Related Experiment Videos
Synthesis of a recombinant DNA-derived HBV e antigen and its application in diagnosis
Insights
Researchers developed a new method to produce Hepatitis B virus e antigen (HBeAg) in E. coli. This recombinant HBeAg serves as a diagnostic tool for detecting HBeAg and anti-HBe in human sera using ELISA.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- The Hepatitis B virus (HBV) genome's C region encodes the HBV e antigen (HBeAg) and core antigen (HBcAg).
- HBeAg is a crucial marker for HBV infection and replication.
- Efficient production of HBeAg is essential for diagnostic development.
Purpose of the Study:
- To construct recombinant plasmids for high-level synthesis of HBeAg in E. coli.
- To purify and characterize recombinant HBeAg.
- To evaluate the utility of recombinant HBeAg as a diagnostic reagent.
Main Methods:
- Cloning of the HBc gene from adw2 HBV DNA into pUR222 and pUC18 vectors.
- Generation of DNA fragments of varying lengths using Bal31 and HapII digestion.
- Expression of recombinant HBeAg in E. coli, followed by purification using DEAE and affinity chromatography.
- Identification and molecular weight determination of recombinant HBeAg via electrophoresis (SDS-PAGE) and immunochemical analysis.
- Application of bacterial extracts in Enzyme-Linked Immunosorbent Assay (ELISA) for HBeAg and anti-HBe detection.
Main Results:
- Two recombinant plasmids capable of high-level HBeAg synthesis in E. coli were successfully constructed.
- Recombinant HBeAg was purified and characterized, showing a molecular weight of approximately 38 kDa, consistent with a dimer of two 19 kDa subunits.
- Bacterial extracts containing recombinant HBeAg demonstrated efficacy as a diagnostic reagent in ELISA for detecting HBeAg and anti-HBe in human sera.
Conclusions:
- The study successfully produced and purified recombinant HBeAg in E. coli.
- The recombinant HBeAg is suitable for use as a diagnostic reagent in ELISA for HBV infection markers.
- This approach offers a viable method for producing diagnostic tools for Hepatitis B virus.
Abstract:
The gene coding for HBV e antigen (HBeAg) and core antigen (HBeAg) are located in the C region of the HBV genome. In this paper 1.8 kb BamHI-EcoRI DNA fragment carrying HBc gene from adw2 HBV DNA was digested with Bal31 and HapII, a series of fragment with different length were produced and inserted into pUR222 and pUC18 vectors to construct various recombinant plasmids. Two new recombinants able to direct high level synthesis of HBeAg in E. coli were obtained. Recombinant HBeAg was then purified by DEAE chromatography and affinity chromatography. The preparation obtained were identified by electrophoresis and immunochemical analysis. Molecular weight of the recombinant HBeAg determined by SDS-PAGE was about 38 kd. It is considered to be a dimer of two fused antigen molecules in size of 19 kd. Bacterial extracts prepared from cells harboring one of the constructed plasmids have been used successfully as a diagnostic reagent for the detection of HBeAg and anti-HBe in human sera by ELISA.