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目的克隆弓形虫RH株致密颗粒蛋白7(GRA7)的全长基因,在大肠杆菌工程菌中重组表达GST-His-GRA7融合蛋白。方法提取弓形虫总RNA,反转录获得cDNA,PCR扩增GRA7基因全长,与pET-41 Ek/LIC载体通过基因重组直接连接,在大肠杆菌RosettaTM(DE3)pLysS工程菌中诱导表达重组融合蛋白,最后采用SDS-PAGE和West ern blotting鉴定重组蛋白产物。结果构建了弓形虫GRA7全基因的重组表达质粒,诱导表达了GST-His-GRA7融合重组蛋白,分子量为62kD,与预测结果相符。结论研究获得了弓形虫GRA7全基因的重组蛋白,为进一步研究弓形虫GRA7抗原性及其与宿主细胞的相互作用奠定了基础。
OBJECTIVE: To clone the full-length gene of GRA7 from RH strain Toxoplasma gondii, and express GST-His-GRA7 fusion protein in E. coli. Methods The total RNA of Toxoplasma gondii was extracted and the cDNA was obtained by reverse transcription. The full length of GRA7 gene was amplified by PCR and ligated with pET-41 Ek / LIC vector through gene recombination to induce expression of recombinant fusion in Escherichia coli RosettaTM (DE3) pLysS Finally, SDS-PAGE and West ern blotting were used to identify the recombinant protein product. Results The recombinant plasmids of GRA7 gene of Toxoplasma gondii were constructed and the fusion protein of GST-His-GRA7 fusion protein was induced with a molecular weight of 62 kD, which was consistent with the predicted results. Conclusion The recombinant protein of GRA7 gene of Toxoplasma gondii was obtained, which laid the foundation for further study on the antigenicity of Toxoplasma gondii GRA7 and its interaction with host cells.