论文部分内容阅读
目的构建A型肉毒毒素轻链表达载体重组质粒,在大肠埃希菌中表达后,利用金属螯合层析柱纯化。方法以p GEM-Bo NT/AL轻链质粒为模板,PCR扩增Bo NT/AL基因,克隆至表达载体p ET-28(a)中,构建重组质粒p ET-28(a)-Bo NT/AL,转化E.coli BL21(DE3),分别于37、30、25℃IPTG诱导表达,表达产物经Chelating Sepharose 4Fast Flow(Cu~(2+))层析柱纯化,纯化产物经尿素梯度复性。结果质粒p ET-28(a)-Bo NT/AL经酶切鉴定及测序证明构建正确;表达的重组蛋白相对分子质量约52 000,主要以包涵体形式存在,37℃诱导表达量最高,占菌体沉淀总蛋白的41%;包涵体经2%Trition-X100和2 mol/L尿素洗涤后,可去除部分杂蛋白,8 mol/L尿素能溶解大部分包涵体;经再次浓缩后,蛋白浓度可达89μg/ml,纯度达90%。结论成功克隆及表达了Bo NT/A轻链基因,为制备抗Bo NT/A轻链单链抗体以及研究肉毒中毒机制和治疗方案奠定了基础。
Objective To construct recombinant plasmid of botulinum toxin type A light chain expression vector and express it in Escherichia coli after purification by metal chelate chromatography. Methods The BoNT / AL gene was amplified by PCR using p GEM-Bo NT / ALV light chain plasmid as a template and cloned into the expression vector p ET-28 (a) to construct the recombinant plasmid p ET-28 (a) -Bo NT / AL, transformed into E.coli BL21 (DE3), induced by IPTG at 37, 30, 25 ℃, respectively. The expressed product was purified by Chelating Sepharose 4 Fast Flow (Cu 2+) Sex. Results The recombinant plasmid p ET-28 (a) -Bo NT / AL was confirmed by restriction enzyme digestion and sequencing. The recombinant protein had a relative molecular mass of about 52 000, which was mainly in the form of inclusion body. The highest expression level was induced at 37 ℃ After the inclusion bodies were washed with 2% Trition-X100 and 2 mol / L urea, some of the extracellular proteins could be removed and 8 mol / L urea could dissolve most of the inclusion bodies. After reconcentration, the protein Concentration of up to 89μg / ml, purity of 90%. Conclusion The BoNT / A light chain gene was successfully cloned and expressed, which laid the foundation for the preparation of anti-BoNT / A light chain single chain antibody and study on the mechanism and treatment of botulism.