重组人平足蛋白基因在中国仓鼠卵巢细胞的稳定表达
发布时间：2018-11-26 13:13
【摘要】：目的构建平足蛋白(PDPN)真核表达质粒PDPN-pEGFP-N1,建立稳定高表达重组人PDPN的中国仓鼠卵巢(CHO)细胞株并对其进行生物学活性研究。方法通过反转录PCR和DNA重组技术从HEK293细胞中克隆PDPN cDNA,插入带有增强型绿色荧光蛋白(EGFP)标记的真核表达载体pEGFP-N1中。通过PCR、酶切及测序等方法鉴定重组载体;进而将该重组载体转染至CHO细胞中,通过荧光显微镜检测EGFP的表达,Western blot法检测PDPN在CHO细胞中的表达,流式细胞术分选高表达PDPN的CHO细胞,运用血小板聚集实验检测分选后细胞株的生物学活性。结果 DNA测序和酶切鉴定证明PDPN基因正确克隆至pEGFP-N1载体中,重组载体稳定转染CHO细胞后,在荧光显微镜下观察到绿色荧光,流式细胞术分选后的细胞高表达PDPN;Western blot结果显示转染后CHO细胞膜表面表达PDPN蛋白;过表达PDPN的CHO细胞可以诱导人血小板聚集。结论成功构建了稳定高表达PDPN蛋白的CHO细胞株,该细胞株可表达PDPN,并诱导血小板聚集。
[Abstract]:Objective to construct eukaryotic expression plasmid PDPN-pEGFP-N1, of flat foot protein (PDPN) to establish Chinese hamster ovarian (CHO) cell line with high expression of recombinant human PDPN and to study its biological activity. Methods PDPN cDNA, was cloned from HEK293 cells by reverse transcription PCR and DNA recombination techniques and inserted into the eukaryotic expression vector pEGFP-N1 with enhanced green fluorescent protein (EGFP) labeling. The recombinant vector was identified by PCR, digestion and sequencing. Then the recombinant vector was transfected into CHO cells. The expression of PDPN in CHO cells was detected by, Western blot method by fluorescence microscopy, and the CHO cells with high PDPN expression were selected by flow cytometry. The biological activity of the sorted cell line was detected by platelet aggregation test. Results DNA sequencing and restriction endonuclease analysis proved that PDPN gene was cloned into the pEGFP-N1 vector correctly. After stable transfection of the recombinant vector into CHO cells, green fluorescence was observed under fluorescence microscope, and high expression of PDPN; was observed in the cells separated by flow cytometry. Western blot results showed that PDPN protein was expressed on the surface of CHO cell membrane after transfection, and that CHO cells with overexpression of PDPN could induce human platelet aggregation. Conclusion A stable CHO cell line with high expression of PDPN protein was successfully constructed, which could express PDPN, and induce platelet aggregation.
【作者单位】： 苏州大学附属第一医院江苏省血液研究所卫生部血栓与止血重点实验室;
【基金】：国家自然科学基金(81270593;81370617)
【分类号】：Q78;R730.2
本文编号：2358659
[Abstract]:Objective to construct eukaryotic expression plasmid PDPN-pEGFP-N1, of flat foot protein (PDPN) to establish Chinese hamster ovarian (CHO) cell line with high expression of recombinant human PDPN and to study its biological activity. Methods PDPN cDNA, was cloned from HEK293 cells by reverse transcription PCR and DNA recombination techniques and inserted into the eukaryotic expression vector pEGFP-N1 with enhanced green fluorescent protein (EGFP) labeling. The recombinant vector was identified by PCR, digestion and sequencing. Then the recombinant vector was transfected into CHO cells. The expression of PDPN in CHO cells was detected by, Western blot method by fluorescence microscopy, and the CHO cells with high PDPN expression were selected by flow cytometry. The biological activity of the sorted cell line was detected by platelet aggregation test. Results DNA sequencing and restriction endonuclease analysis proved that PDPN gene was cloned into the pEGFP-N1 vector correctly. After stable transfection of the recombinant vector into CHO cells, green fluorescence was observed under fluorescence microscope, and high expression of PDPN; was observed in the cells separated by flow cytometry. Western blot results showed that PDPN protein was expressed on the surface of CHO cell membrane after transfection, and that CHO cells with overexpression of PDPN could induce human platelet aggregation. Conclusion A stable CHO cell line with high expression of PDPN protein was successfully constructed, which could express PDPN, and induce platelet aggregation.
【作者单位】： 苏州大学附属第一医院江苏省血液研究所卫生部血栓与止血重点实验室;
【基金】：国家自然科学基金(81270593;81370617)
【分类号】：Q78;R730.2
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