CAJ | 학술논문

目的设计多型别HCV-E1表位复合免疫原,通过免疫小鼠,探讨其在丙型肝炎治疗性疫苗与诊断试剂研究中的应用.方法分析比较HCV-E1包膜糖蛋白B细胞表位序列,选取几个代表性基因型的中和性优势抗原表位,再结合泛DR辅助性T细胞表位(PADRE),构建含有不同HCV型别E1表位抗原基因和通用T辅助细胞表位基因的原核表达质粒.结果成功构建了含有HCV 1a、1b、2a、3a、4a和6a等基因型E1中和性表位以及通用T辅助细胞表位的质粒pBVIL1/E1s-PADRE,该质粒转化大肠杆菌后获得的工程菌,通过诱导培养可以高效表达重组多型别HCV-E1表位复合抗原,所获得的多型别HCV-E1表位复合免疫原可在BALB/c小鼠体内诱发强烈的体液免疫反应,ELISA检测抗体水平可达到1:12 800.结论新构建的HCV-E1表位复合免疫原具有很好的免疫原性,为进一步研究HCV疫苗奠定了基础.
목적 설계다형별HCV-E1표위복합면역원,통과면역소서,탐토기재병형간염치료성역묘여진단시제연구중적응용.방법 분석비교HCV-E1포막당단백B세포표위서렬,선취궤개대표성기인형적중화성우세항원표위,재결합범DR보조성T세포표위(PADRE),구건함유불동HCV형별E1표위항원기인화통용T보조세포표위기인적원핵표체질립.결과 성공구건료함유HCV 1a、1b、2a、3a、4a화6a등기인형E1중화성표위이급통용T보조세포표위적질립pBVIL1/E1s-PADRE,해질립전화대장간균후획득적공정균,통과유도배양가이고효표체중조다형별HCV-E1표위복합항원,소획득적다형별HCV-E1표위복합면역원가재BALB/c소서체내유발강렬적체액면역반응,ELISA검측항체수평가체도1:12 800.결론 신구건적HCV-E1표위복합면역원구유흔호적면역원성,위진일보연구HCV역묘전정료기출.
Objective To design a complex hepatitis C vires(HCV)=E1 antigen,and to search its application in HCV vaccine and diagnosis test.Methods Through consulting the database and widely comparison of sequences from HCV E1 of different genotype,the representative immunodominant epitope sequences were selected from all the six genotypes.Their genes were deduced according to the preference codon in E.coli and three fragments were designed to contain the six epitopes.They were chemically synthesized and cloned into pBVIL1 vector separately.The cloned fragments were conjugated together profiting from pBVIL1's property,and then a doubled pan-DR helper T cell epitopes(PADRE)gene was inserted to form a complex expression plasmid.The transformed E.coli cells with this plasmid were cultured and induced to express recombinant protein and the antigenic activity of the product was tested.Results An expressing plasmid containing 8 epitopes from HCV genotype 1a,1b,2a,3a,4a,6a and a doubled PADRE sequences was constructed successfully and the engineering E.coli transformed with this plasmid highly expressed after inducing culture at 42℃ in an inclusion manner.The immunological activity of the purified recombinsnt multi-epitope antigen shows that:(1)It can react with a great part of sera from HCV positive patients by indirect ELISA.(2)It can induce notable specific humoral immunity in injected mice.Conclusion The novel constructed expressing plasmid and its product may be useful in study of a newly HCV vaccine as well as an antigen for HCV immunoassays.