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[High expression of hepatoma associated antigen HAb18G in prokaryotic cells and study on its function in vitro]
Peng Xu1, Ke-feng Dou, Zhi-nan Chen
1Department of Hepato-Biliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Aim:
To construct prokaryotic expression vector of HAb18G, and express high level of this fusion protein in E. coli and to identify its function and immunogenicity.
Methods:
The HAb18G full length cDNA from pBluescript/HAb18G was obtained by PCR and cloned into prokaryotic expression vector pRSET-C and then transformed into E. coli BL21(DE3) to induce its expression. Expressed products were analyzed by SDS-PAGE and laser thin layer scan. The purified HAb18G protein was identified by gelatin enzymogram and ELISA.
Results:
Endonuclease digestion and DNA sequencing proved that HAb18G cDNA was cloned correctly into the expression vector. Result of SDS-PAGE showed that the relative molecular mass of the expressed product HAb18G fusion protein was 34,600, which was in accordance with predicted relative molecular mass value. Laser thin layer scan showed that the expressed product accounted for 33% of the total bacteria protein. Result of enzymogram was negative whereas the result of ELISA was positive.
Conclusion:
It was testified that the protein HAb18G has immunogenicity but no bioactivity. The high level prokaryotic expression of HAb18G lay the foundation for manufacturing the HAb18G protein in great quantities and proceeding to its relative research.
