论文部分内容阅读
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。
Objective To clone the gene encoding S-layer protein of Bacillus sphaericus and to express it in prokaryotic cells. Methods The genomic DNA of Bacillus sphaericus was extracted and designed and synthesized according to the nucleotide sequence published in GenBank. The sllB gene of S protein was amplified and ligated with pMD19-T vector. After sequencing, the prokaryotic expression plasmid pET28a (+) - sllB, transformed into BL21 (DE3), induced by IPTG, expressed by SDS-PAGE and Western blot, purified recombinant protein by affinity chromatography. Results The full-length sllB gene (3 306 bp) of S-protein gene was amplified and the homology with the reference sequence was 97.5%. The constructed prokaryotic expression plasmid pET28a (+) - sllB transformed BL21 (DE3), SDS-PAGE showed that the molecular mass of the expressed protein was 116ku, consistent with the theoretical value. Western blot showed that the whole protein, soluble protein and insoluble protein were detected by mouse anti-Penta-His antibody. Conclusion SllB, a gene encoding S - layer protein of Bacillus sphaericus, was obtained and its prokaryotic expression was achieved.