcane mosaic virus(SCMV,Potyvirus sacchari)was engineered as a dual gene expression vector for simultaneous expression of two heterologous proteins in maize plants.Inoculation of the full-length cDNA clone of SCMV from agro-infiltrated Nicotiana benthamiana resulted in a rapid systemic infection in maize.To assess the possibility of SCMV as the gene expression vector,the marker gene GFP or GUS was inserted into either the NIb/CP or P1/HC-Pro junction site of SCMV to produce single-gene expression vectors.The results showed that these engineered SCMV vectors permitted efficient gene expression in systemically infected leaves and had the genetic capacity of inserts of more than 1800 bp,suggesting that both junction sites are suitable for heterologous gene insertion and expression.Furthermore,two different genes GFP and mCherry could be expressed simultaneously by engineering them into either NIb/CP or P1/HC-Pro junction sites of the same vector.These results clearly demonstrate the suitability of SCMV as a transient dual gene expression vector for maize plants.展开更多
基金supported by grants from the Independent Innovation Research Fund of Huazhong University of Science and Technology(No.2016YXMS200)Natural Science Foundation of the Science and Technology Department of Hubei Province(No.ZRMS2017000406)
文摘This study aimed to construct the dual-gene expression vector p Hsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. Hep G2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in Hep G2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBs Ag and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection（P〈0.01 vs. control group）. RT-PCR showed that the level of HBV mRNA decreased by 80.2%（t=–99.22, P〈0.01）, the genomic HBV DNA by 92.8%（t=–73.06, P〈0.01）, and the supernatant of HBV DNA copy number by 89.8%（t=–47.13, P〈0.01） in pHsa-miR16-siRNA transfected group. It was suggested that the dual-gene expression vector pHsa-miR16-siRNA can inhibit the replication of HBV more efficiently than a single-gene expression vector.
基金supported by the Biological Breeding-National Science and Technology Major Project(2024ZD04077)the Ministry of Science and Technology of the People’s Republic of China(2021YFD1400400)the National Natural Science Foundation of China(32172360,32130086,32430085).
文摘Virus-based gene expression is a simple and powerful approach for functional genetic studies in plants.Here,sugarcane mosaic virus(SCMV,Potyvirus sacchari)was engineered as a dual gene expression vector for simultaneous expression of two heterologous proteins in maize plants.Inoculation of the full-length cDNA clone of SCMV from agro-infiltrated Nicotiana benthamiana resulted in a rapid systemic infection in maize.To assess the possibility of SCMV as the gene expression vector,the marker gene GFP or GUS was inserted into either the NIb/CP or P1/HC-Pro junction site of SCMV to produce single-gene expression vectors.The results showed that these engineered SCMV vectors permitted efficient gene expression in systemically infected leaves and had the genetic capacity of inserts of more than 1800 bp,suggesting that both junction sites are suitable for heterologous gene insertion and expression.Furthermore,two different genes GFP and mCherry could be expressed simultaneously by engineering them into either NIb/CP or P1/HC-Pro junction sites of the same vector.These results clearly demonstrate the suitability of SCMV as a transient dual gene expression vector for maize plants.
