摘要
本文构建并表达了旋毛虫热休克蛋白70（Ts—Hsp70）与排泄分泌抗原Ts87的融合蛋白，并对融合蛋白进行纯化和免疫学鉴定。本研究采用限制性内切酶EcoRI酶切位点将目的基因Ts—Hsp70与Ts87相连接，插入pET28-a（＋）质粒，转人大肠埃希菌BL21（DE3）中诱导表达、纯化，获得的融合蛋白rTs—Hsp70-Ts87用Western blot的方法鉴定。经PCR、酶切鉴定、测序分析，结果显示，目的基因片段大小为2721bp，构建的重组质粒与预期相符。经IPTG诱导表达，通过镍离子柱层析纯化复性后得到可溶的融合蛋白rTs—Hsp70-Ts87，其相对分子质量约为100kDa；Western blot结果显示，该蛋白可被抗His标签单抗、rTs—Hsp70免疫血清、rTs87免疫血清识别。以上结果表明，本研究成功构建并表达了融合蛋白rTs．Hsp70-Ts87，为进一步研究rTs—Hsp70-Ts87的免疫保护性，开发新的有效抗旋毛虫病的疫苗奠定基础。
To construct and express the fusion protein that combines Trichinella spiralis heat shock protein 70 with excretory- secretory antigen Ts87, restriction enzyme EcoR I site was applied to fuse Ts-HspT0 with Ts87. The target genes were inserted into plasmid pET28-a （ ＋ ） , and transformed into E. coli. BL21 （DE3）. After expression and purification, the fusion protein rTs-Hsp70-Ts87 was characterized by SDS-PAGE and Western blot. The results showed that the fusion gene was 2 721 bp. The results of PCR, enzyme digestion and sequencing analysis showed that the recombinant plasmid was affirmed to be successfully constructed. The fusion protein rTs-Hsp70-Ts87 was expressed with IPTG induction and purified by Ni-affinity chromatography and refolded. The results of SDS-PAGE and Western blot indicated that molecular weight of the protein is about 100 kDa, and it could be recognized by anti His-tag monoclonal antibody, anti-rTs-Hsp70 immune serum and anti-rTs87 immune serum. In conclusion, T. spiralis recombinant fusion protein rTs-Hsp70-Ts87 was constructed and expressed successfully, which had laid the groundwork for further research.
出处
《寄生虫与医学昆虫学报》
CAS
北大核心
2015年第1期1-6,共6页
Acta Parasitologica et Medica Entomologica Sinica
基金
国家传染病重大专项（No.2012ZX10004220-12）
北京市自然科学基金青年基金资助项目（No.7154183）
关键词
旋毛虫
融合蛋白
热休克蛋白70
排泄分泌抗原
疫苗
Trichinella spiralis
Recombinant fusion protein
Heat shock protein 70 （HSP70）
Excretory-secretory antigen
Vaccine
