Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombina

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AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg 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-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 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-β1loop 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-β1not 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-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1immunogenicity and antigenicity.
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