AIM:To clarify the specific roles and mechanisms of long interspersed nuclear element-1 ORF-1 protein [human long interspersed nuclear element-1(LINE-1),ORF-1p] in chemotherapeutic drug resistance and cell proliferati...AIM:To clarify the specific roles and mechanisms of long interspersed nuclear element-1 ORF-1 protein [human long interspersed nuclear element-1(LINE-1),ORF-1p] in chemotherapeutic drug resistance and cell proliferation regulation in hepatocellular carcinoma(HCC) cells.METHODS:MTT assays were performed to identify the effect of the chemotherapeutic drug toxicity on HepG2 cells.Cell proliferation inhibition and the IC 50 were calculated by the Origin 8.0 software.Western blotting assays were performed to investigate whether LINE-1 ORF-1p modulates the expression of some important genes,including p53,p27,p15,Bcl-2,mdr,and p-gp.To corroborate the proliferation and anchor-independent growth results,the HepG2 cells were analyzed by flow cytometry to investigate the effect of LINE-1 ORF1p on the apoptosis regulation.RESULTS:LINE-1 ORF-1p contributed to the resistance to several chemotherapeutic drugs(cisplatin and epirubicin) in HepG2 cells.The IC 50 of the epirubicin and cisplatin increased from 36.04 nmol/L to 59.11 nmol/L or from 37.94 nmol/L to 119.32 nmol/L.Repression of LINE-1 ORF-1p expression by the siRNA could markedly enhance the response of HepG2 cells to the epirubicin and cisplatin.The IC 50 correspondingly decreased from 28.06 nmol/L to 3.83 nmol/L or from 32.04 nmol/L to 2.89 nmol/L.Interestingly,down-regulation of LINE-1 ORF-1p level by siRNA could promote the response of HepG2 cells to the paclitaxel.The IC 50 decreased from 35.90 nmol/L to 7.36 nmol/L.However,overexpression of LINE-1 ORF-1p did not modulate the paclitaxel toxicity in HepG2 cells.Further Western blotting revealed that LINE-1 ORF-1p enhanced mdr and p-gp gene expression.As a protein arrested in the nucleus,LINE-1 ORF-1p may function through modulating transcriptional activity of some important transcription factors.Indeed,LINE-1 ORF-1p promoted HepG2 cell proliferation,anchor-independent growth and protected the cells against apoptosis through modulating the expression of p15,p21,p53,and Bcl-2 genes.CONCLUSION:LINE-1 ORF-1p promotes Hep展开更多
A pair of specific primers were designed and synthesized according to the published sequences of ORF1 gene of PCV 2. The complete DNA fragment of ORF1 gene was obtained by PCR from viral DNA of PCV 2 QD strain.Its nuc...A pair of specific primers were designed and synthesized according to the published sequences of ORF1 gene of PCV 2. The complete DNA fragment of ORF1 gene was obtained by PCR from viral DNA of PCV 2 QD strain.Its nuclectide sequence was determined by the dideoxy mediated chain termination method.The results showed that the complete open reading frame (ORF) of ORF1 gene encoding 314 amino acids was 945 bp in length.A comparison of the nucleotide and amino acid sequences of ORF1 gene with that of other PCV strains showed that the identity of nucleotide with PCV 1 and PCV 2 were 83% and 96 4%～99 2% respectively,and identity of the deduced amino acid with PCV 1 and PCV 2 were 84% and more than 98% respecitively.The DNA fragment of ORF1 gene was subcloned into prokaryotic expression vector pET 28a and pGEX KG;while the specific non fusion and fusion proteins with GST of molecular weight 38 kD and 63 kD were expressed in E.coli BL 21 (DE3).Western blotting assay indicated that the polyclonal antibody against PCV 2 could recognize these two proteins.展开更多
[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively ...[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively and compare them. [Method] According to the relatively＇conserved sequences of PCV20RF1 and ORF2 registered in GenBank, two pairs of specific primers and TaqMan probes were designed and synthesized. Then the recombinant plasmids containing the whole sequences of PCV20RF1 and ORF2 were constructed to draw the standard curves through optimizing the reaction system and conditions. And thus two kinds of TaqMan real-time PCR detection methods based on the whole sequences of ORF1 and ORF2 respectively were constructed for PCV2. [Result] For the two established standard curves, the Ct values showed a good linear relationship with the loga- rithms of copy numbers of templates （F2〉0.99）. The amplification efficiency ranged from 90% to 110%. The amplifications all had a good repeatability with variation coefficients within groups all less than 5%. Moreover, the amplifications all had a good specificity. When the sequences of porcine parvovirus （PPV）, porcine circovirus type 1 （PCV1）, swine pseudorabies virus （PRV）, porcine reproductive and respiratory syndrome virus （PRRSV） were used as templates, the target sequence was not amplified. The amplifications also had a high sensitivity. The ORF1 detection method could reach 1.0x10T copies/;ul, and the ORF2 detection method could reach 1.0×10^2 copies/μl. The two established real-time PCR detection methods were used to detect the 80 clinical samples respectively. The results showed the magnitudes of 72 amplified samples were basically consistent between the 2 detection methods, while the magnitudes of the other 8 amplified samples were inconsistent. Then the 8 samples were detected with SYBR Green I real-time PCR method established based on the sequence of PCV2-1ike factor P1 by Wen et aL The PCV2-1ike factor P1 was amplified in all the 8展开更多
Objective:To analyze the sequence of ORFl protein of Torque teno virus to prepare for the future hyhrid experiments.Methods:The sequence of ORFl protein of Torque teno vims was analyzed by bioinformalics using some we...Objective:To analyze the sequence of ORFl protein of Torque teno virus to prepare for the future hyhrid experiments.Methods:The sequence of ORFl protein of Torque teno vims was analyzed by bioinformalics using some web lools.Results:The most likely cleavage site was between position 14aa and 15aa and signal peptide may be position laa-14aa.Two possible transmembrane helices from inside to outside and three possible transmembrane helices from outside to inside were found.The position 509(NKTN) was the potential:V-glyeosylation site. The speculative molecular weight of TTV ORF1 protein,which may be a kind of unstable protein was 88 705.7 Da.laa-91aa and 278aa-361aa were localized in non-regular secondary structure region.Conclusions:TTV ORF1 protein may be a nuclear protein which contains two nonregular secondary structure region.265aa to 486aa and 510aa to 679aa may be the two approciale fragments to construct the plasmids,which would be prepared for the future hybrid experiments to study the functional positions of the protein and the interactions between TTV and its hosts. Bioinformatics analysis would possibly make it easier to study the protein’s function.展开更多
基金Supported by The Key Scientific and Technological Research Foundation of the National Special Purpose Program,No.2008ZX10002-018
文摘AIM:To clarify the specific roles and mechanisms of long interspersed nuclear element-1 ORF-1 protein [human long interspersed nuclear element-1(LINE-1),ORF-1p] in chemotherapeutic drug resistance and cell proliferation regulation in hepatocellular carcinoma(HCC) cells.METHODS:MTT assays were performed to identify the effect of the chemotherapeutic drug toxicity on HepG2 cells.Cell proliferation inhibition and the IC 50 were calculated by the Origin 8.0 software.Western blotting assays were performed to investigate whether LINE-1 ORF-1p modulates the expression of some important genes,including p53,p27,p15,Bcl-2,mdr,and p-gp.To corroborate the proliferation and anchor-independent growth results,the HepG2 cells were analyzed by flow cytometry to investigate the effect of LINE-1 ORF1p on the apoptosis regulation.RESULTS:LINE-1 ORF-1p contributed to the resistance to several chemotherapeutic drugs(cisplatin and epirubicin) in HepG2 cells.The IC 50 of the epirubicin and cisplatin increased from 36.04 nmol/L to 59.11 nmol/L or from 37.94 nmol/L to 119.32 nmol/L.Repression of LINE-1 ORF-1p expression by the siRNA could markedly enhance the response of HepG2 cells to the epirubicin and cisplatin.The IC 50 correspondingly decreased from 28.06 nmol/L to 3.83 nmol/L or from 32.04 nmol/L to 2.89 nmol/L.Interestingly,down-regulation of LINE-1 ORF-1p level by siRNA could promote the response of HepG2 cells to the paclitaxel.The IC 50 decreased from 35.90 nmol/L to 7.36 nmol/L.However,overexpression of LINE-1 ORF-1p did not modulate the paclitaxel toxicity in HepG2 cells.Further Western blotting revealed that LINE-1 ORF-1p enhanced mdr and p-gp gene expression.As a protein arrested in the nucleus,LINE-1 ORF-1p may function through modulating transcriptional activity of some important transcription factors.Indeed,LINE-1 ORF-1p promoted HepG2 cell proliferation,anchor-independent growth and protected the cells against apoptosis through modulating the expression of p15,p21,p53,and Bcl-2 genes.CONCLUSION:LINE-1 ORF-1p promotes Hep
文摘A pair of specific primers were designed and synthesized according to the published sequences of ORF1 gene of PCV 2. The complete DNA fragment of ORF1 gene was obtained by PCR from viral DNA of PCV 2 QD strain.Its nuclectide sequence was determined by the dideoxy mediated chain termination method.The results showed that the complete open reading frame (ORF) of ORF1 gene encoding 314 amino acids was 945 bp in length.A comparison of the nucleotide and amino acid sequences of ORF1 gene with that of other PCV strains showed that the identity of nucleotide with PCV 1 and PCV 2 were 83% and 96 4%～99 2% respectively,and identity of the deduced amino acid with PCV 1 and PCV 2 were 84% and more than 98% respecitively.The DNA fragment of ORF1 gene was subcloned into prokaryotic expression vector pET 28a and pGEX KG;while the specific non fusion and fusion proteins with GST of molecular weight 38 kD and 63 kD were expressed in E.coli BL 21 (DE3).Western blotting assay indicated that the polyclonal antibody against PCV 2 could recognize these two proteins.
基金Supported by National Natural Science Foundation of China(31302071)Special Fund for Agro-scientific Research in the Public Interest(201303046)+1 种基金Jiangsu Agricultural Science and Technology Innovation Fund(CX(14)2045)"333 High-level Personnel Training Project"of Jiangsu Province(BRA2012194)~~
文摘[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively and compare them. [Method] According to the relatively＇conserved sequences of PCV20RF1 and ORF2 registered in GenBank, two pairs of specific primers and TaqMan probes were designed and synthesized. Then the recombinant plasmids containing the whole sequences of PCV20RF1 and ORF2 were constructed to draw the standard curves through optimizing the reaction system and conditions. And thus two kinds of TaqMan real-time PCR detection methods based on the whole sequences of ORF1 and ORF2 respectively were constructed for PCV2. [Result] For the two established standard curves, the Ct values showed a good linear relationship with the loga- rithms of copy numbers of templates （F2〉0.99）. The amplification efficiency ranged from 90% to 110%. The amplifications all had a good repeatability with variation coefficients within groups all less than 5%. Moreover, the amplifications all had a good specificity. When the sequences of porcine parvovirus （PPV）, porcine circovirus type 1 （PCV1）, swine pseudorabies virus （PRV）, porcine reproductive and respiratory syndrome virus （PRRSV） were used as templates, the target sequence was not amplified. The amplifications also had a high sensitivity. The ORF1 detection method could reach 1.0x10T copies/;ul, and the ORF2 detection method could reach 1.0×10^2 copies/μl. The two established real-time PCR detection methods were used to detect the 80 clinical samples respectively. The results showed the magnitudes of 72 amplified samples were basically consistent between the 2 detection methods, while the magnitudes of the other 8 amplified samples were inconsistent. Then the 8 samples were detected with SYBR Green I real-time PCR method established based on the sequence of PCV2-1ike factor P1 by Wen et aL The PCV2-1ike factor P1 was amplified in all the 8
基金supported by a grant from a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘Objective:To analyze the sequence of ORFl protein of Torque teno virus to prepare for the future hyhrid experiments.Methods:The sequence of ORFl protein of Torque teno vims was analyzed by bioinformalics using some web lools.Results:The most likely cleavage site was between position 14aa and 15aa and signal peptide may be position laa-14aa.Two possible transmembrane helices from inside to outside and three possible transmembrane helices from outside to inside were found.The position 509(NKTN) was the potential:V-glyeosylation site. The speculative molecular weight of TTV ORF1 protein,which may be a kind of unstable protein was 88 705.7 Da.laa-91aa and 278aa-361aa were localized in non-regular secondary structure region.Conclusions:TTV ORF1 protein may be a nuclear protein which contains two nonregular secondary structure region.265aa to 486aa and 510aa to 679aa may be the two approciale fragments to construct the plasmids,which would be prepared for the future hybrid experiments to study the functional positions of the protein and the interactions between TTV and its hosts. Bioinformatics analysis would possibly make it easier to study the protein’s function.
