刚地弓形虫肌动蛋白profilin的原核表达和纯化

发布时间：2018-04-09 08:11

本文选题：刚地弓形虫　切入点：profilin　出处：《吉林大学学报(医学版)》2017年06期

【摘要】：目的:探讨刚地弓形虫肌动蛋白profilin(TgPRF)的原核表达体系和纯化条件,为后续的抗肿瘤免疫佐剂研究提供依据。方法:以弓形虫RH株速殖子的cDNA为模板,采用一对特定的引物扩增TgPRF基因的编码区。PCR产物双酶切后克隆入pET28a(+)载体中。重组的pET28a(+)-TgPRF质粒转化E.coli DH5α感受态细胞。双酶切鉴定阳性克隆,并选取测序正确的质粒转化E.coli BL21(DE3)表达菌,经IPTG诱导表达4 h,SDS-PAGE法检测TgPRF蛋白的表达,Western blotting法检测重组蛋白His-prolilin的表达。结果:PCR扩增产物长度为492bp。经双酶切和测序,重组质粒pET28a-TgPRF连接产物构建成功。SDSPAGE检测,目的蛋白在超声菌液的上清中表达。经Ni-NTA琼脂糖凝胶柱纯化,获得纯化的TgPRF蛋白(纯度90%)。Western blotting检测,重组TgPRF蛋白能被Anti-His抗体识别。结论:成功构建重组质粒pET28a-TgPRF,并实现可溶性原核表达。
[Abstract]:Aim: to investigate the prokaryotic expression system and purification conditions of TgPRF of Toxoplasma gondii actin profilinus, and to provide a basis for the further study of antitumor immune adjuvants.Methods: using cDNA of Toxoplasma gondii RH strain tachyzoites as template, the encoding region of TgPRF gene was amplified by a pair of specific primers and cloned into pET28a () vector.The recombinant plasmid pET28a (TgPRF) was transformed into E.coli DH5 伪 competent cells.The positive clones were identified by double enzyme digestion, and the recombinant plasmid was transformed into E.coli BL21DE3). The expression of TgPRF protein was detected by IPTG induced expression 4 h SDS-PAGE and the expression of recombinant protein His-prolilin was detected by Western blotting method.Results the length of PCR amplification product was 492bp.After double enzyme digestion and sequencing, the recombinant plasmid pET28a-TgPRF ligation product was successfully constructed. SDS-PAGE was used to detect the expression of the protein in the supernatant of ultrasonic bacteria.The purified TgPRF protein was purified by Ni-NTA agarose gel column. The purified TgPRF protein (purity 90%).Western blotting) could be recognized by Anti-His antibody.Conclusion: the recombinant plasmid pET28a-TgPRF was successfully constructed and expressed in soluble prokaryotic expression.
【作者单位】：吉林大学第一医院转化医学研究院移植免疫实验室;吉林大学第二医院检验科;
【基金】：吉林省科技厅自然科学基金资助课题(20150101127JC)
【分类号】：R382.5

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