Expression, purification and polyclonal antibody preparation of the Schistosoma japonicum SjGrpE protein

Abstract

Objective To investigate the biological properties of Schistosoma japonicum SjGrpE protein, and to express and purify the recombinant SjGrpE protein and test its immunogenicity.

Methods The amino acid composition, molecular weight, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, localization, phosphorylation site, ubiquitination site, glycosylation site, secondary and tertiary structures and B cell epitopes of the SjGrpE protein were predicted using bioinformatics analyses. The SjGrpE gene was amplified using PCR assay using S. japonicum cDNA as a template, double enzyme-digested and linked to the pET28a vector to yield the recombinant plasmid pET28a-SjGrpE. The recombinant plasmid pET28a-SjGrpE was transformed into Escherichia coli BL21, and then IPTG was employed to induce the expression of the target protein, which was purified by nickel ion affinity chromatography. After mice were immunized with the recombinant SjGrpE protein, mouse sera were collected, and the polyclonal antibody against the SjGrpE protein was characterized.

Results SjGrpE protein, which was identified as a hydrophilic protein, was predicted to have a molecular weight of approximately 24.3 kDa without transmembrane regions or signal peptides, and locate in the mitochondrion. SjGrpE protein contained 18 phosphorylation sites and 2 ubiquitination sites, but had no glycosylation sites. In addition, SjGrpE protein contained 5 B-cell epitopes. The full length of SjGrpE gene was approximately 660 bp. The recombinant pET28a-SjGrpE plasmid was successfully generated, and the recombinant SjGrpE protein was obtained following the affinity chromatography, which stimulated mice to secrete high-titer antibodies.

Conclusion The recombinant SjGrpE protein has been successfully prepared and this recombinant protein has a high immunogenicity, which provides a basis for evaluating its value as a vaccine candidate for S. japonicum infections.

Abstract

［摘要］ 目的 分析日本血吸虫核苷酸交换因子 (SjGrpE) 蛋白生物学特性, 表达与纯化重组SjGrpE蛋白并测定其免疫原性。 方法 采用生物信息学方法预测SjGrpE蛋白的氨基酸组成、分子量、亲/疏水性、跨膜区、信号肽、定位、磷酸化位点、泛素化位点、糖基化位点、二级和三级结构及B细胞表位。以日本血吸虫cDNA为模板, PCR扩增 SjGrpE 基因, 将其双酶切后连接到pET28a载体得到重组质粒pET28a-SjGrpE。将pET28a-SjGrpE转化大肠埃希菌BL21, 用IPTG诱导目的蛋白表达, 并用镍离子亲和层析法纯化蛋白。将重组SjGrpE蛋白免疫小鼠, 分离血清并鉴定获得的抗多克隆抗体。 结果 SjGrpE蛋白分子量约为24.3 kDa, 是一种亲水性蛋白, 无跨膜区、无信号肽, 定位在线粒体; 该蛋白含有18个磷酸化位点和2个泛素化位点, 无糖基化位点, 含有5个B细胞表位。 SjGrpE 基因全长为660 bp, 成功构建重组质粒pET28a-SjGrpE并纯化获得重组SjGrpE蛋白。重组SjGrpE蛋白能够刺激小鼠分泌高滴度抗体。 结论 成功制备重组SjGrpE蛋白, 该蛋白具有良好免疫原性, 为后续研究其作为日本血吸虫病疫苗候选分子的价值奠定了基础。