球形芽胞杆菌S层蛋白基因的克隆及原核表达

目的克隆球形芽胞杆菌S层蛋白的编码基因,实现其原核表达并纯化表达产物。方法提取球形芽胞杆菌菌体基因组DNA,根据GenBank公布的核酸序列设计并合成引物,扩增S层蛋白编码基因sllB,与pMD19-T载体连接;测序验证后,构建原核表达质粒pET28a(+)-sllB并转化BL21(DE3)中,经IPTG诱导表达,SDS-PAG和Westernblot鉴定表达产物,亲和柱层析纯化重组蛋白。结果扩增获得S层蛋白编码基因sllB全长(3 306bp),与参考序列同源性为97.5%。构建的原核表达质粒pET28a(+)-sllB转化BL21(DE3)后,SDS-PAGE显示表达蛋白的分子质量为116ku,与理论值相符。Western blot显示其全蛋白、可溶性蛋白和不溶性蛋白中均被鼠抗Penta-His抗体识别。结论获得了球形芽胞杆菌S层蛋白编码基因sllB,并实现了其原核表达。

Gene cloning,expression,and purification of S-layer protein from Bacillus sphaericus

Objective To clone,express and purify surface layer protein from Bacillus sphaericus.Methods Genomic DNA was extracted from B.sphaericus with primers designed in accordance with the nucleotide sequence registered in GenBank.The target gene was ligated to a pMD19-T plasmid.The fragment was then sequenced,sub-cloned into the plasmid pET28a(+),expressed in E.coli BL21,and purified by nickel-affinity chromatography.Results Full-length sllB cDNA coding for the surface layer protein of Bacillus sphaericus was amplified(3,306 bp) and had 97.5% homology with the reference sequence(GenBank Accession No.AJ849550).SDS-PAGE and Western blotting showed a specific band of about 116 ku in the whole cell lysate,soluble protein fraction,and insoluble protein fraction.As estimated by chromatography,about 1.8 mg of the recombinant product was obtained.Conclusion The gene sllB coding for surface layer protein was amplified from Bacillus sphaericus and successfully expressed in E.coli BL21