【摘要】：目的 PTD-hBDNF基因的克隆、表達(dá)和表達(dá)產(chǎn)物的純化及生物活性測(cè)定。方法 先從人血白細(xì)胞中提取基因組總DNA,以基因組總DNA為模板,采用PCR技術(shù)擴(kuò)增人BDNF全長(zhǎng)基因,插入到pUC19質(zhì)粒載體,篩選陽(yáng)性克隆,經(jīng)酶切鑒定無(wú)誤后進(jìn)行測(cè)序;以陽(yáng)性克隆重組質(zhì)粒pUC19-hBDNF為模板,再用含PTD編碼序列作為PCR反應(yīng)的5’引物,經(jīng)PCR反應(yīng)擴(kuò)增得PTD-hBDNF融合基因,并插入pUCm-T載體;然后將PTD-hBDNF融合基因克隆至溫控表達(dá)載體pJW2,轉(zhuǎn)化大腸桿菌DH-5 α,42℃誘導(dǎo)表達(dá)。重組蛋白經(jīng)初步復(fù)性純化后,將其加入海馬神經(jīng)元細(xì)胞中進(jìn)行重組蛋白的生物活性測(cè)定。結(jié)果PTD-hBDNF在大腸桿菌中的表達(dá)主要是以包涵體的形式存在。細(xì)菌的菌體蛋白經(jīng)SDS-PAGE分析,在15kD處有一條明顯的蛋白條帶,經(jīng)Western-blot分析表明PTD-hBDNF具有明顯的免疫反應(yīng)。復(fù)性純化后的蛋白質(zhì)純度達(dá)到了90%以上,且具有明顯的生物活性。結(jié)論成功構(gòu)建了含PTD-hBDNF融合基因的表達(dá)載體,并在大腸桿菌中獲得表達(dá)且對(duì)表達(dá)產(chǎn)物進(jìn)行了有效的復(fù)性和純化,得到了具有生物活性的融合蛋白。
[Abstract]:Objective to clone, express and purify PTD-hBDNF gene and determine its biological activity. Methods Total genomic DNA, was extracted from human leukocytes and genomic total DNA was used as template. The full-length gene of human BDNF was amplified by PCR technique and inserted into pUC19 plasmid vector to screen positive clones. The fusion gene of PTD-hBDNF was amplified by PCR reaction and inserted into pUCm-T vector using the positive clone recombinant plasmid pUC19-hBDNF as template and PTD coding sequence as 5 'primer for PCR reaction. Then the PTD-hBDNF fusion gene was cloned into the temperature-controlled expression vector pJW2, and transformed into Escherichia coli DH-5 偽 to induce expression at 42 鈩，

本文編號(hào)：2224596