刚地弓形虫肌动蛋白profilin的原核表达和纯化

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492 bp。经双酶切和测序，重组质粒pET28a-TgPRF连接产物构建成功。SDS-PAGE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。

Prokaryotic expression and purification of Toxoplasma gondii profilin protein

Objective:To discuss the prokaryotic expression system and purification conditions of Toxoplasma gondii profilin-like protein (TgPRF), and to provide basis for the study on anti-tumor immuno-adjuvant. Methods:The coding region of TgPRF gene was amplified with a pair of specific primers which were designed according to the cDNA of tachyzoites of Toxoplasma gondii RH strain.The PCR products were cloned into the pET-28a (+ ) vector after double enzyme digestion. The recombinant pET28a (+ )-TgPRF plasmid was transformed into E.coli DH5αcells.The positive clones were selected by the double restrictive enzyme digestion and sequencing.The correct pET28a (+)-TgPRF plasmid was transformed into E.coli BL21 (DE3)and induced for 4 h by IPTG.The expression of recombinant TgPRF protein was analyzed by SDS-PAGE method;the expression of recombinant protein His-profilin was detected by Western blotting method.Results:The length of product of PCR was 492 bp.The recombinant plasmid pET28a-TgPRF was confirmed by double restriction enzyme digestion and sequencing.The SDS-PAGE results showed that the target protein was expressed in E.coli BL21 (DE3)in bacteria supernatant.The purified TgPRF protein was obtained by Ni-NTA agarose gel column chromatography with the purity>90%.The Western blotting results revealed that the recombinant TgPRF protein could be recognized by Anti-His antibody.Conclusion: The recombinant plasmid pET28a-TgPRF is successfully constructed,and the TgPRF protein is obtained with the soluble prokaryotic expression.