【摘要】：目的構(gòu)建平足蛋白(PDPN)真核表達(dá)質(zhì)粒PDPN-pEGFP-N1,建立穩(wěn)定高表達(dá)重組人PDPN的中國倉鼠卵巢(CHO)細(xì)胞株并對其進(jìn)行生物學(xué)活性研究。方法通過反轉(zhuǎn)錄PCR和DNA重組技術(shù)從HEK293細(xì)胞中克隆PDPN cDNA,插入帶有增強(qiáng)型綠色熒光蛋白(EGFP)標(biāo)記的真核表達(dá)載體pEGFP-N1中。通過PCR、酶切及測序等方法鑒定重組載體;進(jìn)而將該重組載體轉(zhuǎn)染至CHO細(xì)胞中,通過熒光顯微鏡檢測EGFP的表達(dá),Western blot法檢測PDPN在CHO細(xì)胞中的表達(dá),流式細(xì)胞術(shù)分選高表達(dá)PDPN的CHO細(xì)胞,運(yùn)用血小板聚集實(shí)驗(yàn)檢測分選后細(xì)胞株的生物學(xué)活性。結(jié)果 DNA測序和酶切鑒定證明PDPN基因正確克隆至pEGFP-N1載體中,重組載體穩(wěn)定轉(zhuǎn)染CHO細(xì)胞后,在熒光顯微鏡下觀察到綠色熒光,流式細(xì)胞術(shù)分選后的細(xì)胞高表達(dá)PDPN;Western blot結(jié)果顯示轉(zhuǎn)染后CHO細(xì)胞膜表面表達(dá)PDPN蛋白;過表達(dá)PDPN的CHO細(xì)胞可以誘導(dǎo)人血小板聚集。結(jié)論成功構(gòu)建了穩(wěn)定高表達(dá)PDPN蛋白的CHO細(xì)胞株,該細(xì)胞株可表達(dá)PDPN,并誘導(dǎo)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.
【作者單位】： 蘇州大學(xué)附屬第一醫(yī)院江蘇省血液研究所衛(wèi)生部血栓與止血重點(diǎn)實(shí)驗(yàn)室;
【基金】：國家自然科學(xué)基金(81270593;81370617)
【分類號】：Q78;R730.2

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