重组人HER3胞外域Ⅰ区183-227aa的原核表达及大鼠多克隆抗体的制备与鉴定  

Bacterial expression of 183-227aa region of HER3 extracellular domain I and preparation and identification of its polyclonal antibodies

作　　者：朱磊 袁平川[1,2,3] 赵志刚 王鑫 王国栋[1,2,3] 颜亮 ZHU Lei;YUAN Pingchuan;ZHAO Zhigang;WANG Xin;WANG Guodong;YAN Liang(Anhui Provincial Engineering Research Center for Polysaccharide Drugs,Wannan Medical College,Wuhu 241002,China;Drug Research&Development Center,Wannan Medical College,Wannan Medical College,Wuhu 241002,China;Anhui Province Key Laboratory of Active Biological Macromolecules,Wannan Medical College,Wuhu 241002,China;Research Institute for Pharmaceutical Screening&Evaluation,Wannan Medical College,Wuhu 241002,China)

机构地区：[1]安徽省多糖药物工程技术研究中心,安徽芜湖241002 [2]皖南医学院药物研发中心,安徽芜湖241002 [3]活性生物大分子研究安徽省重点实验室,安徽芜湖241002 [4]皖南医学院药物筛选与评价研究所,安徽芜湖241002

出　　处：《南方医科大学学报》2020年第6期806-813,共8页Journal of Southern Medical University

基　　金：国家自然科学基金(81802651);安徽省自然科学基金(1808085QH291,1908085MH248);安徽省高校自然科学基金(KJ2018A0255,KJ2018ZD025);活性生物大分子研究安徽省重点实验室自主研究课题(LAB201801,LAB201802);皖南医学院大学生科研资助金(WK2019S43)。

摘　　要：目的原核表达、纯化由人表皮生长因子受体3(HER3)183-227aa肽段(HER3Ⅰ)和麻疹病毒蛋白288-302aa肽段(MVF)融合构建的重组肽(MVF-HER3Ⅰ),并制备其多克隆抗体。方法将MVF与HER3Ⅰ融合构建重组肽MVF-HER3Ⅰ,用化学合成法合成重组肽基因,并将其与pET21b质粒和含有硫氧还蛋白(Trx)基因的pET32a质粒连接构建重组肽表达质粒,重组质粒用双酶切法鉴定。将重组质粒导入到大肠杆菌BL21(DE3)并进行表达参数优化及诱导表达。融合蛋白依次经镍离子亲和层析、肠激酶酶切和再次镍离子亲和层析制备重组肽。表达和纯化过程中的样品纯度和相对分子质量用SDS-PAGE分析。以纯化的重组肽为抗原免疫大鼠制备多克隆抗体。用ELISA法测定抗重组肽多克隆抗体效价,免疫印迹和免疫沉淀法分析抗重组肽多克隆抗体对重组肽和非变性状态HER3的识别,激光共聚焦法分析抗重组肽多克隆抗体与MCF7细胞的结合,磺酰罗丹明B染色法测定在有无神经调节素刺激下抗重组肽多克隆抗体对高表达HER3的MCF7细胞的增殖抑制作用。结果成功构建了重组肽的两种表达质粒,重组肽基因不能单独表达,但和Trx融合后可在37℃、0.2 mmol/L IPTG条件下呈可溶高表达。融合蛋白经亲和层析、酶切和再次亲和层析后得到重组肽。重组肽可刺激大鼠产生效价达1:512000的抗重组肽多克隆抗体。该抗体不仅可特异性识别重组肽,还可特异性沉淀非变性HER3和结合于MCF7细胞的细胞膜。不管有无神经调节素刺激,该抗体均呈浓度依赖性地抑制HER3高表达的MCF7细胞的增殖(P<0.01)。结论成功进行了重组肽MVF-HER3Ⅰ的原核表达和纯化,制备并鉴定了抗MVF-HER3Ⅰ多克隆抗体,为在体内外深入研究该抗体对HER3信号通路的影响打下基础。Objective To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3(HER3 I)and the measles virus protein 288-302 peptide segment(MVF),and prepare polyclonal antibodies(PcAb)against this recombinant peptide.Methods The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin(Trx)tag gene.The recombinant plasmids were identified by endonuclease digestion.MVF-HER3 I was expressed in E.coli BL21(DE3)cells under an optimal bacterial expression condition.The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography,and the purified protein was digested by enterokinase to remove Trx tag.The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I.The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb.The titer of PcAb was determined using ELISA.The bindings of anti-MVF-HER3 I PcAb to MVF-HER3 I,native HER3 and MCF7 cells were analyzed using immunoblotting,immunoprecipitation and laser confocal microscopy.The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B.Results The recombinant peptide gene could not be expressed alone,but could be efficiently expressed after fusion with Trx gene under optimized conditions.The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I.The anti-MVF-HER3 I PcAb,with a titer reaching 1:512000,specifically bound to MVF-HER3 I,recognized native HER3 and bound to the membrane of MCF7 cells.The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG.Conclusion We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb,which can facilitate further functional analysis of HER3 signaling pathway.

关 键 词：人表皮生长因子受体3 二聚化 融合表达 亲和层析 多克隆抗体 

分 类 号：R392-33[医药卫生—免疫学]