To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino asids in length were used to screen w...To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino asids in length were used to screen with the serum from a hepatitis B virus infected patient in the recovery phase. After 3 rounds of biopanning, the positive phages were confirmed by competitive ELISA using HBsAg/P33. Two phagotopes were identified and one of them was confirmed as mimotope by competition experiment. Based on the mimotpe, a multiple antigenic peptide with four branches was synthesized by solid phase peptide synthesis. The antiginicity and specificity of the synthesized antigen was tested in BALB/c mice compared with the native epitope-based antigen. The results showed that the mimotope-based antigen could evoke higher titer of antibodies with the same specificity of the epitope-based antigen. Those findings indicate mimotopes can be used in antigen and vaccine design.展开更多
AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridizat...AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences展开更多
AIM: To identify the prevalence of pre-S2 start codon mutations and to assess their association with liver disease progression. METHODS: The mutations were identified by direct sequencing from 73 asymptomatic carriers...AIM: To identify the prevalence of pre-S2 start codon mutations and to assess their association with liver disease progression. METHODS: The mutations were identified by direct sequencing from 73 asymptomatic carriers, 66 chronic hepatitis (CH), 66 liver cirrhosis (LC) and 63 hepatocellular carcinoma (HCC) patients. Statistical significances were determined using Fisher's exact test, χ 2 test, and t -test analyses whenever appropriate. Pre-S mutation as a risk factor for advanced liver disease was estimated by unconditional logistic regression model adjusted with age, sex, and hepatitis B e antigen (HBeAg). P < 0.05 was considered significant. RESULTS: Mutation of the hepatitis B virus (HBV) pre-S2 start codon was found in 59 samples from 268 subjects (22.0%), with higher prevalence in patients with cirrhosis 27/66 (40.9%) followed by HCC 18/63 (28.6%), chronic hepatitis 12/66 (18.2%) and asymptomatic carriers 2/73 (2.7%) (P < 0.001). Logistic regression analysis showed that pre-S2 start codon mutation was an independent factor for progressive liver disease. Other mutations, at T130, Q132, and A138, were also associated with LC and HCC, although this was not statistically significant when adjusted for age, sex, and HBeAg. The prevalence of pre-S2 start codon mutation was higher in HBV/B than in HBV/C (23.0% vs 19.1%), whilst the prevalence of T130, Q132, and A138 mutation was higher in HBV/C than in HBV/B. The prevalence of pre-S2 start codon mutation was higher in LC (38.9%) and HCC (40.0%) than CH (5.6%) in HBeAg(+) group, but it was similar between CH, LC and HCC in HBeAg(-) group. CONCLUSION: Pre-S2 start codon mutation was higher in Indonesian patients compared to other Asian countries, and its prevalence was associated with advanced liver disease, particularly in HBeAg(+) patients.展开更多
Enzyme immunoassays were developed to evaluate the clinical significance of anti-pre-S1 and anti-pre-S2 antibodies, using the synthetic oligopeptide analogues of pre-S1 and pre-S2 proteins as antigens, and 104 patient...Enzyme immunoassays were developed to evaluate the clinical significance of anti-pre-S1 and anti-pre-S2 antibodies, using the synthetic oligopeptide analogues of pre-S1 and pre-S2 proteins as antigens, and 104 patients with different categories of hepatitis B virus (HBV) infection were tested. The detection rates of anti-pre-S1 and anti-pre-S2 were 100% and 66.7% in acute hepatitis B; 30% and 24% in asymptomatic HBV carriers; an0d 47.2% and 19.4% in chronic active hepatitis respectively. In case of chronic HBV infections, in HBV DNA negative and anti-HBe positive cases, the rates were 52.9% and 29.4%, while in HBV DNA and HBeAg positive cases, they were 33.3% and 8.3%. The results suggest that in acute hepatitis B virus infection anti-pre-S appeared even earlier than anti-HBs and might be the markers for early prognosis of infection recovery. Anti-pre-S might also reflect viral clearance in chronic infection.展开更多
The human α-interferon(IFNα) is a group of proteins possessing activity to render cells resistant to viral infection. It is one of the most promising and well documented recombinant proteins used in biotherapy. A pr...The human α-interferon(IFNα) is a group of proteins possessing activity to render cells resistant to viral infection. It is one of the most promising and well documented recombinant proteins used in biotherapy. A product of recombinant human α2a-interferon(IFN-α2a) first developed in our laboratory has been put into clinical trials upon the approval of the China National Committee for Drug Administration. Data show that展开更多
基金Grants39789010 and 39700132 from the Natural ScienceFoundat,ion of China and agrant from National Ed-u~ation Ministery und
文摘To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino asids in length were used to screen with the serum from a hepatitis B virus infected patient in the recovery phase. After 3 rounds of biopanning, the positive phages were confirmed by competitive ELISA using HBsAg/P33. Two phagotopes were identified and one of them was confirmed as mimotope by competition experiment. Based on the mimotpe, a multiple antigenic peptide with four branches was synthesized by solid phase peptide synthesis. The antiginicity and specificity of the synthesized antigen was tested in BALB/c mice compared with the native epitope-based antigen. The results showed that the mimotope-based antigen could evoke higher titer of antibodies with the same specificity of the epitope-based antigen. Those findings indicate mimotopes can be used in antigen and vaccine design.
文摘AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences
基金Supported by MRIN Funding, Budget, No. cc041/2010
文摘AIM: To identify the prevalence of pre-S2 start codon mutations and to assess their association with liver disease progression. METHODS: The mutations were identified by direct sequencing from 73 asymptomatic carriers, 66 chronic hepatitis (CH), 66 liver cirrhosis (LC) and 63 hepatocellular carcinoma (HCC) patients. Statistical significances were determined using Fisher's exact test, χ 2 test, and t -test analyses whenever appropriate. Pre-S mutation as a risk factor for advanced liver disease was estimated by unconditional logistic regression model adjusted with age, sex, and hepatitis B e antigen (HBeAg). P < 0.05 was considered significant. RESULTS: Mutation of the hepatitis B virus (HBV) pre-S2 start codon was found in 59 samples from 268 subjects (22.0%), with higher prevalence in patients with cirrhosis 27/66 (40.9%) followed by HCC 18/63 (28.6%), chronic hepatitis 12/66 (18.2%) and asymptomatic carriers 2/73 (2.7%) (P < 0.001). Logistic regression analysis showed that pre-S2 start codon mutation was an independent factor for progressive liver disease. Other mutations, at T130, Q132, and A138, were also associated with LC and HCC, although this was not statistically significant when adjusted for age, sex, and HBeAg. The prevalence of pre-S2 start codon mutation was higher in HBV/B than in HBV/C (23.0% vs 19.1%), whilst the prevalence of T130, Q132, and A138 mutation was higher in HBV/C than in HBV/B. The prevalence of pre-S2 start codon mutation was higher in LC (38.9%) and HCC (40.0%) than CH (5.6%) in HBeAg(+) group, but it was similar between CH, LC and HCC in HBeAg(-) group. CONCLUSION: Pre-S2 start codon mutation was higher in Indonesian patients compared to other Asian countries, and its prevalence was associated with advanced liver disease, particularly in HBeAg(+) patients.
文摘Enzyme immunoassays were developed to evaluate the clinical significance of anti-pre-S1 and anti-pre-S2 antibodies, using the synthetic oligopeptide analogues of pre-S1 and pre-S2 proteins as antigens, and 104 patients with different categories of hepatitis B virus (HBV) infection were tested. The detection rates of anti-pre-S1 and anti-pre-S2 were 100% and 66.7% in acute hepatitis B; 30% and 24% in asymptomatic HBV carriers; an0d 47.2% and 19.4% in chronic active hepatitis respectively. In case of chronic HBV infections, in HBV DNA negative and anti-HBe positive cases, the rates were 52.9% and 29.4%, while in HBV DNA and HBeAg positive cases, they were 33.3% and 8.3%. The results suggest that in acute hepatitis B virus infection anti-pre-S appeared even earlier than anti-HBs and might be the markers for early prognosis of infection recovery. Anti-pre-S might also reflect viral clearance in chronic infection.
基金Project supported by grants from the State Commission of ScienceTechnology for the "863" Project for the Research and Development of Biotechnology.
文摘The human α-interferon(IFNα) is a group of proteins possessing activity to render cells resistant to viral infection. It is one of the most promising and well documented recombinant proteins used in biotherapy. A product of recombinant human α2a-interferon(IFN-α2a) first developed in our laboratory has been put into clinical trials upon the approval of the China National Committee for Drug Administration. Data show that
