本文選題：B同源物 + 真核表達載體　； 參考：《中國病理生理雜志》2017年11期

【摘要】：目的:探討過表達B7同源物6(B7-H6)在自然殺傷(NK)細胞介導的肝細胞凋亡中的作用。方法:設(shè)計針對B7-H6全長的寡核苷酸引物,經(jīng)PCR擴增調(diào)取B7-H6全長,并將其亞克隆入線性化的真核表達載體p IRES2-EGFP,構(gòu)建重組B7-H6過表達載體p IRES2-EGFP-B7-H6,通過雙酶切、PCR及測序進行鑒定。利用脂質(zhì)體將p IRES2-EGFP-B7-H6重組質(zhì)粒轉(zhuǎn)染正常肝細胞系L02,采用熒光顯微鏡觀察EGFP的表達,流式細胞技術(shù)檢測轉(zhuǎn)染效率,qRT-PCR和Western blot檢測B7-H6 mRNA和蛋白的表達水平。將轉(zhuǎn)染p IRES2-EGFP-B7-H6重組質(zhì)粒的L02細胞與NK-92細胞以不同的效靶比共培養(yǎng),利用CCK-8實驗分析檢測NK-92細胞對L02細胞的殺傷效應。結(jié)果:經(jīng)PCR、酶切及測序等方法證實成功構(gòu)建p IRES2-EGFP-B7-H6過表達載體;經(jīng)脂質(zhì)體轉(zhuǎn)染L02細胞48 h后,在熒光顯微鏡下觀察到較強綠色熒光表達,流式檢測顯示轉(zhuǎn)染效率達到92.6%;qRT-PCR和Western blot結(jié)果顯示L02細胞B7-H6的mRNA和蛋白高表達;CCK-8實驗證實相對于轉(zhuǎn)染空載體p IRES2-EGFP,NK-92細胞對轉(zhuǎn)染了p IRES2-EGFP-B7-H6的L02細胞的殺傷活性顯著增強(P0.05)。結(jié)論:成功構(gòu)建過表達B7-H6的真核表達載體p IRES2-EGFP-B7-H6,并進一步證實NK-92細胞對過表達B7-H6的L02細胞具有顯著的殺傷效應。
[Abstract]:Aim: to investigate the role of overexpression of B7 homologue 6B 7 H 6 in natural killer NKK cells mediated hepatocyte apoptosis. Methods: the full-length B7-H6 oligonucleotide primers were designed and amplified by PCR and subcloned into the linear eukaryotic expression vector pIRES2-EGFP.The recombinant B7-H6 overexpression vector pIRES2-EGFP-B7-H6 was constructed and identified by double enzyme digestion and sequencing. The recombinant plasmid of p IRES2-EGFP-B7-H6 was transfected into the normal liver cell line L02 by liposome. The expression of EGFP was observed by fluorescence microscope, the transfection efficiency was detected by flow cytometry and the expression level of B7-H6 mRNA and protein was detected by Western blot and RT-PCR. L02 cells transfected with p IRES2-EGFP-B7-H6 recombinant plasmid were co-cultured with NK-92 cells at different target ratios. CCK-8 assay was used to detect the killing effect of NK-92 cells on L02 cells. Results: the overexpression vector of p IRES2-EGFP-B7-H6 was successfully constructed by PCR, restriction endonuclease digestion and sequencing, and strong green fluorescent expression was observed under fluorescence microscope after 48 h transfection of liposome into L02 cells. Flow cytometry showed that the transfection efficiency was 92.6g / qRT-PCR and Western blot results showed that the high expression of mRNA and protein in B7-H6 of L02 cells showed that the cytotoxicity of pIRES2-EGFPNK-92 cells to L02 cells transfected with p IRES2-EGFP-B7-H6 was significantly enhanced compared with that of pIRES2-EGFPNK-92 cells. Conclusion: the eukaryotic expression vector pIRES2-EGFP-B7-H6 was successfully constructed, and it was further confirmed that NK-92 cells had a significant killing effect on L02 cells over-expressing B7-H6.
【作者單位】： 中山大學附屬第三醫(yī)院輸血科;中山大學附屬第三醫(yī)院血液科;
【基金】：廣東省科技計劃資助項目(No.2014A020212575;No.2016A020215215) 廣東省自然科學基金資助項目(No.2016A030313357)
【分類號】：R512.62

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