本文選題：大鼠β-防御素- + 基因轉(zhuǎn)染�。� 參考：《中國現(xiàn)代醫(yī)學(xué)雜志》2010年13期

【摘要】：目的構(gòu)建大鼠β-防御素-2(rBD2)基因的真核表達(dá)載體pCAGG-rBD2,并轉(zhuǎn)染小鼠纖維母細(xì)胞(L929),為解決細(xì)菌耐藥性問題提供進(jìn)一步的實(shí)驗(yàn)基礎(chǔ)。方法以大鼠肺組織總RNA為模板,采用RT-PCR的方法獲得β-防御素-2基因cDNA,將其克隆到pGEM-T Easy載體并構(gòu)建含有目的片段的真核表達(dá)載體pCAGG-rBD2,經(jīng)脂質(zhì)體介導(dǎo)轉(zhuǎn)染L929細(xì)胞,通過RT-PCR和Western blotting檢測目的基因表達(dá)情況。結(jié)果成功克隆并構(gòu)建了含有目的基因rBD2的真核表達(dá)載體pCAGG-rBD2,轉(zhuǎn)染pCAGG-rBD2的細(xì)胞中檢測到了rBD2基因在轉(zhuǎn)錄水平和蛋白水平的表達(dá),其細(xì)胞培養(yǎng)的上清液對金黃色葡萄球菌具有殺滅作用。結(jié)論防御素在真核表達(dá)系統(tǒng)的成功表達(dá),為進(jìn)一步開發(fā)高效能殺滅耐藥性致病微生物的多肽類新藥提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。
[Abstract]:Objective to construct the eukaryotic expression vector pCAGG-rBD2 of rat 尾 -defensin -2rBD2 gene and transfect it into mouse fibroblast cell line L929 to provide a further experimental basis for solving the problem of bacterial resistance. Methods using total RNA of rat lung tissue as template, 尾 -defensin-2 gene cDNAs were obtained by RT-PCR, cloned into pGEM-T Easy vector, constructed eukaryotic expression vector pCAGG-rBD2containing the target fragment, and were transfected into L929 cells by liposome-mediated transfection. The expression of target gene was detected by RT-PCR and Western blotting. Results the eukaryotic expression vector pCAGG-rBD2 containing the target gene rBD2 was cloned and constructed successfully. The expression of rBD2 gene at transcription and protein levels was detected in the transfected pCAGG-rBD2 cells. The supernatant of the cell culture could kill Staphylococcus aureus. Conclusion the successful expression of defensin in eukaryotic expression system provides experimental and theoretical basis for the further development of new polypeptide drugs with high efficacy in killing drug-resistant pathogenic microorganisms.
【作者單位】： 甘肅省新藥臨床前研究重點(diǎn)實(shí)驗(yàn)室(蘭州大學(xué)基礎(chǔ)醫(yī)學(xué)院病理生理學(xué)研究所);
【基金】：甘肅省新藥臨床前研究重點(diǎn)實(shí)驗(yàn)室開放基金(No:GSKFKT-0703)
【分類號】：Q786
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本文編號：1944460