含鸡源启动子的真核表达载体的构建及IBV S1蛋白抗原表位的表达

Construction of eukaryotic expression vector with a chicken-origin promoter and the expressing of S1 protein epitopes in IBV

利用PCR方法扩增出鸡β-肌动蛋白(β-actin)启动子基因序列,克隆至pcDNA3.1和pCIneo载体中以替代CMV启动子,获得含鸡源启动子的真核表达载体pcDNA-act和pCIneo-act。将IBV ZY3S1蛋白抗原表位基因亚克隆到启动子替换前后的质粒中,然后将重组质粒转染至鸡胚成纤维细胞(CEF)中,采用间接免疫荧光试验检测转染细胞对S1蛋白抗原表位的表达。将重组质粒以150μg/只的剂量肌注免疫SPF雏鸡,同时设立IBV灭活疫苗组及PBS空白对照组,经间接ELISA检测免疫前后血清中IBV特异性抗体,最后使用ZY3强毒株给各组实验动物攻毒,观察鸡群的发病死亡情况,计算攻毒保护率。结果显示,替换启动子后的表达载体在CEF中对S1蛋白抗原表位的表达量以及实验动物的抗IBV抗体水平均高于未更换启动子的相应载体,攻毒后鸡群的发病率和死亡率均比替换前降低,说明外源基因的表达和启动子的来源有密切的关系。

In order to study the influence of promoter for exogenous gene expression in eukaryotic expression system,the chickenβ-actin(β-actin)promoter gene sequence was amplified by PCR from chicken peripheral blood lymphocyte and cloned into eukaryotic expression plasmid pcDNA3.1and pCIneo respectively to replace its CMV promoter,getting eukaryotic expression plasmid pcDNA-act and pCIneo-act with a promoter of chicken origin.The S1 protein epitope gene of infectious bronchitis virus(IBV)ZY3strain was subcloned into the eukaryotic expression vectors with or without the replaced promoter,and then transfected them into chicken embryo fibroblasts(CEF).Indirect immunofluorescence assay was used to detect the expression of S1 protein epitopes in transfected cells.Intramuscularly immunized a SPF chicken with 150μg recombinant plasmids,also set up IBV vaccine group and PBS control group.The specific antibody against IBV in the serum of the immunized chickens was measured by indirect ELISA.Finally,use ZY3 virulent strain to attack the animals in each group,observed the incidence and the mortality of chickens,calculated the protection rate.The results showed that the expression of the S1 protein epitopes in transfected cells in which vectors had replaced the promoter was significantly more than that had not been replaced.The antibody titer was significantly improved while incidence and mortality of chickens were reduced after replacing the vectorspromoter.The result declared that the expression of exogenous gene is close related with the source of promoter.