基于Ii分子的HCV—NS3内源性靶向基因疫苗的构建及免疫应答研究  

Construction and immune responses of HCV-NS3 plasmid vaccine based on invariant chain CLIP substitution

作　　者：高明[1] 王海平[2] 卢媛[1] 周勇[2] 王燕宁[1] 詹林盛[2] 王全立[3] 

机构地区：[1]兰州军区兰州总医院输血科,730050 [2]军事医学科学院输血医学研究所,北京100085 [3]解放军307医院输血科

出　　处：《中华微生物学和免疫学杂志》2007年第3期242-246,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30200245）

摘　　要：目的构建基于Ii分子的HCV内源性靶向基因疫苗并在真核细胞中表达，用构建的基因疫苗免疫BALB／c小鼠，研究免疫后小鼠的体液和细胞免疫应答。方法通过3轮PCR以HCV-NS3的TH1表位（1248～1261AA）取代Ii链CLIP片段编码基因，构建基于Ii分子的HCV内源性靶向基因疫苗，并在Cos-7细胞中表达；30只6～8周龄的雌性BALB／c小鼠随机分为5组，用内源性靶向基因疫苗（pHCV-NS3-TH1）和非靶向疫苗（pHCV-NS3）于股四头肌进行免疫，生理盐水、pCl-neo和pCl-neo-li作为研究对照，经过5次免疫后，取外周血对小鼠的体液免疫进行检测，取小鼠脾脏细胞对细胞免疫进行检测。结果内源性靶向基因疫苗在Cos-7细胞中得到高效表达；对BALB／c小鼠的免疫结果显示，只有HCV-NS3免疫组小鼠可以检测到针对NS3的特异性抗体，抗体滴度达到1／1024；pHCV-NS3和pHCV-NS3-TH1免疫的小鼠都可以检测到CD4^＋细胞的增殖，但pHCV-NS3-TH1免疫的小鼠CD4^＋细胞的增殖强度要明显大于pHCV-NS3免疫的小鼠（P=0．002）；在细胞因子的检测中，只有pHCV-NS3-TH1免疫组小鼠检测到IFN-7，浓度达到33．65pg／ml；只有HCV-NS3免疫的小鼠产生IL-4，浓度达到4．55pg／ml。结论基于Ii分子的HCV内源性靶向基因疫苗能够在真核细胞中表达并刺激小鼠产生CD4^＋TH1类型的细胞免疫，为HCV的疫苗开发提供了一个新思路。Objective To construct a plasmid vaccine based on invariant chain CLIP substitution and explore its immune effect in BALB/c mice. Methods The HCV-NS3 TH 1 plasmid vaccine based on invariant chain CLIP substitution was constructed by three cycles of PCR. The expression of foreign gene was observed in mRNA and protein levels. Thirty female BALB/c mice of 6-8 weeks old were randomly divided into five groups to receive injection with experimental vaccine （ pHCV-NS3, pHCV-NS3-TH 1 ） and experimental controls （ saline, pCI-neo, pCI-nco-li） respectively. After fifth immunization, the humoral and cellular immune responses were esti- mated. Results The HCV-NS3 plasmid vaccine based on ClIP substitution was efficiently expressed in Cos-7 cell line. Specific antibody to HCV-NS3 was detected only in pHCV-NS3 immunized mice and the titers could reach 1/ 1024. CD4^＋ TH cell proliferation was found only in the pHCV-NS3 and pHCV-NS3-TH1 treated groups and the A450 of pHCV-NS3-TH 1 treated group was significant higher than that of pHCV-NS3 treated group （ P =0.002）. Production of IFN-T was examined only in the pHCV-NS3-TH 1 immunized group with the concentration of 33.65 pg/ml. Production of IL-4 was detected only in pHCV-NS3 immunized group tinily with the concentration of 4.55 pg/ml. Conclusion HCV-NS3-TH 1 plasmid vaccine based on CLIP substitution could been expressed efficiently in Mammalia cell line and stimulate CD4^＋ TH1 mediated cellular immune response . It might be a potential candidate for HCV therapeutic vaccine development.

关 键 词：丙型肝炎病毒 质粒疫苗 恒定链 免疫反应 

分 类 号：R686[医药卫生—骨科学]