本文選題：凋亡素 + 雞貧血病毒��； 參考：《中國(guó)生物工程雜志》2017年02期

【摘要】：為獲得高效可溶表達(dá)的雞貧血病毒凋亡素基因(CAV-Apoptin),根據(jù)大腸桿菌密碼子偏愛(ài)性?xún)?yōu)化CAV-Apoptin基因序列,將其連接到含msyB伴侶基因的pBCX載體上,轉(zhuǎn)化至大腸桿菌BL21(DE3)進(jìn)行誘導(dǎo)表達(dá),經(jīng)SDS-PAGE鑒定后應(yīng)用鎳柱親和層析純化蛋白質(zhì),采用顯微鏡觀察、CCK-8實(shí)驗(yàn)與DNA ladders測(cè)定其抗腫瘤細(xì)胞的活性。結(jié)果顯示,成功構(gòu)建大腸桿菌表達(dá)載體pBCX-CAV-Apoptin,在37℃正常培養(yǎng)條件下的大腸桿菌中得到大量可溶性表達(dá)產(chǎn)物,融合蛋白質(zhì)分子量大小約為42 kDa,50 ml菌液即可獲得1.5 mg左右的純化重組蛋白,CCK-8實(shí)驗(yàn)結(jié)果顯示純化后的重組蛋白作用36 h后可對(duì)套氏淋巴瘤JEKO-1、REC-1細(xì)胞生長(zhǎng)產(chǎn)生超過(guò)60%的抑制率;顯微鏡觀察與DNA ladder實(shí)驗(yàn)證明重組蛋白可誘導(dǎo)JEKO-1、REC-1細(xì)胞的凋亡,對(duì)HUVEC沒(méi)有誘導(dǎo)凋亡的作用。免疫印跡分析表明重組凋亡素對(duì)JEKO-1細(xì)胞誘導(dǎo)了Caspase-3的激活,而對(duì)Caspase-8的表達(dá)沒(méi)有影響。研究表明融合蛋白msyB-CAV-Apoptin可達(dá)到高效可溶表達(dá),且表達(dá)產(chǎn)物具有特異抗腫瘤活性。
[Abstract]:In order to obtain the highly soluble chicken anemia virus apoptin gene (CAV-Apoptin), the CAV-Apoptin gene sequence was optimized according to the preference of Escherichia coli codon, and ligated to pBCX vector containing msyB chaperone gene, and transformed into E. coli BL21 (DE3) for induction expression. The protein was purified by nickel column affinity chromatography after SDS-PAGE. The activity of CCK-8 and ladders was observed by microscope. The results showed that the expression vector pBCX-CAV-Apopin was successfully constructed, and a large number of soluble expression products were obtained in E. coli at 37 鈩，

本文編號(hào)：2050492