The DNA of duck plague virus (DPV) thymidine kinase (TK) gene was cloned and sequenced from a vaccine virus in the study. Degenerate oligonucleotide primers for the consensus site of herpesvirus UL24, TK, and glyc...The DNA of duck plague virus (DPV) thymidine kinase (TK) gene was cloned and sequenced from a vaccine virus in the study. Degenerate oligonucleotide primers for the consensus site of herpesvirus UL24, TK, and glycoprotein H(gH) gene were used in the polymerase chain reaction (PCR) to amplify DNA product with 3 741-base-pairs (bp) in size. DNA sequence analysis revealed a 1 077-base-pairs (bp) open reading frame (ORF) encoding a 358 amino acid polypeptide homologous to herpesvirus TK proteins. The predicted TK protein shared 31.2, 41.3, 35.7, 37.4, and 28.4% identity with herpes simplex virus typel, equine herpesvirus type 4, Marek's disease virus 2, herpesvirus turkey, and infectious laryngotracheitis virus, respectively. Comparison of the amino acid sequences of other herpesvirus TK proteins showed that these proteins were not conserved on the whole, otherwise the portion of the TK proteins corresponding to the nucleotide binding domain and the nucleoside binding site were highly conserved among herpesvirus. Comparison with the amino acid sequences of the conserved nucleotide and nucleoside binding domains of other eleven herpesvirus TK proteins to the predicted DPV peptide confirmed its identity as the DPV TK protein.展开更多
A retroviral vector(LNHcTL)containing the herpes simplex virus type 1 thymldine kinase(HSVI-tk)gene was constructed and used for transduction of the gene into human hepatocellular carcinoma cells(SMMC-7721).Xenografte...A retroviral vector(LNHcTL)containing the herpes simplex virus type 1 thymldine kinase(HSVI-tk)gene was constructed and used for transduction of the gene into human hepatocellular carcinoma cells(SMMC-7721).Xenografted tumor on nude mice was produced with the injection of the transduced cells(SMMC- 7721/LN HcTL) inoculated subcutaneously and showed regression when treated with Acyclovir.The mean weight of the residual tumors was six times less than that of the controls'tumors. Patients with liver carcinoma were given an intratumoral injection of ampbotropic packing cells(PA317/LNHcTL)producing HSV1-tk recombinant retroviral particles,and then treated with Acyclovir intravenously, which showed a marked regression of the tumor.Our preliminary data suggest that HSV1-tk gene/Acyclovir system might be a useful therapeutic approach for the treatment of hepatic carcinoma in humans.展开更多
AIM: To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase (HSV-tk) targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. METHODS: Recombinant adenovirus c...AIM: To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase (HSV-tk) targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. METHODS: Recombinant adenovirus containing kinase domain insert with receptor (KDR) or cytomegalovirus (CMV) promoter-controlled HSV-tk gene (AdKDR-tk and AdCMV-tk) was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells (HUVEC) and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir (GCV), the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups: ganciclovir group (Ⅰ), Ad group (Ⅱ), AdCMV-tk group (Ⅲ) and AdKDR-tk group (Ⅳ). Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir (GCV) was given at a dose of 100 mg·kg-1·d-1 (ip) started on the following day and lasted 10 d. Microvessel density (MVD) of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and after the last GCV dose.RESULTS: Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk- transfected HUVEC and HepG2 decreased from 100% to (28.94 ± 5.67)% and (75.45 ± 2.91)% at proper order, respectively (P < 0.01), while the survival rate of AdCMV- tk-transfected HUVEC and HepG2 declined from 100% to (17.56 ± 2.48)% and (23.15 ± 5.72)%, respectively (P > 0.05). Compared with groupⅠ, there was a decrease of tumor weight by 14.7% in group Ⅲ and by 23.6% in group Ⅳ. And there was a distinct difference between group Ⅲ and Ⅳ (P < 0.05). The median MVD for all groups was 37.4 ± 8.6, 30.6 ± 7.8, 27.6 ± 7.1, and 10.7 ± 4.1 (microvessels/mm2) in groupⅠ, Ⅱ, Ⅲ and Ⅳ, respectively. And there was a marked difference between group Ⅲ and Ⅱ (P < 0.05), Ⅳ and Ⅱ (P < 0.01), and Ⅳ and Ⅲ (P < 0.01). CONCLUSION: KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV.展开更多
The therapeutic effect of herpes simplex virus thymi-dine kinase/ganciclovir (HSV-tk/GCV) system on hepa-tocellular carcinoma was studied in this experimeflt. Thetk-containing retroviral recombinants were used to infe...The therapeutic effect of herpes simplex virus thymi-dine kinase/ganciclovir (HSV-tk/GCV) system on hepa-tocellular carcinoma was studied in this experimeflt. Thetk-containing retroviral recombinants were used to infecthepatoma cells (BEL-7402) and the cells were treated withganciclovir (0-1000 pg/ml). The results showed that HSV-tk gene could be efficielltly transferred in Vitro into hep-atoma cells and stably expressed. The growth potentialof the tk-containing cells was significantly inhibited byGCV (P<0.01) as compared to the non-tk-containing cells.The antitumor effect of HSV-tk/GCV system was also pro-duced ex vivo in tk-containing tumor of nude mice as char-acterized by a marked decrease in tumor growth after GCVtreatment contrary to a progressive enlargement of non-tk-containing tumors. Although the histological examinationdemonstrated that the efficiency of the gene transfer wasless than 30%, the killing effect of HSV-tk/GCV systemon hepatocellular carcinoma was still significantIy gener-ated. The proper mechanism of HSV-tk gene therapy onhepatic tumor referred as "bystander effect" in therapeu-tic approach has not been found in this study and requiredto be explored further.展开更多
The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC)...The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC) cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction (RT-PCR) and strept avidin-biotin complex (SABC) im- munohistochemical method. The killing effect of GCV, cisplatin (CDDP), etoposide (VP-16), vincristine (VCR), methotrexate (MTX), 5-fluorouracil (5-Fu), and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry (FCM). The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38 % and 35.51%, and the proportion of necrosis being 33.05 % and 28.87 %, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment. Our results highlight the potential for such new combination therapies for future treatments of HRPC.展开更多
Objective: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Her...Objective: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage. Methods: A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase (hTERT), the coding sequence of E1A gene, an internal ribosome entry site sequence (IRES) and the coding sequence of herpes simplex virus-thymidine kinase (HSV-TK) was constructed. Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK. The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured. The infection efficiency was determined by flow cytometry. Results: Ad-ETK deliverys E1A and HSV-TK gene, which selectively replicates in hTERT-positive tumor cells, and the progeny virus can reach up to 150 IU/cell. Our in vitro study showed that Ad-ETK plus ganciclovir (GCV) induced an obvious cell death. Conclusion: An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.展开更多
The long-term success of gene therapy for cancer relies heavily on the development of effective targeting systems. We investigate the possibility of targeted gene therapy using promoter of carcinoembryonic antigen (CE...The long-term success of gene therapy for cancer relies heavily on the development of effective targeting systems. We investigate the possibility of targeted gene therapy using promoter of carcinoembryonic antigen (CEA) gene. By using luciferase reporter gene, we found that CEA promoter exhibit 16 times high activity in CEA-producing lung cancer cells, A549 than in nonproducing cells, Hela. We also constructed a recombinant expression plasmid pCEATK, in which CEA promoter drives the effector gene, thymidine kinase gene of Herpes Simplex Virus (HSVTK). A549 cells transfected with pCEATK became 865 times more sensitive to ganciclovir (GCV) than the control cells. However, Hela cells transfected with this plasmid remained resistant to GCV. These data indicate the potential for targeted gene therapy using the CEA promoter against CEA-producing tumor cells, such as lung cancer cells.展开更多
Recent research showed that the transfer of the Herpes simplex virus thymidine kinase (HSV tk) gene into malignant tumor cells could confer the tumor cells susceptibility to the antiviral drug ganciclovir (GCY), thus ...Recent research showed that the transfer of the Herpes simplex virus thymidine kinase (HSV tk) gene into malignant tumor cells could confer the tumor cells susceptibility to the antiviral drug ganciclovir (GCY), thus produce "killing" effect selectively on the tumor cells exposed to GCV. We constructed a recombinant retroviral vector LHyTK/N by inserting HyTK gene into the retroviral vector LXSN and cutting out the SV4O early promoter. The HyTK gene was transferred into mouse melanoma cell line B16 mediated by a recombinant virus. PCR analysis showed that the HyTK gene was successfully transferred and replication-competent virus was absent. The "killing" effect on B16/HyTK+ cells exposed to GCV (>0. 1 μmol/L) was evident when investigated under light microscope and by live cell counting.展开更多
Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a ki...Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses. Methods pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E. coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease Pad and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with Pacl and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000. Results Comet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk. Conclusion The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.展开更多
To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virule...To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virulence, thymidine kinase (TK)-deficient mutant of pseudorabies virus strain Hubei (PRV HB) was isolated by selection for resistance to 5-bromodeoxyuridine. The tk genes of PRV HB and its TK mutant were cloned and sequenced. 1587 base pairs of the tk gene and flanking regions of wild-type (wt) virus were sequenced, which included an open reading frame (ORF) of 1098 bp encoding a protein of 366 amino acids. The ORF contained two 137-bp repeated sequences, which were connected by an adenosine. 1458 bp of the tk and flanking regions of TK- mutant were sequenced. Analysis of the tk gene sequence of TK mutant indicated that one of 137 bp repeated sequence and the connecting adenosine in the tk gene of the wt virus was deleted and a repeated sequence of 8 nucleotides (GCGCGCC) was inserted. All展开更多
文摘The DNA of duck plague virus (DPV) thymidine kinase (TK) gene was cloned and sequenced from a vaccine virus in the study. Degenerate oligonucleotide primers for the consensus site of herpesvirus UL24, TK, and glycoprotein H(gH) gene were used in the polymerase chain reaction (PCR) to amplify DNA product with 3 741-base-pairs (bp) in size. DNA sequence analysis revealed a 1 077-base-pairs (bp) open reading frame (ORF) encoding a 358 amino acid polypeptide homologous to herpesvirus TK proteins. The predicted TK protein shared 31.2, 41.3, 35.7, 37.4, and 28.4% identity with herpes simplex virus typel, equine herpesvirus type 4, Marek's disease virus 2, herpesvirus turkey, and infectious laryngotracheitis virus, respectively. Comparison of the amino acid sequences of other herpesvirus TK proteins showed that these proteins were not conserved on the whole, otherwise the portion of the TK proteins corresponding to the nucleotide binding domain and the nucleoside binding site were highly conserved among herpesvirus. Comparison with the amino acid sequences of the conserved nucleotide and nucleoside binding domains of other eleven herpesvirus TK proteins to the predicted DPV peptide confirmed its identity as the DPV TK protein.
文摘A retroviral vector(LNHcTL)containing the herpes simplex virus type 1 thymldine kinase(HSVI-tk)gene was constructed and used for transduction of the gene into human hepatocellular carcinoma cells(SMMC-7721).Xenografted tumor on nude mice was produced with the injection of the transduced cells(SMMC- 7721/LN HcTL) inoculated subcutaneously and showed regression when treated with Acyclovir.The mean weight of the residual tumors was six times less than that of the controls'tumors. Patients with liver carcinoma were given an intratumoral injection of ampbotropic packing cells(PA317/LNHcTL)producing HSV1-tk recombinant retroviral particles,and then treated with Acyclovir intravenously, which showed a marked regression of the tumor.Our preliminary data suggest that HSV1-tk gene/Acyclovir system might be a useful therapeutic approach for the treatment of hepatic carcinoma in humans.
基金Supported by the National Natural Science Foundation of China, No. 30371386the Natural Science Foundation of Guangdong Province, No. 31010
文摘AIM: To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase (HSV-tk) targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. METHODS: Recombinant adenovirus containing kinase domain insert with receptor (KDR) or cytomegalovirus (CMV) promoter-controlled HSV-tk gene (AdKDR-tk and AdCMV-tk) was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells (HUVEC) and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir (GCV), the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups: ganciclovir group (Ⅰ), Ad group (Ⅱ), AdCMV-tk group (Ⅲ) and AdKDR-tk group (Ⅳ). Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir (GCV) was given at a dose of 100 mg·kg-1·d-1 (ip) started on the following day and lasted 10 d. Microvessel density (MVD) of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and after the last GCV dose.RESULTS: Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk- transfected HUVEC and HepG2 decreased from 100% to (28.94 ± 5.67)% and (75.45 ± 2.91)% at proper order, respectively (P < 0.01), while the survival rate of AdCMV- tk-transfected HUVEC and HepG2 declined from 100% to (17.56 ± 2.48)% and (23.15 ± 5.72)%, respectively (P > 0.05). Compared with groupⅠ, there was a decrease of tumor weight by 14.7% in group Ⅲ and by 23.6% in group Ⅳ. And there was a distinct difference between group Ⅲ and Ⅳ (P < 0.05). The median MVD for all groups was 37.4 ± 8.6, 30.6 ± 7.8, 27.6 ± 7.1, and 10.7 ± 4.1 (microvessels/mm2) in groupⅠ, Ⅱ, Ⅲ and Ⅳ, respectively. And there was a marked difference between group Ⅲ and Ⅱ (P < 0.05), Ⅳ and Ⅱ (P < 0.01), and Ⅳ and Ⅲ (P < 0.01). CONCLUSION: KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV.
文摘The therapeutic effect of herpes simplex virus thymi-dine kinase/ganciclovir (HSV-tk/GCV) system on hepa-tocellular carcinoma was studied in this experimeflt. Thetk-containing retroviral recombinants were used to infecthepatoma cells (BEL-7402) and the cells were treated withganciclovir (0-1000 pg/ml). The results showed that HSV-tk gene could be efficielltly transferred in Vitro into hep-atoma cells and stably expressed. The growth potentialof the tk-containing cells was significantly inhibited byGCV (P<0.01) as compared to the non-tk-containing cells.The antitumor effect of HSV-tk/GCV system was also pro-duced ex vivo in tk-containing tumor of nude mice as char-acterized by a marked decrease in tumor growth after GCVtreatment contrary to a progressive enlargement of non-tk-containing tumors. Although the histological examinationdemonstrated that the efficiency of the gene transfer wasless than 30%, the killing effect of HSV-tk/GCV systemon hepatocellular carcinoma was still significantIy gener-ated. The proper mechanism of HSV-tk gene therapy onhepatic tumor referred as "bystander effect" in therapeu-tic approach has not been found in this study and requiredto be explored further.
文摘The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC) cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction (RT-PCR) and strept avidin-biotin complex (SABC) im- munohistochemical method. The killing effect of GCV, cisplatin (CDDP), etoposide (VP-16), vincristine (VCR), methotrexate (MTX), 5-fluorouracil (5-Fu), and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry (FCM). The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38 % and 35.51%, and the proportion of necrosis being 33.05 % and 28.87 %, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment. Our results highlight the potential for such new combination therapies for future treatments of HRPC.
文摘Objective: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage. Methods: A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase (hTERT), the coding sequence of E1A gene, an internal ribosome entry site sequence (IRES) and the coding sequence of herpes simplex virus-thymidine kinase (HSV-TK) was constructed. Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK. The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured. The infection efficiency was determined by flow cytometry. Results: Ad-ETK deliverys E1A and HSV-TK gene, which selectively replicates in hTERT-positive tumor cells, and the progeny virus can reach up to 150 IU/cell. Our in vitro study showed that Ad-ETK plus ganciclovir (GCV) induced an obvious cell death. Conclusion: An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.
文摘The long-term success of gene therapy for cancer relies heavily on the development of effective targeting systems. We investigate the possibility of targeted gene therapy using promoter of carcinoembryonic antigen (CEA) gene. By using luciferase reporter gene, we found that CEA promoter exhibit 16 times high activity in CEA-producing lung cancer cells, A549 than in nonproducing cells, Hela. We also constructed a recombinant expression plasmid pCEATK, in which CEA promoter drives the effector gene, thymidine kinase gene of Herpes Simplex Virus (HSVTK). A549 cells transfected with pCEATK became 865 times more sensitive to ganciclovir (GCV) than the control cells. However, Hela cells transfected with this plasmid remained resistant to GCV. These data indicate the potential for targeted gene therapy using the CEA promoter against CEA-producing tumor cells, such as lung cancer cells.
文摘Recent research showed that the transfer of the Herpes simplex virus thymidine kinase (HSV tk) gene into malignant tumor cells could confer the tumor cells susceptibility to the antiviral drug ganciclovir (GCY), thus produce "killing" effect selectively on the tumor cells exposed to GCV. We constructed a recombinant retroviral vector LHyTK/N by inserting HyTK gene into the retroviral vector LXSN and cutting out the SV4O early promoter. The HyTK gene was transferred into mouse melanoma cell line B16 mediated by a recombinant virus. PCR analysis showed that the HyTK gene was successfully transferred and replication-competent virus was absent. The "killing" effect on B16/HyTK+ cells exposed to GCV (>0. 1 μmol/L) was evident when investigated under light microscope and by live cell counting.
基金the National Natural Science Foundation of China(No.30070808).
文摘Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses. Methods pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E. coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease Pad and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with Pacl and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000. Results Comet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk. Conclusion The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.
文摘To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virulence, thymidine kinase (TK)-deficient mutant of pseudorabies virus strain Hubei (PRV HB) was isolated by selection for resistance to 5-bromodeoxyuridine. The tk genes of PRV HB and its TK mutant were cloned and sequenced. 1587 base pairs of the tk gene and flanking regions of wild-type (wt) virus were sequenced, which included an open reading frame (ORF) of 1098 bp encoding a protein of 366 amino acids. The ORF contained two 137-bp repeated sequences, which were connected by an adenosine. 1458 bp of the tk and flanking regions of TK- mutant were sequenced. Analysis of the tk gene sequence of TK mutant indicated that one of 137 bp repeated sequence and the connecting adenosine in the tk gene of the wt virus was deleted and a repeated sequence of 8 nucleotides (GCGCGCC) was inserted. All