Cloning and prokaryotic expression of a novel binding protein 1 of HBeAg / 解放军医学杂志

ABSTRACT

Objective To clone the human gene of Hepatitis B virus e antigen binding protein 1 (HBEBP1), which was screened with yeast two-hybrid system and bioinformatics techniques, to construct prokaryotic expression vector of pET-32a(+)-HBEBP1, and to induce the expression of recombinant protein in E. coli BL21. Methods The DNA fragment of HBEBP1 of about 372bp was amplified by reverse transcription PCR (RT-PCR), in which the mRNA was taken from HepG2 cells as the template, and cloned into pGEM-T vector. After restriction enzyme digestion identification and sequencing, the correct target DNA fragment was inserted into inducible prokaryotic expression vector pET-32a(+) and then transformed into competent E. coli BL21. By restriction enzyme digestion and PCR, the positive transformed clones were identified and induced with IPTG to obtain fusion protein. The HBEBP1 fusion protein was analyzed by Western blotting hybridization. Results The 372bp DNA fragment of HBEBP1 was amplified by RT-PCR. The recombinant expression vector pET-32a(+)-HBEBP1 was constructed successfully. After transformation with pET-32a(+)-HBEBP1 and induction with IPTG, the recombinant target protein of about 33kD was obtained, which was consistent with our anticipation. Western blotting assay showed that the protein had good specificity. Conclusions The recombinant prokaryotic expression vector pET-32a(+)-HBEBP1 is constructed, and the HBEBP1 gene is cloned successfully. The HBEBP1 fusion protein could be expressed in prokaryotic expression system of E. coli. These results lays a foundative for studying the immunogenicity and the biological characteristics of the HBEBP1 protein.