Brief Reports
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World J Gastroenterol. Oct 28, 2005; 11(40): 6389-6394
Published online Oct 28, 2005. doi: 10.3748/wjg.v11.i40.6389
Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity
Yong-Hong Guo, Zhi-Ming Hao, Jin-Yan Luo, Jun-Hong Wang
Yong-Hong Guo, Jin-Yan Luo, Jun-Hong Wang, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an710004, Shannxi Province, China
Zhi-Ming Hao, The Post Doctor Xijing Hospital of Fourth Military Medical University, Xi’an 710004, Shannxi Province, China
Author contributions: All authors contributed equally to the work.
Supported by the JRC Research Fund
Correspondence to: Dr. Yong-Hong Guo, Department of Infectious Diseases, the Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an 710004, China. xiaoqing9759@sina.com
Telephone: +86-029-87679274; 13891866147
Received: March 10, 2005
Revised: April 9, 2005
Accepted: April 12, 2005
Published online: October 28, 2005
Abstract

AIM: To insert the constructed TGF-β1 epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1 antigenicity in its prokaryotic expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1 vaccine.

METHODS: The TGF-β1 encoding epitope gene (the mature TGF-β1 from 78-109 amino acid residues, TGF-β132) was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-β132 vector. The HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β132 and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T4 ligase. The fusion gene fragments HBcAg1-71-TGF-β132- HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.

RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1 epitope gene was inserted into the el loop of C-terminus of truncated hepatitis B core antigen. SDS-PAGE analysis showed that relative molecular mass (Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein, mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1 polyclonal IgG but not with anti-HBcAg. The purity of protein was about 90 % and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1 antigenicity and could be used as anti-TGF-β1 vaccine.

CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF-β1 epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1. The expressed TGF-β1 epitope gene shows good immunogenicity and antigenicity.

Keywords: Prokaryotic expression system; TGF-β1 epitope; Immunogenicity; Antigenicity