基于黏蛋白1的非小细胞肺癌基因疫苗的构建及免疫原性评价  

Construction and immunogenicity evaluation of non-small cell lung cancer gene vaccine based on mucin

作　　者：岳红红[1] 赵亮[2] 姜威[2] 

机构地区：[1]武警总医院过敏反应科,北京市100039 [2]武警总医院医务部,北京市100039

出　　处：《广西医学》2016年第9期1197-1200,共4页Guangxi Medical Journal

摘　　要：目的构建基于黏蛋白1(MUC1)的非小细胞肺癌基因疫苗,并对其免疫原性进行评价。方法利用弗林蛋白酶(furin)/2A序列将MUC1基因与粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因进行连接,克隆入真核表达载体p VAX1,构建基因疫苗p VAX1-MUC1-F2A-GM-CSF。对基因疫苗进行鉴定后将该疫苗体外转染COS7细胞,利用流式细胞术和酶联免疫吸附测定法(ELISA)分别对MUC1、GM-CSF的表达情况进行检测。将32只Balb/c小鼠分为空白对照组、空质粒p VAX1组、p VAX1-MUC1组和p VAX1-MUC1-F2A-GM-CSF组,每组8只。除空白对照组外,其余实验组每只小鼠肌肉注射相应质粒100μg,并进行电穿孔刺激,末次免疫后14 d采用ELISA和酶联免疫斑点法分别检测其诱导的体液免疫和细胞免疫反应。结果成功克隆并构建了非小细胞肺癌基因疫苗p VAX1-MUC1-F2A-GM-CSF,流式细胞检测结果显示MUC1在COS7细胞中的阳性表达率为10%,ELISA检测结果显示p VAX1-MUC1-F2A-GM-CSF组GM-CSF表达水平高于空载体p VAX1组(P<0.05)。将疫苗免疫接种小鼠模型后,p VAX1-MUC1-F2A-GM-CSF组诱导的MUC1抗体水平高于p VAX1-MUC1组(P<0.05),p VAX1-MUC1-F2A-GM-CSF组的小鼠脾细胞分泌干扰素-γ的斑点数多于p VAX1-MUC1组(P<0.05)。结论 p VAX1-MUC1-F2A-GM-CSF能够同时诱导较高的体液免疫和细胞免疫反应。Objective To construct the non-small cell lung cancer gene vaccine based on mucin 1 （MUC1）, and to evaluate its immunogenicity. Methods MUC1 gene was fused with granulocyte-macrophage colony-stimulating factor （GM-CSF） by furin/2A sequence, and the fused gene was cloned into pVAXI to construct the gene vaccine pVAX1-MUCI-F2A-GM-CSF. The gene vaccine was transfected into COS7 cells after identification, and then the expressions of MUC1 and GM-CS were detected by flow cytometry and enzyme-linked immunosorbent assay（ELISA） respectively. Thirty-two Balb/c mice were divided into blank control group, pVAX1 group, pVAX1-MUC1 group and pVAX1-MUC1-F2A-GM-CSF group,with 8 mice in each group. Except the blank control group,the corresponding plasmids（ 100 ~g） were injected into the mice of the experimental groups and electroporation stimulation was also performed in each mice. After 14 days of last immunization, the induced humoral immune and cellular immune responses were detected by ELISA and enzyme-linked immunospot assay respectively. Results Non-small cell lung cancer gene vaccine pVAX1-MUC1-F2A-GM-CSF was successfully cloned and constructed. The result of flow cytometry detection showed that MUC1 expressed in COS7 cell and the positive expression rate was 10%. And the result of ELISA showed that the GM-CSF expression level of pVAX1-MUC1-F2A-GM-CSF group was higher than that of pVAX1 group（ P 〈 0.05）. After the mice models injected with the vaccine, the expression of MUC1 antigen induced by pVAX1-MUC1-F2A-GM-CSF group was higher than that induced by pVAX1-MUC1 group（P 〈0.05）,and the spots of interferon-7 secreted by mice splenocytes in the pVAX1-MUC1- F2A-GM-CSF group was more than that in the pVAX1-MUC1 group （P 〈 0. 05 ）. Conclusion The pVAX1-MUC1-F2A-GM-CSF can induce either high humoral immune response or cellular immune response.

关 键 词：非小细胞肺癌 基因疫苗 黏蛋白1 粒细胞-巨噬细胞集落刺激因子 体液免疫 细胞免疫 

分 类 号：R392.11[医药卫生—免疫学]