人乳头瘤病毒L1基因与丹毒丝菌SpaA-N优势抗原表位序列的嵌合重组质粒构建及活性鉴定  

Construction and Active Identification of Recombinant Plasmid with Chimeric Human Papillomavirus Type 16 L1 Expressing an Epitope of Erysipelothrix rhusiopathiae SpaA-N

作　　者：庆格乐图[1] 杨薇薇[1] 晏鹏飞[1] 苏幼红[1] 李江伟[1] 

机构地区：[1]新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《生物技术通报》2011年第10期145-150,共6页Biotechnology Bulletin

基　　金：新疆自治区高校科研计划重点项目(XJEDU2005105)

摘　　要：为确定丹毒丝菌表面保护性抗原A的N-末端(SpaA-N)优势抗原表位,研发新型表位DNA嵌合疫苗,利用生物信息学软件对丹毒丝菌SpaA-N的优势B细胞抗原表位进行预测,以重叠PCR将优势表位插入人乳头瘤病毒16型的主要衣壳蛋白(HPV-16 L1)HI环结构的编码序列中,构建获得重组嵌合质粒pcDNA3-HPV-L1-ΔspaA。该重组质粒经体内、外瞬时转染后,RT-PCR均扩增获得了1 500 bp左右的目的片段。免疫印迹试验显示,体外转染嵌合质粒的细胞总蛋白能够与GST-SpaA-N重组蛋白免疫血清在56 kD处产生特异性结合。ELISA分析显示嵌合质粒可在小鼠体内产生差异显著的特异性抗体(P<0.01),抗体滴度为1∶1 000。此外,pcDNA3-HPV-L1-ΔspaA制备的抗血清至少可在1∶10稀释度条件下,介导外源补体对半数以上的菌体进行杀伤。该研究表明,获得的SpaA-N表位DNA嵌合疫苗具有免疫活性,为研发安全有效的丹毒丝菌新型DNA疫苗提供了一个新思路。To comfirm the B cell epitope of N-terminal of surface protective antigen A（SpaA-N） from Erysipelothrix rhusiopathiae and develop an efficient DNA vaccine,an experimental DNA plasmid was constructed based on the predicted B cell epitope derived from SpaA-N,and then the predominant epitope was inserted into the coding sequence of human papillomavirus type 16 major capsid protein（HPV-16 L1）HI loop by overlap PCR.pcDNA3-HPV-L1-ΔspaA were transfected in vitro and in vivo,the results of RT-PCR showed that the 1 500 bp round products were amplified,while Western blotting illustrated the total proteins of cells which transfected pcDNA3-HPV-L1-ΔspaA plasmid can bound with mouse against GST-SpaA-N antiserum at 56 kD specifically.Antiserum from mice immunized with pcDNA3-HPV-L1-ΔspaA showed could bind to SpaA-N recombinant protein in ELISA detection,and the titer of antibody was 1∶ 1 000（P0.01）.In addition,in vitro bactericidal assay showed the antiserum from pcDNA3-HPV-L1-ΔspaA immunized mice mediated efficient killing of Erysipeolothrix rhusiopathiae by addition of complements at lest in dilution of 1∶ 10.This results indicated that the DNA vaccine with chimeric human papillomavirus L1 expressing an epitope of SpaA could induce efficient immune response to Erysipeolothrix rhusiopathiae infection in mouse,and provided a selective strategy for developing a safer and more efficient DNA vaccine to control Erysipeolothrix rhusiopathiae infection.

关 键 词：丹毒丝菌 SpaA-N 表位预测 HPV-16 L1嵌合质粒 瞬时表达 杀菌活性 

分 类 号：R392.1[医药卫生—免疫学]