5种腺病毒通用型抗原表位融合蛋白的设计制备及检测试剂的研发

A Chimetric Protein of Universal Antigenic Epitopes from Five Adenovirus and its Application for Development of Diagnostic Reagent

人腺病毒(Human adenovirus,HAd Vs)可引起包括急性呼吸道感染、胃肠炎、流行性角结膜炎等多种人群疾病。其中儿童、新兵及免疫力低下人群是最易感人群。到目前为止,HAd V-B3、B4、B7和新近出现的14型、55型等基因型,常导致严重肺炎。目前美国FDA批准在17~50岁的军人中使用口服4型和7型腺病毒活疫苗,大量的临床试验和正在进行的发热性呼吸道疾病监测均证明此口服活疫苗是安全和高效的。然而,目前没有批准在儿童和成人中普遍使用的疫苗。六邻体蛋白是腺病毒主要的抗原蛋白,该蛋白前中段含有大量的保守区,有很好的抗原性,是制备通用型腺病毒疫苗的重要候选蛋白,也是研制抗体检测试剂的理想抗原。本研究通过计算机分析,从六邻体蛋白内筛选出高度保守的抗原表位,将多个抗原表位串联成融合蛋白。利用基因工程技术,构建了高表达抗原表位融合蛋白的工程菌,纯化获得了表达的融合蛋白,并建立了融合蛋白的复性方法。复性的融合蛋白配伍不同的免疫佐剂分别免疫小鼠,检测抗血清免疫效价及相关细胞因子水平,评价其体液免疫和细胞免疫特性,结果,免疫后的小鼠产生了高效价、高特异性的血清抗体,并引起了较强的细胞免疫,显示表达制备的抗原表位融合蛋白可以引起体液免疫和细胞免疫,有希望用于研制通用型腺病毒疫苗。用制备的表位融合蛋白作抗原,研制的腺病毒抗体免疫层析检测试剂,具有很好的检出率和特异性。

Human adenovirus(Human adenovirus,HAd Vs)can cause a variety of human diseases,including acute respiratory infection,gastroenteritis,epidemic kerato conjunctivitis and so on.Among them,children,recruits and people with low immunity are the most susceptible groups.So far,there are HAd V-B3,B4,B7 and the newly emerging 14 and 55,which often lead to severe pneumonia.At present,FDA of the United States has approved the use of live oral adenovirus vaccine types 4 and 7 in soldiers aged 17~50.A large number of clinical trials and ongoing surveillance of febrile respiratory diseases have proved that this live oral vaccine is safe and effective.However,there is currently no approved vaccine for universal use in children and adults.Hexon protein is the main antigen protein of adenovirus.The anterior and middle part of the protein contains a large number of conservative regions and has good antigenicity,so it is an important candidate protein for the preparation of universal adenovirus vaccine.In this study,highly conserved antigenic epitopes are screened from Hexon proteins by computer analysis,and multiple antigenic epitopes are connected to form fusion proteins.By using genetic engineering technique,the engineering bacteria with high expression of antigen epitope fusion protein are constructed,the expressed fusion protein is purified,and the renaturation method of the fusion protein is established.Mice were immunized with renatured fusion protein combined with different immune adjuvants,the immune titer of antiserum and the content of related cytokines are detected,and the characteristics of humoral and cellular immunity are evaluated.The immunized mice produced high titer and high specificity of serum antibodies and caused strong cellular immunity,indicating that the expressed antigen epitope fusion protein could induce humoral immunity and cellular immunity.It is expected to be used in the development of universal adenovirus vaccine.The prepared epitope fusion protein is used to develop adenovirus antibody immunochromatographic detection reagent with high sensitivity and specificity.