[Gene optimization, protein expression, purification and characterization of the HDV antigen for diagnosis].

OBJECTIVE: To prepare HDAg with biological activities as a candidate of diagnostic reagent. METHODS: 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: RESULTS: 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. CONCLUSION: HDAg was obtained successfully and it will pave the road to the research of HDV diagnostic reagent.

Development of a hepatitis delta virus antibody assay for study of the prevalence of HDV among individuals infected with hepatitis B virus in China.

Co-infection with hepatitis delta virus (HDV) and hepatitis B virus (HBV) has been shown to be associated with a more severe form of acute and chronic hepatitis. Cloning and expression of recombinant HDV antigen (rHDAg) in Escherichiacoli are described. Using purified rHDAg, a cost-effective indirect anti-HDV enzyme-linked immunosorbent assay (ELISA) kit was developed. Direct comparison of 15 known HDV-positive sera and 15 HDV-negative sera showed concordance agreement between the new assay kit and the Abbott Murex Anti-Delta (total) kit. In addition, 1,486 hepatitis B surface antigen (HBsAg) positive blood samples collected from various areas of China were tested using this indirect anti-HDV ELISA. It was found that 1.2% (95% CI: 0.7-1.9%) of the samples were anti-HDAg positive. It is suggested that the prevalence of HDV and HBV co-infection in China is relatively low.

[Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

OBJECTIVE: To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Seroprevalence of hepatitis delta virus infection among subjects with underlying hepatic diseases in Chennai, southern India.

Four hundred million people are carriers of hepatitis B virus (HBV) worldwide and approximately 5% of these are reportedly positive for hepatitis delta virus (HDV). Several reports indicate a declining trend in the occurrence of HDV infection in the north of tropical India. To our knowledge, no study has been conducted to evaluate whether a similar epidemiological change is occurring in southern India. Therefore we evaluated the seroprevalence of HDV among 153 individuals with HBV-related liver diseases in Chennai, and assessed any change in epidemiological pattern by comparing the results with seroprevalence figures reported previously. Of the 153 patients screened, nine (5.9%) were reactive to anti-delta antibodies, six (3.9%) presented an evidence of past infection (IgG anti-delta positive) and three (2.0%) showed anti-HDV IgM, suggestive of recent HDV infection. Alanine transaminase elevation was not significant in HDV-associated infection compared with HBV alone-infected acute viral hepatitis (AVH) (P=0.82) and chronic liver disease (P=0.77) patients. The anti-HDV positivity in AVH was considerably low (6.6%), compared with previous Indian reports varying from 10.7% to >30%. HDV infection was relatively low and seems to play a minor determining factor of liver diseases in the tropical south Indian population.

Transcription of hepatitis delta virus RNA by RNA polymerase II.

Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

High prevalence of hepatitis delta virus infection detectable by enzyme immunoassay among apparently healthy individuals in Mongolia.

A previous study revealed a high prevalence of hepatitis B surface antigen (HBsAg) and hepatitis delta virus (HDV) RNA among 249 apparently healthy individuals (mean+/-standard deviation age, 48.4+/-13.9 years; 126 males and 123 females) in Ulaanbaatar, Mongolia. To investigate further the prevalence of HDV infection there, the same serum samples obtained from the cohort were tested for the presence of immunoglobulin G (IgG) class antibody to HDV (anti-HDV) by a newly developed enzyme-linked immunosorbent assay using recombinant hepatitis delta antigen protein expressed in the pupae of silkworm as the antigen probe. Anti-HDV was detected in 42 persons (16.9%), among whom 22 (52.4%) were positive for HBsAg and 20 (47.6%) had detectable HDV RNA. Among 170 persons with anti-HBc in the absence of HBsAg, 20 (11.8%) tested positive for anti-HDV, and 1 of the 20 subjects was positive for HDV RNA. Of note, none of 55 anti-HBc-negative persons had anti-HDV, supporting the specificity of the anti-HDV assay. The optical density (OD) value of anti-HDV was significantly higher among HDV RNA-positive subjects (n=21) than among HDV RNA-negative subjects (n=21) (2.513+/-0.514 vs. 0.836+/-0.550, P<0.0001). The present study confirmed the extremely high prevalence of HDV infection in Mongolia, and identified a person who was positive for both anti-HDV and HDV RNA despite negativity for HBsAg and HBV DNA probably due to viral interference. The anti-HDV assay may be useful for further epidemiological studies on HDV infection in larger cohorts in urban and rural areas of Mongolia, where elucidation of the transmission route of HDV is required urgently.

Purification of recombinant proteins with high isoelectric points.

The use of recombinant antigens is essential for the construction of robust and sensitive diagnostic assays. A critical step in the preparation of recombinant antigens is protein purification. Purification problems may be very different for related structural proteins expressed in the same host or for the same protein expressed in different hosts, because the biochemical characteristics of a recombinant protein, expressed in a heterologous system, are unique. In this chapter we make a brief introduction to protein purification procedures and we present a quick purification process suitable for the isolation of recombinant protein having high isoelectric points encoding non-conformational epitopes.