本文選題：委內(nèi)瑞拉馬腦炎病毒 + 原核表達(dá)��； 參考：《中國(guó)獸醫(yī)學(xué)報(bào)》2017年11期

【摘要】：原核表達(dá)并純化委內(nèi)瑞拉馬腦炎病毒(Venezuelan equine encephalitis virus,VEEV)E2蛋白胞外區(qū),并以此為抗原免疫動(dòng)物制備多克隆抗體。利用PCR擴(kuò)增VEEV E2蛋白胞外區(qū)基因并將其插入到原核表達(dá)載體pET-30a(+),構(gòu)建重組質(zhì)粒pET-30a(+)-VEEV-E2,轉(zhuǎn)化至大腸桿菌BL21(DE3),IPTG誘導(dǎo)目的蛋白表達(dá)。優(yōu)化誘導(dǎo)條件并對(duì)目的蛋白進(jìn)行親和純化,用純化后的蛋白免疫BALB/c小鼠制備多克隆抗體,通過(guò)間接免疫熒光方法檢測(cè)抗體的結(jié)合能力。通過(guò)PCR擴(kuò)增出約為1 023bp的VEEV E2基因胞外區(qū),成功構(gòu)建重組表達(dá)質(zhì)粒pET-30a(+)-VEEV-E2,在37℃,0.6mmol/L IPTG誘導(dǎo)3h條件下目的蛋白表達(dá)量最高且主要以包涵體形式存在;將純化的目的蛋白免疫BALB/C小鼠成功制備了抗VEEV E2蛋白胞外區(qū)鼠源多克隆抗體,經(jīng)間接免疫熒光鑒定制備的抗體能夠特異性識(shí)別真核表達(dá)的VEEV E2蛋白。VEEV E2基因胞外區(qū)在大腸桿菌中獲得穩(wěn)定表達(dá),且表達(dá)的目的蛋白具有良好的免疫原性,為VEEV快速診斷方法及新型疫苗的研究奠定了基礎(chǔ)。
[Abstract]:The extracellular domain of Venezuela equine encephalitis virus (Venezuelan equine encephalitis virus) VEEV E 2 protein was expressed and purified in prokaryotic cells and used as antigen to immunize animals to prepare polyclonal antibodies. The extracellular domain gene of VEEV E2 protein was amplified by PCR and inserted into the prokaryotic expression vector pET-30a.Recombinant plasmid pET-30a (-VEEV-E2) was constructed and transformed into E. coli BL21DE3 / IPTG to induce the expression of the target protein. The BALB/c mice were immunized with the purified protein to prepare polyclonal antibody and the binding ability of the antibody was detected by indirect immunofluorescence method. The extracellular region of VEEV E2 gene was amplified by PCR, and the recombinant expression plasmid pET-30a (-VEEV-E2) was successfully constructed. The expression of the target protein was the highest at 37 鈩，

本文編號(hào)：1919794