幽门螺旋杆菌尿素酶纳米抗体UreNb重组质粒的构建及其产物的表达、纯化与鉴定  

Construction of recombinant plasmid of Urease Nanobody UreNb against Helicobacter pylori and expression,purification and identification of its product

作　　者：刘文婧 吕慧玲 王顺娟 冯琳[1] 汤锋[4] 李润乐[1] 胥瑾[1] Liu Wenjing;Lu Huiling;Wang Shunjuan;Feng Lin;Tang Feng;Li Runle;Xu Jin(Department of Basic Medicine,Qinghai University,Xining 810001,China;Tibetan Hospital of Qinghai Province,Xining 810007,China;Qinghai Provincial People's Hospital,Xining 810007,China;Research Center for High Altitude Medicine,Qinghai University,Xining 810001,China)

机构地区：[1]青海大学医学部,西宁810001 [2]青海省藏医院,西宁810007 [3]青海省人民医院,西宁810007 [4]青海大学高原医学研究中心,西宁810001

出　　处：《中国高原医学与生物学杂志》2023年第3期203-210,共8页Journal of Chinese High Altitude Medicine & Biology

基　　金：宁夏回族自治区重点研发计划项目(2020BFG02012);青海省科技厅自然科学基金青年项目(2020-ZJ-963Q)。

摘　　要：目的构建幽门螺旋杆菌尿素酶纳米抗体UreNb重组质粒,在大肠杆菌系统中通过诱导表达目的蛋白并予以纯化,再对表达产物及纯化产物进行鉴定。方法利用DNA重组技术,先后使用两种原核表达载体pET22b和pSumo-mut表达目的蛋白UreNb,构建幽门螺旋杆菌尿素酶纳米抗体UreNb,重组质粒。将构建成功的纳米抗体UreNb重组质粒分别转化至大肠杆菌BL-21、Arctic Express、Rosetta菌株中,采用不同温度经IPTG诱导表达目的蛋白。将成功表达的重组蛋白扩大培养,通过SDS-PAGE法分析其表达蛋白,表达蛋白在沉淀物中即确定为包涵体蛋白。将包涵体蛋白变、复性后,通过镍柱亲和层析法纯化蛋白并用Western Blot法鉴定。结果纳米抗体UreNb目的序列成功插入质粒中,在大肠杆菌表达系统BL-21中以包涵体形式表达(经SDS-PAGE法验证)。经过包涵体复性后纯化,获得大量高纯度目的蛋白pSumo-mut-UreNb,纯化蛋白的分子量约为27 kDa(经Western Blot鉴定)。结论使用pSumo-mut质粒构建的幽门螺旋杆菌尿素酶纳米抗体UreNb能够在原核系统中得到高效表达,通过纯化获得具有活性的纯化蛋白。经鉴定,其大小与理论大小基本相符,可用于后续研究。Objective The prokaryotic expression plasmid of Helicobacter pylori Urease Nanobody UreNb was constructed and purified after the induced expression in Escherichia coli system.The expression product and purified product were identified.Methods Two prokaryotic expression vectors pET22b and pSumo-mut were used to express the target protein UreNb by DNA recombination technique.The recombinant plasmid UreNb was transformed into E.coli BL-21,Arctic Express and Rosetta.The target proteins were induced by IPTG at different temperatures.The successfully-expressed recombinant protein was expanded and cultured.The expression of the protein was analyzed by SDS-PAGE and identified as inclusion body protein.After the inclusion body protein transformation,the protein was purified by nickel column affinity chromatography and identified by Western Blot.Results The target sequence of UreNb Nanobody was successfully inserted into the plasmid and expressed as inclusion bodies in the Escherichia coli expression system BL-21(verified by SDS-PAGE).A large amount of high purity target protein pSumo-mut-UreNb was obtained after the inclusion body purification.The molecular weight of the purified protein was about 27kDa(identified by Western Blot).Conclusion The Helicobacter pylori Urease Nanobody UreNb is constructed by pSum-mut plasmid can be efficiently expressed in the prokaryotic system,and the purified protein with activity can be obtained by purification.The size of the purified protein is basically consistent with the theoretical size,which can be used for subsequent studies.

关 键 词：尿素酶 纳米抗体 原核表达 基因重组 

分 类 号：R392.11[医药卫生—免疫学]