AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, a...AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.展开更多
AIM: To determine the frequencies of HGV and TTV infections in blood donors in Hangzhou. METHODS: RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nu...AIM: To determine the frequencies of HGV and TTV infections in blood donors in Hangzhou. METHODS: RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nucleotide sequence analysis were performed. RESULTS: Thirty-two (15.8%) and 30 (14.8%) of the 203 serum samples were positive for HGV RNA and TTV DNA, respectively. And 5 (2.5%) of the 203 serum samples were detectable for both HGV RNA and TTV DNA. Homology of the nucleotide sequences of HGV RT-nested PCR products and TTV semi-nested PCR products from 3 serum samples compared with the reported HGV and TTV sequences was 89.36%, 87.94%, 88.65% and 63.51%, 65.77% and 67.12%, respectively. CONCLUSION: The infection rates of HGV and/or TTV in blood donors are relatively high, and to establish HGV and TTV examinations to screen blood donors is needed for transfusion security. The genomic heterogeneity of TTV or HGV is present in the isolates from different areas.展开更多
[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activi...[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 （DE3） and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 （DE3）. Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.展开更多
AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein. METHODS: The complete PreS region (P...AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein. METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6× histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization. RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in E.coli. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain. CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in E.coli can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.展开更多
AIM:To determine the prevalences of TTV and HGV infections among blood donors and patients with chronic liver disease in Korea,to investigate the association of TTV and HGV infections with blood transfusion,and to ass...AIM:To determine the prevalences of TTV and HGV infections among blood donors and patients with chronic liver disease in Korea,to investigate the association of TTV and HGV infections with blood transfusion,and to assess the correlation between TTV and HGV viremia and hepatic damage. METHODS:A total of 391 serum samples were examined in this study.Samples were obtained from healthy blood donors(n=110),hepatitis B surface antigen(HBsAg)-positive donors(n=112),anti-hepatitis C virus(anti-HCV)-positive donors(n=69),patients with type B chronic liver disease (n=81),and patients with type C chronic liver disease(n=19). Trv DNA was detected using the hemi-nested PCR.HGV RNA was tested using RT-PCR.A history of blood transfusion and serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were also determined. RESULTS:TTV DNA was detected in 8.2%of healthy blood donors,16.1%of HBsAg-positive donors,20.3%of anti- HCV-positive donors,21.0%of patients with type B chronic liver disease,and 21.1%of patients with type C chronic liver disease.HGV RNA was detected in 1.8%of healthy blood donors,1.8%of HBsAg-positive donors,17.4%of anti-HCV-positive donors,13.6%of patients with type B chronic liver disease,and 10.5%of patients with type C chronic liver disease.The prevalence of TTV and HGV infections in HBV- or HCV-positive donors and patients was significantly higher than in healthy blood donors(P<0.05), except for the detection rate of HGV in HBsAg-positive donors which was the same as for healthy donors.There was a history of transfusion in 66.7%of TTV DNA-positive patients and 76.9%of HGV RNA-positive patients(P<0.05).No significant increase in serum ALT and AST was detected in the TTV or HGV-positive donors and patients. CONCLUSION:TTV and HGV infections are more frequently found in donors and patients infected with HBV or HCV than in healthy blood donors.However,there is no significant association between TTV or HGV infections and liver injury.展开更多
AIM: To investigate the state of infection, replication site, pathogenicity and clinical significance of transfusion transmitted virus (TTV) in patients with hepatitis, especially in patients of unknown etiology. METH...AIM: To investigate the state of infection, replication site, pathogenicity and clinical significance of transfusion transmitted virus (TTV) in patients with hepatitis, especially in patients of unknown etiology. METHODS: Liver tissues taken from 136 cases of non-A non-G hepatitis were tested for TT virus antigen and nucleic acid by in situ hybridization (ISH) and nested-polymerase chain reaction (PCR). Among them, TT virus genome and its complemental strand were also detected in 24 cases of autopsy liver and extrahepatic tissues with ISH. Meanwhile, TTV DNA was detected in the sera of 187 hepatitis patients by nested-PCR. The pathological and clinical data of the cases infected with TTV only were analyzed. RESULTS: In liver, the total positive rate of TTV DNA was 32.4% and the positive signals were located in the nuclei of hepatocytes. In serus, TTV DNA was detected in 21.4% cases of hepatitis A-G, 34.4% of non-A non-G hepatitis and 15% of healthy donors. The correspondence rate of TTV DNA detection between liver tissue with ISH and sera with PCR was 63.2% and 89.3% in the same liver tissues by ISH and by PCR, respectively.Using double-strand probes and single-strand probes designed to detect TTV genome, the correspondence rate of TTV DNA detected in liver and extrahepatic tissues was 85.7%. Using single-strand probes, TTV genome could be detected in liver and extrahepatic tissues by PCR, but its complemental strands (replication strands) could be observed only in livers. The liver function of most cases infected with TTV alone was abnormal and the liver tissues had different pathological damage such as ballooning, acidophilia degeneration, formation of apoptosis bodies and focus of necrosis, but the inflammation in the lobule and portal area was mild. CONCLUSION: The positive rate of TTV DNA among cases of hepatitis was higher than that of donors, especially in patients with non-A non-G hepatitis, but most of them were coinfected with other hepatitis viruses. TTV can infect not only hepatocytes, but also extrah展开更多
基金Supportod ty the State Key Basic Research Program Grant G1998051211 the Fund for Leading Specialty of Shanghai Metropolitan Bureau of Public Health.
文摘AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.
文摘AIM: To determine the frequencies of HGV and TTV infections in blood donors in Hangzhou. METHODS: RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nucleotide sequence analysis were performed. RESULTS: Thirty-two (15.8%) and 30 (14.8%) of the 203 serum samples were positive for HGV RNA and TTV DNA, respectively. And 5 (2.5%) of the 203 serum samples were detectable for both HGV RNA and TTV DNA. Homology of the nucleotide sequences of HGV RT-nested PCR products and TTV semi-nested PCR products from 3 serum samples compared with the reported HGV and TTV sequences was 89.36%, 87.94%, 88.65% and 63.51%, 65.77% and 67.12%, respectively. CONCLUSION: The infection rates of HGV and/or TTV in blood donors are relatively high, and to establish HGV and TTV examinations to screen blood donors is needed for transfusion security. The genomic heterogeneity of TTV or HGV is present in the isolates from different areas.
文摘[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 （DE3） and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 （DE3）. Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.
基金Supported by the Basic Research Program from Ministry of Science and Technology of China, No. G1999054105special funds for Sino-France Center for Life Science and Genome Research from Chinese Academy of Sciences and Pasteur Institute in France
文摘AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein. METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6× histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization. RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in E.coli. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain. CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in E.coli can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.
文摘AIM:To determine the prevalences of TTV and HGV infections among blood donors and patients with chronic liver disease in Korea,to investigate the association of TTV and HGV infections with blood transfusion,and to assess the correlation between TTV and HGV viremia and hepatic damage. METHODS:A total of 391 serum samples were examined in this study.Samples were obtained from healthy blood donors(n=110),hepatitis B surface antigen(HBsAg)-positive donors(n=112),anti-hepatitis C virus(anti-HCV)-positive donors(n=69),patients with type B chronic liver disease (n=81),and patients with type C chronic liver disease(n=19). Trv DNA was detected using the hemi-nested PCR.HGV RNA was tested using RT-PCR.A history of blood transfusion and serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were also determined. RESULTS:TTV DNA was detected in 8.2%of healthy blood donors,16.1%of HBsAg-positive donors,20.3%of anti- HCV-positive donors,21.0%of patients with type B chronic liver disease,and 21.1%of patients with type C chronic liver disease.HGV RNA was detected in 1.8%of healthy blood donors,1.8%of HBsAg-positive donors,17.4%of anti-HCV-positive donors,13.6%of patients with type B chronic liver disease,and 10.5%of patients with type C chronic liver disease.The prevalence of TTV and HGV infections in HBV- or HCV-positive donors and patients was significantly higher than in healthy blood donors(P<0.05), except for the detection rate of HGV in HBsAg-positive donors which was the same as for healthy donors.There was a history of transfusion in 66.7%of TTV DNA-positive patients and 76.9%of HGV RNA-positive patients(P<0.05).No significant increase in serum ALT and AST was detected in the TTV or HGV-positive donors and patients. CONCLUSION:TTV and HGV infections are more frequently found in donors and patients infected with HBV or HCV than in healthy blood donors.However,there is no significant association between TTV or HGV infections and liver injury.
基金the National Natural Science Foundation of China,No.39900133Beijing Natural Science Foundation,No.7992023
文摘AIM: To investigate the state of infection, replication site, pathogenicity and clinical significance of transfusion transmitted virus (TTV) in patients with hepatitis, especially in patients of unknown etiology. METHODS: Liver tissues taken from 136 cases of non-A non-G hepatitis were tested for TT virus antigen and nucleic acid by in situ hybridization (ISH) and nested-polymerase chain reaction (PCR). Among them, TT virus genome and its complemental strand were also detected in 24 cases of autopsy liver and extrahepatic tissues with ISH. Meanwhile, TTV DNA was detected in the sera of 187 hepatitis patients by nested-PCR. The pathological and clinical data of the cases infected with TTV only were analyzed. RESULTS: In liver, the total positive rate of TTV DNA was 32.4% and the positive signals were located in the nuclei of hepatocytes. In serus, TTV DNA was detected in 21.4% cases of hepatitis A-G, 34.4% of non-A non-G hepatitis and 15% of healthy donors. The correspondence rate of TTV DNA detection between liver tissue with ISH and sera with PCR was 63.2% and 89.3% in the same liver tissues by ISH and by PCR, respectively.Using double-strand probes and single-strand probes designed to detect TTV genome, the correspondence rate of TTV DNA detected in liver and extrahepatic tissues was 85.7%. Using single-strand probes, TTV genome could be detected in liver and extrahepatic tissues by PCR, but its complemental strands (replication strands) could be observed only in livers. The liver function of most cases infected with TTV alone was abnormal and the liver tissues had different pathological damage such as ballooning, acidophilia degeneration, formation of apoptosis bodies and focus of necrosis, but the inflammation in the lobule and portal area was mild. CONCLUSION: The positive rate of TTV DNA among cases of hepatitis was higher than that of donors, especially in patients with non-A non-G hepatitis, but most of them were coinfected with other hepatitis viruses. TTV can infect not only hepatocytes, but also extrah
