小鼠Foxp3融合蛋白的原核表达及纯化

Cloning Construction and Purification of Recombinant Mouse Foxp3 Gene

目的克隆小鼠Foxp3(MFoxp3)基因,构建含该基因的原核表达载体,并表达、纯化出小鼠的Foxp3融合蛋白。方法RT-PCR方法从小鼠淋巴细胞扩增出小鼠Foxp3基因,连入T Easy Vector进行测序,然后将序列正确的小鼠Foxp3基因用酶切的方法从T Easy vector切下,并将其连接到用相同酶切,含有硫氧还蛋白(含6个组氨酸“标签”)的pET 32a(+)原核表达载体中构建pET 32a(+)-MFoxp3表达载体。用IPTG诱导转化pET 32a(+)-MFoxp3表达载体的大肠杆菌BL21(DE3),并以镍螯合层析法纯化MFoxp3融合蛋白。结果经RT-PCR成功地克隆了1,290 bp的小鼠Foxp3基因,测序正确后转化大肠杆菌原核表达载体pET 32a(+),重组质粒在BL21(DE3)中成功表达出分子质量约为63 kD的融合蛋白,运用Ni-NTA方法纯化出目的蛋白,纯化后的蛋白纯度达80%,并用Western Blotting检测蛋白的灵敏度和特异性。结论构建了小鼠Foxp3的融合蛋白原核表达载体并成功表达与纯化出小鼠Foxp3的融合蛋白。

Objective To clone Mouse Foxp3 gene （MFoxp3） and construct the prokaryotic expression vector of the protein, and to express and purify mouse Foxp3 fusion protein.Methods Using RT-PCR technique, the gene encoding Mouse Foxp3 was amplified from the spleen of the mouse, and the PCR product was ligated with T Easy vector and sequenced. Then the correct clone was digested by endonuclease for releasing the fragment of mouse Foxp3, and was inserted into pET 32a（＋） prokaryotic expression vector which has Trx protein with His-tag and was digested by the same endonuclease.The E.coli BL21（DE3） transformed with pET 32a（＋）-Mfoxp3 were induced by IPTG for expression of MFoxp3 fusion protein.The protein was purified by Ni^2＋ affinity chromatography. Results The Mouse Foxp3 gene was 1,290bp and the fusion protein was expressed in BL21（DE3） at a high level by cloned into pET 32a（＋）.SDS-PAGE showed its molecular mass was about 63KD. Recombinant protein was purified by Ni-NTA purification column and the purity of the fusion protein was 80%. The immunogenicity and specificity were tested by Western blot assays.Conclusion The prokaryotic expression vector of mouse Foxp3 fusion protein has been constructed, and the fusion protein has been successfully expressed and purified.