Cloning, Expression And Antibody Preparation Of The Polypeptide Fragments Of ERF3b

Objective: Hepatitis B is caused by HBV and very popular in the world. It is the most serious hepatitis of all types of hepatitis. HBV infection of our country is even more serious. More than 700 million individuals have been infected and the infection rate is almost 57% throughout the country. There are 23 million chronic hepatitis B patients and 93 million HBV carriers in China now. At present, there are 230 thousand deaths every year. In the report, the Chinese government had to spend over 50 billion RMB for prevention and treatment every year. Unfortunately, there have been no effective methods of eliminating this problem, so it has been both a health and a national economic disaster. In the view of this situation, it is critically important to find a new method to diagnose and classify hepatitis B accurately and sensitively, and a new biomarker which can be used in detecting hepatitis B and associated-disease.It is momentous significance to research the mechanism of hepatitis B and prepare the antibody. In our lab, it had been found that 4210Da peptide was"GSPT2,eukaryon release factor GTP integrate subunit(eRF)"by MALDI-TOF mass spectrometry analysis. It was a part of the split of eRF3b―37 peptide. eRF3b was very important in the immune process of hepatitis B. 4210Da peptide as a part of eRF3b changed in different stage of hepatitis B. Therefore, it should to be a biomarker from acute hepatitis B to chronic hepatitis B, and screening liver cancer from chronic hepatitis B.Because eRF3b coding 111bp, so eRF3b gene fragment was called GSPT2-111, the corresponding peptide was named as eRF3b-37. The objective of this study is that to prepare the polyclonal antibody of GST-eRF3b-37 fusion protein. It provided the clues of the biomarker, at the same time laid the foundation for molecular epidemiological studies of the hepatitis B.Methods:(1) Molecular cloning of GSPT2-111. Homo-genome DNA was obtained from the peripheral blood using citrulline method. GSPT2-111 was cloned by PCR. The amplified fragment was inserted into pGEX-4T-1 vector by BamH I and EcoR I. After extraction of plasmid DNA, the GSPT2-111 in plasmid was identified by PCR and sequenced.(2) The expression of GST-eRF3b-37 fusion protein. The GSPT2-111 was subcloned into the expression vector pGEX-4T-1. The GST-eRF3b-37 fusion protein was expressed in DH5αE. coli under the 1.0mM IPTG induction. The expression and the solubility of the fusion protein were analyzed on 10% SDS–PAGE.(3) The purification of GST-eRF3b-37 fusion protein. The GST-eRF3b-37 fusion protein was expressed on a large scale, after that it was purified by affinity chromatography (GST SefinoseTM Resin). The purification of fusion protein was analyzed on 10% SDS–PAGE.(4) Preparation of the polyclonal antibodies. To immune the New Zealand white rabbits and rats with GST-eRF3b-37 fusion protein, including four times immunity. Every time the animals should be injected six different parts, each part of rabbit should be injected 300μl antigen solution and each part of rat should be injected 30μl antigen solution. The animals were immuned once every two weeks and the cycle of immunity was two months. The animals were immuned with completely Freund adjuvant for the first immunity, and they were immuned with incompletely Freund adjuvant for the others.(5) Detection and purification of the polyclonal antibodies. It was confirmed that the immuned animals weather had the polyclonal antibodies by protein dot blot. The antisera of animals were purified by protein A affinity chromatography. The purified antibody titer was determined by indirect ELISA and the specificity of the antibodies was identified by Western-blot.Results:(1) Molecular cloning of GSPT2-111. The GSPT2-111 was cloned by PCR from peripheral blood. GSPT2-111 encodes 37 amino acids with molecular weight of approximately 4.1kDa.(2) The expression of GST-eRF3b-37 fusion protein. The recombinant plasmid, pGEX-4T-1-GSPT2-111, induced by 1.0mM IPTG was expressed in DH5αEscherichia coli. The molecular weight of GSPT2-111 was approximately 4.1kDa. The fusion protein was dissoluble and purified it with GST SefinoseTM Resin.(3) Detection and purification of the polyclonal antibodies. It was confirmed that the immuned animals had the antibody of GST-eRF3b-37 fusion protein by protein dot blot. The antibody titer was determined by indirect ELISA. The rabbit antibody titer was approximately 1:51,200, the rat antibody titer was approximately 1:12,800 before purified the antibody. Then we purified the antibody of GST-eRF3b-37 fusion protein by Protein A affinity chromatography. The rabbit antibody titer was approximately 1:384,000, the rat antibody titer was approximately 1:25,600. Western blotting was also confirmed that the antibody could detect the 30kDa band of fusion protein.Conclusions: (1) The gene fragment of GSPT2-111 was successfully cloned, and the recombinant plasmid, pGEX-4T-1-GSPT2-111, was successfully constructed.(2) The GST-eRF3b-37 fusion protein was expressed in DH5αE. coli, and it was purified by GST SefinoseTM Resin.(3) The rabbit anti-human GST-eRF3b-37 polyclonal antibody and rat anti-human GST-eRF3b-37 polyclonal antibody were successfully prepared, and the antiserum of rabbits and rats was successfully purified.