抗多房棘球蚴亮氨酸氨基肽酶纳米抗体筛选及原核表达纯化  

Screening and prokaryotic expression purification of nano antibody against Leucine aminopeptidase of Echinococcus multilocularis

作　　者：戴瑶 刘文婧 肖杨 李润乐[4] 汤锋[1] DAI Yao;LIU Wenjing;XIAO Yang;LI Runle;TANG Feng(Research Center for High Altitude Medicine,Qinghai University,Xining 810001,China;Graduate School of Qinghai University,Xining 810016,China;Tso-Ngon(Qinghai)ProvincialTibetan Medical Hospital,Xining 810007,China;Department of Basic Medicine,Qinghai University,Xining 810001,China)

机构地区：[1]青海大学高原医学研究中心,西宁810001 [2]青海大学研究生院,西宁810016 [3]青海省藏医院,西宁810007 [4]青海大学基础医学部,西宁810001

出　　处：《中国高原医学与生物学杂志》2024年第3期167-178,共12页Journal of Chinese High Altitude Medicine & Biology

基　　金：国家自然科学基金地区项目(81860299,32360192);青海省科技厅应用基础研究项目(2024-ZJ-909)。

摘　　要：目的筛选抗多房棘球蚴亮氨酸氨基肽酶(Em-LAP)纳米抗体并构建该纳米抗体原核表达系统,表达纯化纳米抗体蛋白,获得高特异性抗多房棘球蚴亮氨酸氨基肽酶的纳米抗体。方法利用噬菌体文库展示技术(phage library display technology)筛选亮氨酸氨基肽酶纳米抗体,应用phage-ELISA法检测纳米抗体与靶蛋白的亲和力。将亲和力高的抗体测序后,串联目的序列并优化密码子,合成后接入表达载体pET30a做酶切鉴定。将重组质粒转入大肠杆菌BL21(DE3)菌株,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的蛋白表达,表达的蛋白经金属螯合亲和层析纯化获得目的蛋白LAPNb。结果筛选出抗Em-LAP纳米抗体,构建了重组pET30a-LAPNb质粒,得到了一个大小约为28KD的LAPNb蛋白。结论本研究成功筛选获得了抗Em-LAP纳米抗体,构建了LAPNb原核表达系统,表达纯化了LAPNb蛋白。Objective To screen an anti-leucine aminopeptidase of Echinococcus multilocularis(Em-LAP)nano antibody so as to construct a prokaryotic expression system of the antibody.Therefore,the nano antibody protein was expressed and purified to obtain the nano antibody against leucine aminopeptidase of Echinococcus multilocularis with high specificity.Methods The phage library display technology was utilized for screening a nano antibody against leucine aminopeptidase.The affinity of nano antibody to the target protein was verified by phageELISA.After sequencing the antibody with high affinity,the target sequence was concatenated and the codon was optimized.After synthesis,it was inserted into the expression vector pET30a for enzyme digestion identification.The recombinant plasmid was transferred into Escherichia coli BL21(DE3)strain,and isopropyl-β-D-thiogalactopyranoside(IPTG)was used to induce the expression of the target protein.The recombinant target protein LAPNb was purified by metal-chelating affinity chromatography.Results The anti-Em-LAP nano antibody was screened,and the recombinant pET30a-LAPNb plasmid was constructed.A protein with a size of about 28 KD was obtained.Conclusion The study screened and obtained anti-Em-LAP nano antibody,and constructed a LAPNb prokaryotic expression system.The protein LAPNb was expressed and purified.

关 键 词：多房棘球蚴 纳米 抗体 亮氨酸氨基肽酶 噬菌体文库展示技术 

分 类 号：R392.11[医药卫生—免疫学]