乳糖酶在大肠杆菌中的高效表达与纯化

High-level expression of galactosidase gene in Escherichia Coli

目的克隆并分析乳酸菌LacZ基因,构建其原核表达质粒,在大肠杆菌中诱导融合蛋白表达,并纯化得到纯度较高的目的蛋白。方法采用PCR方法从乳酸菌中扩增出LacZ基因,测序和序列分析。将其克隆至克隆载体pGM-Tvector中,筛选阳性克隆后回收目的片段,将其克隆入原核表达质粒pGEX-6p-1,构建其重组表达质粒pGEX-6p-1/LacZ。以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,聚丙烯酰胺凝胶电泳及Western blot分析鉴定,最后通过纯化得到单一的目的蛋白。结果 PCR扩增获得3024bp的LacZ基因,测序得知序列与GeneBank报道的序列一致,序列分析表明,该序列开放读码框有3024bp,推测肽链具有1008个氨基酸,蛋白分子量为114KDa,等电点为4.9。构建了重组表达质粒pGEX-6p-1/LacZ,IPTG诱导其高效表达出融合蛋白,聚丙烯酰胺凝胶电泳及Western blot分析鉴定出所表达蛋白的分子量为140KD,与融合蛋白的分子量一致。最后利用Glutathione Sepharose 4B对表达产物进行纯化,得到单一的目的蛋白。结论成功地构建了乳酸菌LacZ基因的原核表达质粒,通过序列分析并诱导表达,最后纯化得到单一的目的蛋白,为进一步生产低乳糖奶奠定基础。

Aim To clone and analyze lactic acid bacteria LacZ gene,construct its prokaryotic expression plasmid and induce the expression of fusion protein in E.coli. Methods The LacZ gene fragments was amplified by PCR from the lactic acid bacteria,sequenced and cloned into the pGM-T vector,the positive danes were screened after cloned into prokaryotic expression Plasmid pGEX-6p-1, the recombinant expression plasmid of pGEX-6p-1/LacZ was constructed. After recombinant plasmid was induced by IPTG,polyaerylamide gel electrophoresis and western blot analysis,and finally find a single target protein by purified. Results The sequence of LacZ gene amplified by PCR was the same as the sequence in gene map of Genebank. Sequence analysis showed that the open reading frame sequences was 3024bp, polypeptide chain that consisted of 1008 amino acids,protein molecular weight of 114KDa,isoeleetric point 4.9. Conclusion The recombinant expression plasmid pGEX-6p-1/LacZ has been successfully constructed , the fusion protein was induced to express efficiently by IPTG. Polyaerylamide gel