【摘要】：通過RT-PCR從PK-15細(xì)胞系中擴增克隆MAVS(mitochondrial antiviral signaling protein)基因,構(gòu)建原核表達載體p ET-MAVS220,轉(zhuǎn)化感受態(tài)細(xì)胞Rosetta(DE3),利用IPTG誘導(dǎo)表達,重組MAVS經(jīng)純化后免疫4周齡昆明系小鼠制備抗線粒體抗病病毒信號蛋白(MAVS)多克隆抗體。誘導(dǎo)表達的最佳條件為IPTG 0.05 mmol/L,37℃誘導(dǎo)6 h,重組MAVS以可溶性蛋白和包涵體兩種形式表達。應(yīng)用該重組蛋白免疫小鼠獲得的抗MAVS多克隆抗體與純化的重組MAVS蛋白反應(yīng)效價可達1∶16 000;該抗體與Poly(I∶C)刺激PK-15細(xì)胞產(chǎn)生的MAVS及與轉(zhuǎn)染了重組MAVS基因真核表達載體pcDNA3.0-MAVS的BHK-21細(xì)胞表達的MAVS蛋白發(fā)生特異性反應(yīng),效價可達1∶1 000,特異性良好。
[Abstract]:The MAVS (mitochondrial antiviral signaling protein) gene was amplified and cloned from PK-15 cell line by RT-PCR, and the prokaryotic expression vector p ET-MAVS220, was constructed to transform the receptive cell Rosetta (DE3). The recombinant MAVS was induced by IPTG and immunized with recombinant MAVS for 4 weeks old Kunming mice to prepare polyclonal antibody against mitochondrial antiviral signal protein (MAVS). The best conditions for inducing expression were IPTG 0.05 mmol/L,37 鈩，

本文編號：2500667