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Objective@#The aim was to investigate the genotype distribution of two major epitopes of large surface protein (PreS1) of hepatitis B in Chinese patients and to explore the association between the genotypes of these two epitopes, and to determine whether PreS1 full-length genotype could be revealed according to the polypeptide sequence of key epitopes. @*Methods@#HBV DNA was extracted from the serum of patients for PCR amplification. 278 samples amplified successfully were sequenced and compared with the known HBV sequences in Genbank to determine the two key epitopes of HBV PreS1 genotype (amino acid epitope 21-47 and 94-117, abbreviated as P21 and P94) and PreS1 full-length genotypes. The correlation among three genotyping approaches was analyzed by Cohen’s kappa coefficient to verify the consistency between the key-epitope genotyping and the full-length preS1 genotyping. @*Results@#232 samples were successfully sequenced. The genotyping based on the kind of P21 epitope protein sequence, 201 cases for genotype C, 23 cases for genotype B and 8 cases for uncertain genotypes and genotyping based on the form of P94 epitope protein sequence, 199 cases for genotype C, 25 cases for genotype B and 8 cases for indeterminate genotypes. Lastly, the genotyping based on sequence of the full-length PreS1 sequence, 207 and 25 cases for genotype C and B. P21 or P94 epitope genotyping and PreS1 full length genotyping were highly consistent, respectively, 96.55% and 96.12%, and the two epitopes (P21and P94) genotyping have parallel consistency (93.10%). @*Conclusion@#In this study, an innovatively genotyping method based on the amino acid sequence of key epitopes was proposed. The genotypes of HBV in china were mainly B and C genotypes, and the genotypes of key conserved epitopes of HBV PreS1 were highly consistent with the full-length genotyping ( > 96%). Moreover, genotyping with one or two key epitopes can be used in place of the full-length genotyping.
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Objective To explore the application value of enzyme linked immunosorbent assay for detection of hepatitis B PreS1 antigen and antibody in the diagnosis of hepatitis B.MethodsSeveral common patterns in the results of hepatitis Btwo halfcheck were selected from August 2014 to August 2016 for the sample, the use of hepatitis Btwo half7 commonly used detection model for 140 cases of PreS1 antigen, antibody detection.Enzyme linked immunosorbent assay for detection of PreS1 antigen and antibody.ResultsIn patients with HBeAg in PreS1 antigen positive rate was 87.5%, in patients with positive PreS1 antigen positive rate was 8.6%, HBsAg, HBcAb in patients with positive PreS1 antigen positive 13.3%, in a pure HBsAg in patients with positive PreS1 antigen 4.3%.There was no significant difference in the total positive rate of HBV-DNA and the total positive rate of PreS1, and the total coincidence rate was higher.PreS1 antigen positive correlated with HBsAg, HBeAg and HBcAb positive(P<0.05), and has nothing to do with HBsAb, HBeAb positive.ConclusionHepatitis B PreS1 antigen and antibody detection, can effectively complement the diagnosis of hepatitis B, early detection of hepatitis B infection.
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Objective To verify the interaction between asialoglycoprotein receptor (ASGPR)and hepatitis B virus (HBV)preS1 protein in vivo and in vitro ,and identify ASGPR as a cell-surface receptor for HBV,which could elucidate the molecular mechanism of HBV infection.Methods The preS1-ASGPR interaction was examined in mammalian two-hybrid and coimmunoprecipitation system by strictly following the manufacturer’s instructions.Results ASGPR interacted specifically and directly with the preS1 domain of HBV in vivo and in vitro .Conclusion ASGPR may be a candidate receptor for HBV that mediates further step of HBV entry.
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Objective To investigate the correlations on hepatitis B virus (HBV) preS1‐antigen (pre‐S1Ag) with HBV‐DNA , HBV markers(HBV M) and liver function in the patients with hepatitis B .Methods The markers ,preS1‐Ag ,HBV‐DNA and liver function were determined by CLIA and PCR in 905 patients with hepatitis B (HBV infection group ) and 100 healthy persons (healthy control group) .Results Among 905 samples ,the positive rates of preS1‐Ag and HBV DNA were 68 .51% (620/905) and 67 .96% (615/905) ,there was no statistically significant difference between them (χ2 =30 .064 ,P>0 .05);the positive rates of pre‐S1Ag in 570 patients with HBeAg positive were 85 .08% (485/570) ,which was significantly higher than 40 .30% (135/335) in 335 patients with HBeAg negative ,the difference was statistically significant (χ2 =108 .881 ,P<0 .01) .The abnormal rates of ALT and AST in the Pre‐S1 Ag positive and negative groups were 53 .22% ,25 .96% and 51 .29% ,32 .98% ,respectively ,the differences be‐tween them were statistically significant (χ2ALT =53 .148 ,P<0 .001 ,χ2AST =66 .635 ,P<0 .001) .Conclusion Pre‐S1Ag is a reliable index of the HBV infection and duplication and is highly correlated with HBV‐DNA positive ,which is important supplement and strengthening and can provide more timely and reliable experiment basis for guiding the clinical treatment .
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Objective To investigate the similarities and differences of 3 methods for detecting hepatitis B virus S 1(PreS1) ,and select the appropriate method for detecting clinical samples .Methods The PreS1 of the serum samples from chronic hepatitis B pa-tients were tested with enzyme immunoassay analyzer ,time-resolved method and the manual method .To compare the repetition rate ,select PreS1 antigen strongly positive serum and weak positive serum were detected 15 times by three methods ;To compare the explicit rate ,the reaction temperature was raised or lowered by 3 ℃ .Results The positive rate of three methods was 93 .53% , 92 .81% ,92 .81% .Automated ELISA reproducibility CV strong positive CV3 .62% ,weakly positive CV was 13 .42% ,CV of time-resolved method for the 2 kinds of samples were 5 .10% ,7 .92% ,manual methods CV 11 .10% 29 .88% ;changing the reaction incu-bation temperature 3 ℃ ,automatic detection ELISA all specimens S/CO value decreased ,increasing the chance of a false negative . The manual methods and time-resolved detection method for all specimens S/CO values increased ,increasing the chance of a false positive .Conclusion The detection rate and repeatability of automated ELISA were better .The time-resolved method followed and the manual methods were poor .
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Objective To seek the specific receptors associated with hepatitis B virus (HBV) adhesion by separating the binding protein of the HBV preS1 region in HepG2 and performing the mass spectrometry .Methods The immunomagnetic bead method was adopted to separate HepG2 membrane protein combined with preS1 peptide fragment and the binding protein was separated by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ,then the destination strips was analyzed by LC-MS/MS mass spectrometry and retrieved by the database .Results 16 bands were separated from HepG2 membrane proteins combined with preS1 by SDS-PAGE ;14 kinds of proteins were identified from 6 bands with better repeatability separated from HepG 2 membrane proteins combined with preS1 .Conclusion Protein analyzed by the mass spectrometry is mainly related with the material transport , cellular signal transduction ,antigen presentation ,immune regulation and energy metabolism .
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A região PreS1 da proteína L é importante na adesão celular e, consequentemente, o vírus da hepatite B (HBV) infectividade. Para identificar novas proteínas que interagem PreS1 , realizamos Glutationa -S -transferase (GST) suspenso, electroforese bidimensional do gel ( 2-DE) e os testes de espectrometria de massa. proteínas Glucose- regulamentado (GRP ) 78 e 75 foram encontrados para vincular PreS1 . As interações entre PreS1 e GRP75 foram confirmados por uma experiência de co - imunoprecipitação . GRP78 e GRP75 pode desempenhar um papel importante na mediação HBV virion entrar em hepatócitos e regulação dobradura apropriada da proteína L , devido às suas funções críticas de enovelamento de proteínas e tráfico. A descoberta do romance PreS1 proteína enriquece nosso conhecimento sobre o mecanismo molecular da infecção pelo HBV.
The PreS1 region of the L protein is important in cell attachment and consequently in hepatitis B virus (HBV) infectivity. To identify novel PreS1 interacting protein, we performed Glutathione-S-transferase (GST) pull-down, two-dimensional gel electrophoresis (2-DE) and mass spectrometry assays. Glucose-regulated proteins (GRP) 78 and 75 were found to bind PreS1. The interactions between PreS1 and GRP75 were confirmed by a co-immunoprecipitation experiment. GRP78 and GRP75 may play important roles in mediating HBV virion entering into hepatocyte and regulating proper folding of the L protein due to their critical functions in protein folding and trafficking. The finding of novel PreS1 binding protein enriches our knowledge about molecular mechanism of HBV infection.
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Humans , Glutathione S-Transferase pi , Hepatocytes , In Vitro Techniques , Mass Spectrometry , Polymerase Chain Reaction , Proteomics , Hepatitis B virus/isolation & purification , Electrophoresis , Methods , MethodsABSTRACT
Objective To investigate the clinical significance and relationship of preS1, HBV DNA and HBV-M. Methods PreS1 and HBV-M was detected by ELISA method,and HBV DNA was detected by PCR. Then the results were analyzed. Results In HBV patients,the positive rates of preS1 and HBV DNA had no statistically significant ,they had fine dependability. The detection rate of preS1 in HBeAg(+) group(80.3%) and HBeAg(+)group( 56.3% ) had statistically significant. In some patients,though HBeAg had become negative, HBV still replicated. In HBV DNA replicated patients(≥103 copies/ml) ,the detection rate of HBeAg and preS1 were 51.5% and 70.9% ,they had statistically significant. Conclusion HBV DNA and PreS1 had fine dependability,preSl could reflect the replication of HBV sensitively than HBeAg,it could be used as a reliable new marker of HBV replication in vivo.
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Objective: To screen for peptides that specifically bind to PreS1 antigen from a phage-displayed peptide library. Methods: The PreS1 antigen was used as the target molecule to screen the binding peptide from the Ph. D. -7 peptide library with phage display technique, and the positive clones were identified by ELISA. Results: After three rounds of biopanning, the binding peptides were screened from the peptide library by ELISA and competitive inhibiting ELISA. Sequencing result showed that the binding peptides had high affinity and specificity. Conclusion: A peptide binding PreS1 antigen has been successfully obtained by screening the phage display library, which paves a way for the diagnosis and treatment of hepatitis B infection.
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OBJECTIVE To investigate the correlations on hepatitis B virus(HBV)preS1-antigen(preS1-Ag)and HBV-DNA,HBV markers in patients with hepatitis B.METHODS The markers,preS1-Ag and HBV-DNA were determined by ELISA and PCR in 406 patients with hepatitis B and 80 healthy persons.RESULTS The positive rates of preS1-Ag in 160 patients with HBsAg(+)and HBeAg(+)were 84.4%.The positive rates of preS1-Ag in 246 patients with HBsAg(+)and HBeAg(-)were 42.3%;the difference between them was significant(?2=70.9,P
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OBJECTIVE To detect and analyze the results and significance of HBV-DNA,HBeAg and PreS1 of HBV infective sera.METHODS Routine detection and PreS1 antigen of 450 sera were tested by ELISA,and HBV-DNA was detected by fluorescent quantitation PCR.RESULTS The positive rates of HBV-DNA,HBeAg and PreS1 were 74.4%,48.9% and 63.3%,respectively,in 450 cases of HBV infective sera.Among 285 PreS1-positive samples,the positive cases of HBeAg and HBV-DNA were 190 and 270,respectively.There were significant difference(P0.05)among PreS1,HBV-DNA and PreS1,and they three were in positive correlation.CONCLUSIONS PreS1 and HBV-DNA are more sensitive than HBeAg,and PreS1 is better coincided with HBV-DNA.They can reflect the infection and replication condition of HBV.Therefore,it has important clinical meaning for the diagnosis and therapy of HBV to simultaneously used HBV-DNA,HBeAg and PreS1.
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OBJECTIVE To study the relativity among PreS1-Antigen,HBV markers and HBV-DNA. METHODS The HBV markers,PreS1-antigen and HBV-DNA were determined by ELISA and PCR in 102 patients with chronic hepatitis B and 73 healthy persons. RESULTS Among 102 patients with chronic hepatitis B,the concordance rate of PreS1-antigen and HBeAg with HBV-DNA was 70.6% and 75.5%.The sensitivity of PreS1 was better than HBeAg but the specificity was contrary.It represented some patients with HBeAg(-) still had viral replication.On the increase in the level of HBV-DNA,the positive rates of PreS1-antigen and HBV markers increased. CONCLUSIONS The detection of PreS1-antigen can well reflect the HBV replication.The synchronous dynamic detection of PreS1-Antigen,HBV markers and HBV-DNA has its important clinical meaning.
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To construct, express and purify fusion protein containing HBcAg and HBsAg preS1 epitope peptide for the purpose of investigating a novel HBV vaccine with both prophylactic and therapeutic functions. Using DNA recombinant technology, prokaryotic expression plasmid pBTcs1 expressing HBcAg and HBsAg pre-S1 epitope peptide fusion protein was constructed. After expressed in E.coli. HB101, the production BTcs1 was purified by sucrose density gradient ultracentrifugation and identified by SDS-PAGE, SEC, Western-blot and electron microscope. The results indicated that expression plasmid pBTcs1 was constructed successfully, and 20~25 mg purified BTcs1 fusion protein was obtained from 1L LB culture. Result of DOT-BLOT indicated that the distribution of BTcs1 was mainly in 30~50% sucrose, the purity of BTcs1 was greater than 95% by SDS-PAGE and SEC analysis. BTcs1 could probe with specific antibodies at 28 kDa by Western-blot, BTcs1 could also self assemble VLP by electron microscope analysis, its diameter was 30~34 nm approximately. The present study lay a foundation for further research functions and applications of BTcs1.
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Objective:To screen for peptides that specifically bind to PreS1 antigen from a phage-displayed peptide library.Methods: The PreS1 antigen was used as the target molecule to screen the binding peptide from the Ph.D.-7 peptide library with phage display technique,and the positive clones were identified by ELISA.Results: After three rounds of biopanning,the binding peptides were screened from the peptide library by ELISA and competitive inhibiting ELISA.Sequencing result showed that the binding peptides had high affinity and specificity.Conclusion: A peptide binding PreS1 antigen has been successfully obtained by screening the phage display library,which paves a way for the diagnosis and treatment of hepatitis B infection.
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No abstract available.
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Animals , Cricetinae , Female , Humans , Asian People , CHO Cells , Cricetulus , Hepatitis B Surface Antigens , Hepatitis B Vaccines , OvaryABSTRACT
Objective:To screen and identify proteins that interact with the hepatitis B virus PreS1 protein by means of T7-phage display system. Method:Hepatitis B virus PreS1 protein was expressed by prokaryotic expression and used as selected molecule to biopan the T7 select human liver cDNA library,the selected positive clones were identified by DNA sequence and analyzed with BLAST program in GenBank. Result:After BLAST in all positive clones,one protein:Cytochrome c Oxidase Subunit I(COX1)was found to interact with the hepatitis B virus PreS1 protein. Conclusion:T7-phage display system is a convenient,rapid and effective method for screening interacting proteins. This results will provide important evidences for studying the pathogenesis and mechanism of HBV.
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A peptide was synthesized based on the 2l-47 amino acid sequence of Hepatitis B Virus PreSl protein. This peptide (p21-47) was cross-linked to tetanus toxoid and used to immunize rabbit. Rabbit P21-47 not only reacted with the peptide but also reacted with preSl (Large portein of HBV envelope) in Western blotting. Since preSl seems to be involved in attachment of HBV to target cells, this anti P21-47 has the potentiality in studying virus-receptor intereaction as well as for assaying PreS (?)antigen.
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Objective T7 cDNA phage display system and bioinformatics methods were employed to find the binding protein to the PreS1 protein of hepatitis B virus (HBV). Methods PreS1 protein was coated in ELISA plate as the target protein, and then T7 cDNA library phage display system was used to scan the binding protein or peptide. A piece of cDNA was found to have the function to bind the PreS1 protein, and the product was named as PreS1 binding protein (PreS1BP). Using BLAST in GenBank, the amino acid sequence of PreS1BP was compared in the protein sequence database. Results The amino acid sequence of PreS1BP was identified as a piece of glioma tumor suppressor candidate region gene 2 (GLTSCR2), and the length of cDNA of PreS1BP was proved to be 1436 nt. The gene was located at chromosome 19q arm (19q13.3) with a length of 11445 base pair between 10403483 and 10414989, containing 13 exons and 12 introns. Conclusion HBV PreS1BP gene could be obtained by T7 cDNA phage display system in combination with bioinformatics methods.