Expression, Purification And Functional Identification Of P32, A Hypothetical Adhesin From Mycoplasma Genitalium

Objectives:To construct a recombinant plasmid contamting p32 gene of Mycoplasma genitalium, to express a fusion protein in E.coli BL21(DE3) and purify it, and to identify its role in adhesion of M. genitalium.Methods:The p32 gene was amplified by PCR from the genome of M. genitalium and cloned into pET-30a(+) after digestion with BamHI and HindIII. A codon TGA encoding Tryptophan was changed to TGG by site-directed mutagenesis. The mutagenized recombinant expression vector pET-30a(+)/P32 was transformed into E.coli XL 1-Blue. Recombinants were selected by enzymes digestion and sequencing. One clone with mutated p32 gene was transformed into E.coli BL21(DE3) and induced to express P32. The obtained fusion protein was analyzed by SDS-PAGE and Western blotting, purified by Ni-NTA affinity chromatography. The purified protein was used to immune rabbits. Then the polyclonal antibody against the purified protein was acquired and used for adhesion and inhibition assay.Results:The p32 gene was successfully cloned, whose sequence was compatible with that published by GenBank. The TGA triplet within p32 gene was successfully mutagenized to TGG SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 37.6kDa which was consistent with the theoretically predicted value, and the specificity of expressed protein was confirmed by Western blotting. The target protein was satisfactorily purified by affinity...