Construction of prokaryotic expression system of TGF-beta1 epitope gene and identification of recombinant fusion protein immunity.
OBJECTIVE
To insert the constructed TGF-beta(1) epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-beta(1) 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-beta(1) vaccine.


METHODS
The TGF-beta(1) encoding epitope gene (the mature TGF-beta(1) from 78-109 amino acid residues, TGF-beta(1)(32)) was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-beta(1)(32) 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-beta(1)(32) and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T(4) ligase. The fusion gene fragments HBcAg1-71-TGF-beta(1)(32)- 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-beta(1) 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-beta(1) 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-beta(1) antigenicity and could be used as anti-TGF-beta(1) vaccine.


CONCLUSIONS
A recombinant prokaryotic expression system with high expression efficiency of the target TGF-beta(1) epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-beta(1). The expressed TGF-beta(1) epitope gene shows good immunogenicity and antigenicity.
Guo
Yong-Hong
YH
Department of Infectious Diseases, Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an 710004, China. xiaoqing9759@sina.com
Hao
Zhi-Ming
ZM
Luo
Jin-Yan
JY
Wang
Jun-Hong
JH
D000939:Epitopes; D015870:Gene Expression; D005822:Genetic Vectors; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000276:immunology; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000276:immunology; D053773:Transforming Growth Factor beta1
D000939:Epitopes; D006512:Hepatitis B Core Antigens; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1