日本血吸虫PDIA3的原核表达和多抗血清制备

摘要

为获得日本血吸虫（Schistosoma japonicum, Sj）PDIA3（SjPDIA3）重组蛋白，并制备多克隆抗体血清，本研究以日本血吸虫cDNA文库为模版，利用PCR技术扩增得到PDIA3 ORF全长，并克隆到pET28a (+)载体中，将得到的重组质粒pET28a-SjPDIA3转化至BL21（DE3）感受态细胞，经IPTG诱导后His-Ni柱亲和层析纯化得到SjPDIA3重组蛋白，利用重组蛋白免疫BALB/c小鼠，制备多克隆抗体，Western blot分析重组蛋白的免疫原性。扩增得到长为1482 bp日本血吸虫PDIA3的ORF序列，该序列编码493个氨基酸，含两个硫氧还原蛋白结构域和一段信号肽，无扩膜结构。在原核表达系统中表达得到大小为55 kDa的SjPDIA3重组蛋白，分析显示其具有良好的抗原性。%The objective of the present study was to express recombinant SjPDIA3 protein in E.coli and prepare polyclonal antibodies. The PDIA3 gene was amplified in PCR using the genome of Schistosoma japonicum as a template. The PCR products obtained were inserted into prokaryotic expression vector pET28a(+). The recombinant plasmid pET28a(+)-SjPDIA3 was transformed into E.coli BL21(DE3). Subsequently, SjPDIA3 protein was expressed with induction of IPTG and purified using NI-NTA resin. The BALB/c mice were immunized with recombinant SjPDIA3 protein to generate polyclonal antibodies. The results showed that a cDNA of 1482 bp was amplified. Bioinformatics analysis determined that the full-length ORF was 1482 bp encoding 493 amino acids and SjPDIA3 protein had a thiol-disulfide oxidoreductase domain and a signal peptide with no transmembrane peptide. Western blot demonstrated prokaryotic expression of the recombinant SjPDIA3 protein with molecular mass of 55 kDa and its efficient antigenicity and immunogenicity as expected.