粪类圆线虫丝氨酸蛋白酶抑制剂1蛋白生物信息学分析及原核表达

Bioinformatics analysis and prokaryotic expression of Strongyloides stercora⁃lis serine protease inhibitor 1

目的采用生物信息学方法预测粪类圆线虫丝氨酸蛋白酶抑制剂1(Strongyloides stercoralis serine protease in⁃hibitor 1,Ss⁃SRPN⁃1)蛋白结构和抗原表位,构建原核表达载体并进行重组蛋白表达,为阐明Ss⁃SRPN⁃1蛋白功能奠定基础。方法自NCBI数据库中获取Ss⁃SRPN⁃1氨基酸序列,使用ExPASY、SWISS⁃MODEL和Protean等生物信息学软件对Ss⁃SRPN⁃1蛋白的理化性质、结构和抗原表位等进行预测。根据Ss⁃SRPN⁃1核苷酸序列设计引物,以粪类圆线虫感染性三期幼虫DNA为模板,对Ss⁃SRPN⁃1基因进行扩增、克隆和测序。将重组质粒pET28a(+)/Ss⁃SRPN⁃1转入大肠埃希菌BL21(DE)中诱导重组Ss⁃SRPN⁃1蛋白表达,纯化重组蛋白并进行Western blotting及质谱鉴定。结果生物信息学分析显示,Ss⁃SRPN⁃1蛋白由372个氨基酸组成,分子式为C1948H3046N488O575S16,是一种稳定的亲水性蛋白,预测其亚细胞定位在细胞外;预测该蛋白存在11个优势B细胞抗原表位、20个T细胞表位。成功扩增长度为1119 bp的Ss⁃SRPN⁃1基因,构建了重组质粒pET28a(+)/Ss⁃SRPN⁃1并转化至大肠埃希菌BL21(DE)进行表达,表达的重组Ss⁃SRPN⁃1蛋白分子量约43 kDa,鉴定结果显示该重组蛋白为Ss⁃SRPN⁃1蛋白。结论成功表达了重组Ss⁃SRPN⁃1蛋白,预测该蛋白可作为一种潜在的粪类圆线虫病疫苗候选分子。

Objective To predict the structure and antigenic epitope of the Strongyloides stercoralis serine protease inhibitor 1(Ss⁃SRPN⁃1)protein using bioinformatics tools,and to construct prokaryotic expression plasmids for expression of recombinant Ss⁃SRPN⁃1 protein,so as to provide the basis for unraveling the function of the Ss⁃SRPN⁃1 protein.Methods The amino acid se⁃quence of the Ss⁃SRPN⁃1 protein was downloaded from the NCBI database,and the physicochemical properties,structure and an⁃tigenic epitopes of the Ss⁃SRPN⁃1 protein were predicted using bioinformatics tools,including ExPASy,SWISS⁃MODEL and Pro⁃tean.Primers were designed according to the nucleotide sequences of Ss⁃SRPN⁃1,and the Ss⁃SRPN⁃1 gene was amplified,cloned and sequenced with genomic DNA extracted from the infective third⁃stage larvae of S.stercoralis as a template.The Ss⁃SRPN⁃1 protein sequence was cloned into the pET28a(+)expression vector and transformed into Escherichia coli BL21(DE)cells for in⁃duction of the recombinant Ss⁃SRPN⁃1 protein expression.The recombinant Ss⁃SRPN⁃1 protein was then purified and identified using Western blotting and mass spectrometry.Results Bioinformatics analysis showed that the Ss⁃SRPN⁃1 protein,which was composed of 372 amino acids and had a molecular formula of C1948H3046N488O575S16,was a stable hydrophilic protein,and the sub⁃cellular localization of the protein was predicted to be extracellular.The Ss⁃SRPN⁃1 protein was predicted to contain 11 dominant B⁃cell antigenic epitopes and 20 T⁃cell antigenic epitopes.The Ss⁃SRPN⁃1 gene with a length of 1119 bp was successfully ampli⁃fied,and the recombinant plasmid pET28a(+)/Ss⁃SRPN⁃1 was constructed and transformed into E.coli BL21(DE)cells.The ex⁃pressed recombinant Ss⁃SRPN⁃1 protein had a molecular weight of approximately 43 kDa,and was characterized as a Ss⁃SRPN⁃1 protein.Conclusions The recombinant Ss⁃SRPN⁃1 protein has been expressed successfully,and this recombinant protein may be a potential vaccine candidate against strongyloidiasis.