scholarly journals OPTIMAL CONDITIONS OF FERMENTATION OF THE PICHIA PASTORIS YEAST EXPRESSING THE RECOMBINANT PRES2-S PROTEIN (M-HBsAg) OF HEPATITIS B VIRUS (HBV)

2020 ◽  
Vol 2020 (3) ◽  
pp. 56-59
Keyword(s):  
Hepatitis B Virus ◽  
Pichia Pastoris ◽  
Hepatitis B ◽  
Yeast Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
S Protein ◽  
Wet Biomass ◽  
B Virus ◽  
Pichia Pastoris Yeast

The aim of this study is determination of optimal fermentation conditions of the Pichia pastoris yeast expressing the recombinant Pres2-S protein of hepatitis B virus (HBV). For this purpose we investigated the main factors influencing for the cultivation of recombinant strain of the yeast Pichia pastoris pPIC3,5-S-HBsAg - the composition of the nutrient medium, pH and dissolved oxygen (DO). As a result, by enzyme-linked immunosorbent assay (ELISA) the optimal concentration of methanol ≈1.0 %, which is the initiator and the only source of carbon for protein expression in the Mut+ phenotype Pichia pastoris yeast cells was determined. In the selected conditions at the end of the fermentation process, the wet biomass of yeast cells was 420 g/l.

scholarly journals Expression Level of Small Envelope Protein in Addition to Sequence Divergence inside Its Major Hydrophilic Region Contributes to More Efficient Surface Antigen Secretion by Hepatitis B Virus Subgenotype D2 than Subgenotype A2

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 967
Author(s):  
Qianru Wang ◽  
Shuwen Fu ◽  
Jing Zhang ◽  
Quan Yuan ◽  
Jisu Li ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Surface Antigen ◽  
Envelope Protein ◽  
Hepatitis B Surface Antigen ◽  
Site Directed Mutagenesis ◽  
Enzyme Linked Immunosorbent Assay ◽  
S Protein ◽  
Huh7 Cells ◽  
B Virus

Hepatitis B surface antigen (HBsAg) promotes persistent hepatitis B virus (HBV) infection. It primarily corresponds to small (S) envelope protein secreted as subviral particles. We previously found that genotype D clones expressed less S protein than genotype A clones but showed higher extracellular/intracellular ratio of HBsAg suggesting more efficient secretion. The current study aimed to characterize the underlying mechanism(s) by comparing a subgenotype A2 clone (geno5.4) with a subgenotype D2 clone (geno1.2). Five types of full-length or subgenomic constructs were transfected to Huh7 cells at different dosage. HBsAg was quantified by enzyme linked immunosorbent assay while envelope proteins were detected by Western blot. We found that ratio of extracellular/intracellular HBsAg decreased at increasing amounts of DNA transfected. Conflicting findings from two types of subgenomic construct confirmed stronger secretion inhibitory effect of the genotype D-derived large envelope protein. Chimeric constructs followed by site-directed mutagenesis revealed geno1.2 specific V118/T127 and F161/A168 in the S protein as promoting and inhibitory of HBsAg secretion, respectively. In conclusion, more efficient HBsAg secretion by subgenotype D2 than subgenotype A2 is attributed to lower level of S protein expression in addition to V118 and T127 in S protein, although its F161 and A168 sequences rather reduce HBsAg secretion.

Fine Mapping of Neutralization Epitopes on Duck Hepatitis B Virus (DHBV) pre-S Protein Using Monoclonal Antibodies and Overlapping Peptides

Virology ◽  
1993 ◽  
Vol 192 (1) ◽  
pp. 217-223 ◽  
Author(s):  
Sylvie Chassot ◽  
Véronique Lambert ◽  
Alan Kay ◽  
Catherine Godinot ◽  
Bernard Roux ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Fine Mapping ◽  
Duck Hepatitis B Virus ◽  
S Protein ◽  
Duck Hepatitis ◽  
B Virus

scholarly journals Genomic variability in the preS1 region and determination of routes of transmission of hepatitis B virus

1992 ◽  
Vol 73 (11) ◽  
pp. 3005-3009 ◽  
Author(s):  
A. Uy ◽  
G. Wunderlich ◽  
D. B. Olsen ◽  
K.-H. Heermann ◽  
W. H. Gerlich ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Genomic Variability ◽  
B Virus

scholarly journals Development and evaluation of an in-house ELISA to detect hepatitis B virus surface antigen in resource-limited settings

2014 ◽  
Vol 39 (2) ◽  
pp. 65-68 ◽  
Author(s):  
K Fatema ◽  
S Tabassum ◽  
A Nessa ◽  
M Jahan
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Surface Antigen ◽  
Hbv Infection ◽  
Health Concern ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virus Surface ◽  
B Virus ◽  
Commercial Kits ◽  
Checkerboard Titration

Hepatitis B virus (HBV) infection is of global public health concern. Among various serological tests used for the diagnosis and screening of HBV infection, the enzyme-linked immunosorbent assay (ELISA) to detect hepatitis B surface antigen (HbsAg) is most widely used. The present study was designed to develop and standardize a cost effective in-house ELISA for the detection of HbsAg and compare its performance with two established commercial kits. The concentrations of coating antibody, conjugates and sera were fixed by checkerboard titration. Using known HBsAg positive and negative sera, four different concentrations (1, 0.5, 0.25 and 0.125 ?g/well) of coating anti-HBs were applied. Similarly, serial dilutions of patients’ sera (1 in 2, 1 in 3, 1 in 5 and 1 in 9) and conjugates (1 in 2, 1 in 3, 1 in 5, 1 in 9 and 1 in 17) were evaluated by checkerboard titration. The optimal concentration of coating antibody was determined at 0.25 ?g/well and 1 in 9 dilution for both conjugates and sera. The performance comparison of our in-house ELISA showed excellent correlation with two commercial kits (Pearson 0.957, P=0.001 for monoclonal antibody coated kit and Pearson 0.929, P=0.000 for polyclonal antibody coated kit) when OD values were compared. All commercial kit proven positive samples was positive while all negative samples were negative with the in-house ELISA resulting in 100% sensitivity and specificity. The results of our study demonstrated that our inhouse ELISA for detection of HBsAg was equally as sensitive and specific as two well-known commercial kits. Thus, this system may be a useful tool for diagnostic and screening purposes, as well as outbreak investigations. DOI: http://dx.doi.org/10.3329/bmrcb.v39i2.19644 Bangladesh Med Res Counc Bull 2013; 39: 65-68

Prevalence and genotyping of hepatitis B virus: a cross-sectional study conducted with female sex workers in the Marajó Archipelago, Brazil

2019 ◽  
Vol 30 (9) ◽  
pp. 902-910 ◽  
Author(s):  
Paula CR Frade ◽  
Nairis C Raiol ◽  
Luana M da Costa ◽  
Luiz ML Pinheiro ◽  
Gláucia C Silva-Oliveira ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Sex Workers ◽  
Female Sex Workers ◽  
Hbv Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hepatitis D ◽  
Female Sex ◽  
Hbv Genotypes ◽  
B Virus

In South America, the Amazon basin is considered an endemic area of hepatitis B virus (HBV) infection. However, epidemiological studies with vulnerable groups are scarce. Female sex workers (FSWs) are highly vulnerable to sexually transmitted infections due to a combination of their sexual behavior and socio-economic conditions. Thus, this study investigated the prevalence of HBV infections and HBV–hepatitis C virus, HBV–hepatitis D virus, HBV–HIV, and HBV–human T-lymphotropic virus co-infections among FSWs in the Marajó Archipelago, northern Brazil, as well as identifying the HBV genotypes circulating in this population. A total of 153 FSWs in 5 towns and 18 riverside communities were included in the study. The HBV infection and co-infections were diagnosed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. The HBV genotypes were detected by sequencing and were then analyzed phylogenetically. Most of the FSWs surveyed were single, young, heterosexual, and born locally, with low levels of education. Overall, 21 (13.7%) had been exposed to HBV, and HBV-DNA was detected in 13 (8.5%). Genotypes A (69.2%), D (23.1%), and F (7.7%) were detected. Seven cases of co-infections with other viruses were detected. These findings indicate a clear need for urgent measures to control the spread of HBV and other pathogens, and to promote the health of the local FSWs.

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