【摘要】：根據(jù)NCBI中的人胰島素原基因序列及大腸桿菌密碼子偏愛性設(shè)計(jì)特異引物,PCR擴(kuò)增得到人胰島素原基因,構(gòu)建該基因的原核表達(dá)質(zhì)粒p ET32-PI,重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3)。對(duì)重組菌進(jìn)行溫度和IPTG濃度優(yōu)化,發(fā)現(xiàn)重組菌在30℃和終濃度為0.6 mmol/L的IPTG條件下表達(dá)效果最好且沒有包涵體,經(jīng)SDS-PAGE和Westem blot檢測(cè),表達(dá)蛋白的相對(duì)分子質(zhì)量與理論相對(duì)分子質(zhì)量一致且具有胰島素的免疫原性,證明胰島素原基因得到了正確表達(dá)。對(duì)重組菌進(jìn)行流加發(fā)酵,發(fā)酵液離心收集菌體,破菌后上清液過(guò)親和層析柱和離子交換柱分離純化目的蛋白,透析后的樣品用腸激酶和羧肽酶酶切,利用親和柱去除融合蛋白與His標(biāo)簽,最終分離胰島素樣品,利用免疫酶標(biāo)法,l m L樣品測(cè)得活性92μIU,證明實(shí)驗(yàn)所得樣品具有人胰島素活性。
[Abstract]:According to the sequence of human proinsulin gene in NCBI and the preference of Escherichia coli codon, a specific primer was designed, and the proinsulin gene was amplified by PCR. The prokaryotic expression plasmid p ET32-PI, recombinant plasmid was constructed and transformed into Escherichia coli BL21 (DE3). By optimizing the temperature and IPTG concentration of the recombinant bacteria, it was found that the recombinant strain expressed best at 30 鈩，

本文編號(hào)：2376028