SARS冠状病毒spike蛋白受体结合域的表达及S1抗原表位多肽多克隆抗体制备

发布时间：2018-02-24 02:20

  本文关键词： SARS Spike蛋白 受体结合域 表达 多肽 抗体　出处：《广州医学院》2010年硕士论文　论文类型：学位论文

【摘要】：目的 利用大肠杆菌BL21原核表达系统对spike蛋白的受体结合域(RBD)片段进行融合表达,为进一步研究spike蛋白免疫原性及抗原特性提供了物质基础,同时建立HIS融合表达实验室系统;运用Fmoc技术合成纯化抗原多肽,免疫小鼠获得多克隆抗体,为进一步研究抗体特性并应用于SARS疾病的预防、预后诊断及治疗提供了物质基础。 方法 1、构建PET32(a)-RBD重组质粒进行原核表达依赖高效T4连接酶连接PET32(a)和RBD,转化感受态BL21大肠杆菌。提取质粒酶切电泳初步确定获得PET32(a)-RBD重组质粒,送TaKaRa公司测序。以IPTG诱导BL21大肠杆菌对融合蛋白进行表达,运用聚丙烯酰胺凝胶电泳及Western-blotting来检测表达产物,保存菌种。 2.SARS-CoV的S1蛋白抗原表位多肽合成及其抗体的制备 以DNAstar软件对S1蛋白RBD片段的原始序列进行软件分析,选择三段亲水性强,抗原指数高的多肽,运用Fmoc固相法合成三条肽链:CQ19(CFSNVYADSFVVK GDDVRQ)、TY-14(TRNIDATSTGNYNY)和LV-11 (LRPFERDISNV),分别与KLH和BSA偶联成三条具有免疫原性多肽:KLH(BSA)-CQ19 KLH(BSA)-LV11 KLH(BSA)-TY14,免疫BABL/c小鼠剪尾取血检测抗体产生情况(ELISA法)。小鼠在第一次免疫注射之前,首先剪尾取血,该血清作为阴性对照加0.05% NaN3-70℃保存。 结果 1、PET32(a)-RBD重组质粒构建及其表达: (1)重组质粒构建及鉴定:琼脂糖凝胶电泳及测序表明重组质粒PET32(a)-RBD构建成功。 (2)IPTG诱导融合蛋白表达:SDS-PAGE及Western-blotting实验表明融合蛋白在IPTG诱导下大量表达,未经IPTG诱导的细菌对融合蛋白几乎不表达。(见图1.3)。 2、合成多肽制备抗体: (1)分析S蛋白:根据DNAstar软件分析筛选S蛋白三段多肽:CQ19(378～396AA)、TY14(425～438AA)、LV11(448～458AA)具有较强抗原性和亲水性。 (2)多肽合成:氨基酸组分析表明合成正确序列CQ19、TY14、LV11多肽。 (3) KLH-CQ19免疫小鼠获得抗CQ19多克隆抗体:ELISA检测血清结果表明抗体产生为弱阳性。1:10~3和1:10~4滴度OD值约等于阴性对照2倍。 (4) KLH-LV11、KLH-TY14免疫小鼠均无抗体产生。 结论 1、本研究构建了PET32(a)-RBD重组质粒,获得融合蛋白在IPTG诱导下的稳定表达,建立本实验室PET32(a)载体融合的平台,为进一步对SARS-CoVspike蛋白的研究奠定了基础,但其纯化效果及蛋白在纯化后所具有的免疫特性还有待进一步研究。 2、应用软件分析技术,合成高纯度抗原多肽片断,免疫动物实验可获得抗CQ19多克隆抗体,为抗spike蛋白抗体的后续研究及临床应用抗体治疗及预防SARS疾病提供了可能。
[Abstract]:Purpose. E. coli BL21 prokaryotic expression system was used to express the receptor binding domain of spike protein, which provided a material basis for the further study of immunogenicity and antigenic characteristics of spike protein. At the same time, the HIS fusion expression laboratory system was established. Fmoc technique was used to synthesize and purify antigenic polypeptides and to immunize mice to obtain polyclonal antibodies, which provided a material basis for further study of antibody characteristics and application in the prevention, prognosis diagnosis and treatment of SARS diseases. Method. 1. The PET32(a)-RBD recombinant plasmid was constructed for prokaryotic expression dependent on high efficient T4 ligase ligation with PET32A) and RBD. the recombinant plasmid was obtained by restriction endonuclease digestion, and the recombinant plasmid was obtained by restriction endonuclease electrophoresis, and the recombinant plasmid was transformed into BL21 Escherichia coli. The fusion protein was expressed in BL21 Escherichia coli induced by IPTG. Polyacrylamide gel electrophoresis and Western-blotting were used to detect the expressed product and preserve the strain. 2. Synthesis of SARS-CoV S _ 1 protein epitope peptide and preparation of its antibody. The original sequence of S1 protein RBD fragment was analyzed by DNAstar software. Three segments of polypeptides with strong hydrophilicity and high antigen index were selected. Three peptide chains: CQ19, CFSNVYADSFVVK GDDVRQK TY-14 TRNIDATSTGNYYNYY) and LV-11 LRPFERDISNVY were synthesized by Fmoc solid state method. Three immunogenicity polypeptides, KLHBSA-CQ19 KLH(BSA)-LV11 KLHSABSA-TY14, were coupled with KLH and BSA respectively. First, the blood was taken from the tail, and the serum was stored as a negative control at 0.05% NaN3-70 鈩，

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