以CotX为分子载体在枯草芽胞杆菌芽胞表面展示绿色荧光蛋白

芽胞衣壳蛋白CotB、CotC、CotG等可作为芽胞表面展示外源蛋白的分子载体,制备口服重组疫苗或具有催化活性的重组酶。CotX为枯草芽胞杆菌Bacillussubtilis芽胞衣壳中的另一种结构蛋白。为证明CotX能否作为分子载体将外源蛋白展示在芽胞表面,本研究将cotX基因与绿色荧光蛋白基因gfp的编码序列进行基因重组,构建融合表达CotX-GFP的整合型重组质粒,将该质粒转化枯草芽胞杆菌,筛选重组菌株并诱导产生芽胞,观察到重组芽胞表面具有GFP绿色荧光。结果表明枯草芽胞杆菌的芽胞衣壳蛋白CotX位于芽胞衣壳外层,可作为芽胞表面展示外源蛋白的载体分子。

Surface display of GFP using CotX as a molecular vector on Bacillus subtilis spores

Spore coat proteins,such as CotB,CotC,CotG et al,are able to efficiently display exogenous protein on spore surface for preparing oral vaccines or enzymes.CotX is another structural protein of spore coats of Bacillus subtilis.To investigate whether CotX could carry target protein onto the spore surface,we constructed a recombinant integrative plasmid,designated as pJS749,which carries a recombinant cotX-gfp gene under the control of the cotX promoter.We transformed pJS749 into Bacillus subtilis 168(trp-),an α-amylase inactivated mutant DRJS749 was selected and confirmed to be a double crossover integrant,where cotX-gfp fragment was integrated into the chromosome.After induction of spore formation,significant green fluorescence was observed on spore surface of strain DRJS749 under fluorescent microcopy.This suggests that CotX is associated with the outer part of the coat.CotX can therefore be used as a molecular vehicle for spore surface display of exogenous proteins.