Expression and Purification of Recombinant Hepatitis Delta Virus (HDV) Antigen for Use in a Diagnostic ELISA for HDV Infection Using the High-Density Fermentation Strategy in Escherichia coli / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Hepatitis Delt a Virus (HDV) antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose.</p><p><b>METHODS</b>Reconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method.</p><p><b>RESULTS</b>The expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified (98%) by immobilized metal ion affinity chromatography (IMAC) using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity (97% for IgM and 100% for IgG) and specificity (100% for IgG and IgM) for the detection of anti-HDV antibodies.</p><p><b>CONCLUSION</b>Expression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy.</p>

Gene optimization, protein expression, purification and characterization of the HDV antigen for diagnosis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare HDAg with biological activities as a candidate of diagnostic reagent.</p><p><b>METHODS</b>To synthesize HDV gene fragment after codon optimization. To construct a thio-fused recombinant plasmid based on M48 expression vector. To express in E. coli induced by IPTG. To purify the protein by affinity chromatography followed by characterization in ELISA:</p><p><b>RESULTS</b>Plasmid construction was verified by enzyme digestion. SDS-PAGE indicated the molecular weight of the protein was the same as we expectation. ELISA proved its affinity with HDV antibodies.</p><p><b>CONCLUSION</b>HDAg was obtained successfully and it will pave the road to the research of HDV diagnostic reagent.</p>

Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering.</p><p><b>METHODS</b>Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software.</p><p><b>RESULTS</b>SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene.</p><p><b>CONCLUSION</b>Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.</p>

Expression of hepatitis delta antigen Inner Mongolian strain in prokaryotic cell and analysis of its antigenicity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain high yield and good antigenic activity of HDV L-Ag and to detect different regional patients' sera to test the purified antigen's antigenicity.</p><p><b>METHODS</b>Hepatitis delta virus' sequence was obtained from Inner Mongolian patient by using RT-PCR and PCR methods, PET43a was used and His-tag was added at the HDV L-Ag 5' and 3' to construct the recombinant expression plasmid, transform the plasmid into host bacterium BL21 and induce it with IPTG. The expression supernatant was purified by saturated (NH4)2SO4 and affinity chromatography. The activity and antigenicity of the expressed product were analyzed by using EIA.</p><p><b>RESULTS</b>Comparison of results obtained with detection by using the expressed protein coated plate and ABBOTT Murex anti-Delta (total) of 15 positive and 10 negative sera, the consistency was good (100%).</p><p><b>CONCLUSION</b>EIA proved that the purified antigen had good antigenicity, no serological difference was found in detection between different region's sera, therefore the purified delta antigen may be useful in diagnostic and other research.</p>

Expression of the hepatitis Delta antigen in prokaryotic cell and evaluation of its application as an EIA diagnostic reagent / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To construct the pRSETB-HDAg recombinant expression plasmid and to obtain soluble hepatitis D virus antigen with high biological and antigenic activity.</p><p><b>METHODS</b>HDAg gene fragment was inserted into fusion expression pRSET B vector that includes T7 promoter and a polyhistidine tag. The recombinant plasmid was transformed into host bacterium BL21 after induction with IPTG. The expression supernatant was purified by chelating affinity chromatography and the recombinant HDAg antigenic activity was detected by EIA.</p><p><b>RESULTS</b>EIA detection using the recombinant HDAg showed strong positive reaction with hepatitis D patients sera. The positive rates of the EIA, compared with HDAg from USA and Hua Mei EIA kit in detecting 26 cases of anti-HDV positive reference sera, were 100%, 96.15% and 100%, respectively.</p><p><b>CONCLUSION</b>Recombinant plasmid for HDAg with good antigenicity was successfully constructed and could be used as hepatitis D antibody detection reagent.</p>

The Prevalence and Clinical Characteristics of Hepatitis-delta Infection in Korea / 대한간학회지

BACKGROUND/AIMS: The prevalence of hepatitis delta virus (HDV) infection has been estimated as being approximately 5% among global HBsAg carriers. The anti-delta positive rate in Koreans had been reported as being 0.85% in 1985. While the prevalence of HBV has been decreased from nearly 10% to 5% during the past twenty years, there have been no more studies on the anti-delta prevalence in Koreans. The aim of this study was to estimate the anti-delta prevalence in Koreans and to study the clinical characteristics of anti-delta positive patients in a single center. METHODS: Serum anti-delta was measured in one hundred ninety four HBsAg-positive patients who were admitted to our hospital from February 2003 to August 2003. We checked the genotypes of the HBV in the anti-delta positive patients. The clinical features of the anti-delta positive patients were compared to those clinical features of the anti-delta negative patients from the aspect of age, gender, mode of transmission, the positivity of HBeAg and serum HBV DNA. RESULTS: Serum anti-delta was positive in seven patients among the 194 subjects, giving a 3.6% positive rate. Among these seven patients, six had hepatocellular carcinoma (HCC) and the other one had cholangiocarcinoma. All of the anti-delta positive patients had the C genotype of HBV. The anti-delta positive patients showed significantly suppressed HBV DNA replication compared to the anti-delta negative patients. CONCLUSIONS: In Koreans, anti-delta was positive mainly in HCC patients with an approximate prevalence of 4%, and this rate has not changed much for the past twenty years. HBV DNA replication was suppressed by HDV infection.

Delta hepatitis in Malaysia.

Sera from one hundred and fifty nine Malaysian individuals were screened for the prevalence of delta markers. These included 15 HBsAg positive homosexuals, 16 acute hepatitis B cases, 9 chronic hepatitis B patients, 13 healthy HBsAg carriers and 106 intravenous (i.v.) drug abusers, of whom 27 were positive for HBsAg only and the rest were anti-HBc IgG positive but HBsAg negative. The prevalence of delta markers in the homosexuals was found to be 6.7%, in the HBsAg positive drug abusers 17.8%, in acute hepatitis B cases 12.5%. No evidence of delta infection was detected in healthy HBsAg carriers, chronic hepatitis B cases and HBsAg negative i.v. drug abusers. With reference to i.v. drug abusers, the prevalence of delta markers was higher in Malays (23%) than in Chinese (7%) although the latter had a higher HBsAg carrier rate. Although the HBsAg carrier rate in the homosexuals was high, their delta prevalence rate was low as compared to drug abusers. In Malaysia, as in other non-endemic regions, hepatitis delta virus transmission appeared to occur mainly via the parenteral and sexual routes. This is the first time in Malaysia that a reservoir of delta infection has been demonstrated in certain groups of the population at high risk for hepatitis B.