本文選題：基因沉默 + 狨猴��； 參考：《中國比較醫(yī)學雜志》2017年05期

【摘要】：目的在細胞水平篩選狨猴B2m基因的有效沉默靶點,并進行驗證。方法查詢?nèi)嗽碆2m驗證過的有效siRNA靶位點序列,與狨猴B2m基因序列進行同源性比較,選擇匹配靶點合成shRNA序列。將體外合成的2條干擾序列分別與慢病毒載體FUGW-TDT連接,構建FUGW-TDT-shb2m干擾表達質(zhì)粒,在聚乙烯亞胺(polyethylenimine,PEI)介導下轉(zhuǎn)染293T細胞,轉(zhuǎn)染后48h,用實時熒光定量法檢測轉(zhuǎn)染細胞中B2m基因mRNA的水平。結果篩選出2個與狨猴完全同源的B2m沉默靶位點,分別位于B2m mRNA的290~310 bp,665~685 bp;B2m兩個靶點在轉(zhuǎn)錄水平的沉默效率分別為(46.54±7.91)%(P0.05)和(83.22±4.37)%(P0.0001),差異有顯著性。結論成功構建成FUGW-TDT-shb2m重組質(zhì)粒;在細胞水平篩選得到2個有效的B2m基因沉默靶點;為后續(xù)有關介導狨猴B2m基因沉默的研究奠定了基礎。
[Abstract]:Objective to screen and verify the effective silencing target of marmoset B 2m gene at cell level.Methods the sequence of effective siRNA site verified by human B2m was searched, and the homology of B2m gene sequence of marmoset was compared with that of marmoset. The matching target was selected to synthesize shRNA sequence.The two interference sequences were ligated with lentivirus vector FUGW-TDT in vitro, and FUGW-TDT-shb2m interference expression plasmid was constructed and transfected into 293T cells mediated by polyethylene polyethylenimine (PEI). The mRNA level of B2m gene in transfected cells was detected by real-time fluorescence quantitative method at 48h after transfection.Results two B2m silencing sites homologous to marmosets were screened. The silencing efficiency of the two targets at the transcription level was 46.54 鹵7.91 (P 0.05) and 83.22 鹵4.3737 (P 0.0001), respectively. The silencing efficiency of the two targets was 46.54 鹵7.91 and 83.22 鹵4.37P0.0001, respectively.Conclusion the recombinant plasmid of FUGW-TDT-shb2m was successfully constructed, and two effective targets of B2m gene silencing were obtained at the cell level, which laid a foundation for the further study on the mediation of marmoset B2m gene silencing.
【作者單位】： 中國醫(yī)學科學院醫(yī)學實驗動物研究所;北京大學生命科學學院生物膜及膜生物工程國家重點實驗室北京大學麥戈文腦研究所;
【基金】：國家科技支撐計劃(2014BAI03B01)
【分類號】：R-332

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