中国图书分类号R322.47R512.63R392.1

摘要目的：探讨HCV核心区基因疫苗在Balb/c小鼠的免疫应答。方法：将与HCV核心区基因互补的cDNA定向克隆于真核表达载体pcDNA3(pcDNA-HCV)，免疫Balb/c小鼠，观测小鼠血清抗-HCV抗体及小鼠脾细胞对HCV核心抗原的特异性增殖反应。结果：5只经皮下注射的免疫鼠中有4只出现抗-HCV抗体，经肌肉注射的免疫鼠全部出现抗体阳转，而对照组全阴性，肌肉注射免疫组抗体水平高于皮下注射免疫组，动态观察发现追加免疫2 w后抗体即可阳转，4～8 w达高峰，可持续12 w以上。免疫组小鼠脾细胞对HCV核心抗原的刺激指数明显高于对照组。结论：pcDNA-HCV在Balb/c小鼠不仅可诱导体液免疫应答，而且可诱导细胞免疫应答。

Plasmid hepatitis C virus core protein:DNA-based immunization for immune responses in Balb/c mice

DENG Tao,CHEN Nai-Ling,CHEN Tian-Bao et al

(Institute of Liver Disease ,Beijing 100700)

AbstractObjective：DNA-based immunization has been proved to be an effective means of vaccination in animal models and may be promising candidates as antiviral agents.There is a need to develope new antiviral agents in an attempt to eradicate persistent HCV infection from liver.In this study,the immune responses to HCV core gene DNA-bassed immunization in Balb/c mice were evaluated.Methods：The HCV core gene cDNA(genotype:II)was inserted into the eukaryotic expression vector under the CMV promoter between EcoRI and Xbal I site (pcDNA-HCV).The Balb/c mice were immunized by multiple sites intramuscular or subcutantous injection with pcDNA-HCV.The anti-HCV Ab in serum were detected by ELISA,meanwhile, the proliferative reponses of spleencytes to HCV core antigens were measured by MTT method.Results：The anti-HCV Ab were detected in 4 out of 5 immunized by subcutaneous injection and in all mice immunized by intramuscular injection.Peek titer were reached by 4～6 weeks after immunization and were maintained for at lease 3 months.The stimulation index of spleencytes from mice immunized with pcDNA-HCV to HCV core antigen were significantly higher than that of mice injected with pcDNA3(P＜0.01).Conclusion：The study demonstrated that both humoral and cellular immune responses could be elicited in mice by pcDNA-HCV.Gene immunization can aid the development of HCV vaccines.The pcDNA-HCV might be promising candidates as antiviral agents.

Key wordsHepatitis C virusDNA-based immunizationGene immunization

目前HCV疫苗研究受两大因素制约，首先，HCV体外细胞培养系统尚未建立；其次，HCV至少有6个基因型，而且HCV膜蛋白区具有高度的变异性，易发生免疫逃逸和耐受，采用传统研究方法很难获得对不同HCV株型具有交叉免疫功效的疫苗^［1］。基因免疫，亦称核酸疫苗，既可诱导体液免疫应答，也可诱导细胞免疫应答，这种特异性的细胞免疫应答不仅对预防不同株型的HCV感染具有重要意义，而且对慢性病毒感染可能具有治疗作用^［1-3］，本文对HCV高度保守的核心区基因免疫进行了实验研究。

1材料与方法

1.1质粒DNA的构建从含Ⅱ型HCV核心区基因片段的质粒pTZ19经EcoRI及XbaI双酶切，电泳回收392 bp的含有ATG和TAG的HCV核心区基因片段(cDNA),定向克隆于已预先用EcoRI及 XbaI双酶消化好的真核表达载体pcDNA3(Invitrogene CO.USA)的CMV启动子下游，经氯化钙转化大肠杆菌JM109(北医马大龙教授惠赠)，经LB培养基扩增，按碱裂解法提取质粒，经紫外分光光度计测定浓度，按1μg/μl溶于无菌PBS，-20℃保存备用。

1.2免疫接种选用4～6周龄的Balb/c小鼠，用1 ml的OT注射器(25号针头)行双后肢多点肌肉注射及尾部多点皮下注射，肌肉注射深度严格控制在0.2 cm^±。每只小鼠注射总量100 μg，为加强免疫，2 w后追加免疫1次。实验中设对照组，注射等量空白质粒pcDNA3。

1.3血清收集及抗HCV抗体检测采用割尾或眼眶静脉丛穿刺连续采血，每次采血量0.3～0.5 ml，离心收集上清置-20℃冰箱，一次性检测。用酶联免疫试验(ELISA)检测血清抗-HCV抗体，试剂盒购自北京生物制品研究所，二抗为羊抗鼠IgG，按试剂盒说明进行检测。

1.4T细胞抗原特异性增殖反应测定通过摘眼球放血处死肌肉免疫组小鼠，制备小鼠脾细胞悬液，细胞浓度为1×10⁶ml^-1，加入96孔板，每孔100μl，平行3孔，分别加入HCV核心抗原(HCVcAg)5 μg/孔，37℃、5%CO₂，孵育72 h，加MTT10μl、37℃、6h，再加盐酸异丙醇100μl/孔，混匀，静置15 min，测各孔A570值，计算增殖指数。增殖指数=实验组OD值/对照组OD值^［4］ 。

2结果

2.1pcDNA-HCV质粒的鉴定pcDNA-HCV经EcoRI和XbaI双酶切后，电泳可见392 bp(见图1)的条带，而空载质粒 pcDNA3则无此条带，证明HCVcore基因已被插入pcDNA-HCV。

图1pcDNA-HCV，ptz19的限制性内切酶图谱分析

Fig.1Restriction analysis of pcDNA-HCV,ptz19

Note:1.DNA standard:PBR322DNA/BstNI;2.pcDNA3 cut by EcoRI/XbaI;3.pcDNA-HCV cut by EcoRI/XbaI;4.ptz19 cut by EcoRI/XbaI

2.2血清抗-HCV抗体检测结果5只经皮下注射的免疫鼠中有4只出现抗-HCV抗体，5只经肌肉注射的免疫鼠全部出现抗体阳转，而对照组全阴性，抗体水平及动态改变见图2、3，同样肌肉免疫注射组高于皮下注射组，加强免疫后2 w出现抗-HCV，4～8 w达高峰，可持续12 w以上。而注射空载质粒的对照组无1只抗-HCV抗体阳性。

图2基因免疫小鼠血清抗-HCV抗体水平

Fig.2Comparison of antibody response in mice immunized using different methods

Note：1～5 means the codes of the mice

图3pcDNA-HCV基因免疫Balb/c小鼠抗-HCV抗体动态改变

Fig.3Kinetics of apperance of anti-HCV Ab in sera from mice injected with pcDNA-HCV.Sera were used at a 1∶20 dilution

Note:No.1～5 means the mice's codes of the muscular group

2.3T细胞抗原特异性增殖反应T细胞抗原特异性增殖反应见图4，可见pcDNA-HCV经肌肉注射

免疫小鼠后对HCVAg增殖反应明显高于对照组，增殖指数为1.527，明显高于对照组(P＜0.01)。

图4免疫Balb/c小鼠脾细胞对HCV核心抗原的增殖反应结果

Fig.4The results of proliferation response of T cell from mice immunized by intramuscular injection to HCV core Ag

Note:(1)=pcDNA3 immunization group (No antigen stimulation):(2)=pcDNA3 immunization group(HCV antigen stimulation )(3)=pcDNA-HCV immunization group (No antigen stimulation):(4)=pcDNA-HCV immunization group (HCV antigen stimulation )1、2、3、4、5 means the mice's codes

3讨论

基因免疫是近年发展起来的新型生物技术，其先进的理论构想和众多的动物实验初步表明，DNA疫苗具有众多优点：不仅可诱导体液免疫应答，而且可诱导细胞免疫应答；抗原蛋白的空间构象与天然蛋白更接近，免疫原性更强。

基因免疫的作用机制尚不清楚，其抗原呈递可能存在以下三种途径：①肌纤维细胞被质粒DNA转染，表达的抗原蛋白在肌纤维细胞内被加工后形成小分子抗原多肽，继而与肌纤维细胞内的MHC-I分子结合，激活T淋巴细胞；②肌肉组织中的抗原呈递细胞如树突状细胞(dendritic cells)在免疫接种过程中可能被质粒DNA转染而进行抗原呈递；③抗原蛋白从肌细胞分泌出来后，被树突状细胞吞噬处理，然后与MHC-Ⅱ分子结合，激活T细胞、B<