关键词: 新生隐球菌, 漆酶, 原核表达, 包涵体

Abstract:

Objective To construct a prokaryotic expression vector of Cryptococcus neoformans laccase containing His-tag and to express and identify it.Methods The coding sequence of LAC1 gene was amplified by PCR and correctly inserted into pET-28a (+) vector to obtain the recombinant plasmid. The recombinant plasmid was transformed into E.coli DH5α competent cells for PCR identification and gene sequencing. The correct recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. Induction expression was carried out under different conditions, and the target protein was detected and identified by SDS-PAGE electrophoresis and MALDI-TOF mass spectrometry. Inclusion body protein was denatured by urea and identified by SDS-PAGE electrophoresis and MALDI-TOF mass spectrometry.Results The prokaryotic expression vector of Cryptococcus neoformans laccase containing His-tag was constructed and confirmed by PCR, and the result of sequencing was consistent with the target sequence. The SDS-PAGE showed that the target protein with molecular mass(MR) about 68 000 was successfully induced to express in E. coli BL21(DE3). And identification by MALDI-TOF mass spectrometry demonstrated that the target protein is insoluble in this expression system and may exist in the form of inclusion body. Conclusion The prokaryotic expression vector of Cryptococcus neoformans laccase containing His-tag was successfully constructed. This laid the foundation for further purification and analysis of the crystal structure of Cryptococcus neoformans laccase.

Key words: Cryptococcus neoformans, laccase, prokaryotic expression, inclusion protein