Balb/C小鼠及HBV转基因鼠对不同基因疫苗的免疫应答实验研究

【作者】 邓涛；

【导师】 王孟薇；

【作者基本信息】 军医进修学院 ， 老年消化系疾病， 2001， 博士

【副题名】Immune Responses to Different Constructs in Balb/C Mice and in Hepatitis B Virus Transgenic Mice

【摘要】 目的：为寻求高效的HCV/HBV联合基因免疫策略，研究免疫刺激DNA序列联合 基因免疫在Balb/C小鼠的免疫应答及在HBV转基因鼠的抗病毒效应。材料与方法： 1．分别将与HCV核心区基因互补的cDNA和HBV核心区基因克隆于含两套表达单元的真核双表达载体的 CMV启动子和 RSV启动子下游(pRSC-HBV/HCV)，转染SP2/0细胞，通过免疫荧光和Western印迹法观测蛋白的表达，免疫Balb/c 小鼠，用酶联免疫法[ELISA]检测血清抗-HBc及抗-HCV抗体的转换，通过LDH释放法观察小鼠脾细胞对表达HBcAg及HCV核蛋白的SP2/0细胞的特异性杀伤活性，并通过荷瘤试验观察小鼠注射表达HBcAg及HCV核蛋白的SP2/0细胞后肿瘤生长情况以判断体内CTL活性。 2．通过PCR扩增在HBV核心区3’端引入4个拷贝的CpG序列，定向克隆于卡拉霉素抗性基因的 VR1012为载体(V-HBc/CpG)。同时从质粒pcDNA-HBV经酶切，回收其中1KB的不含CpG序列的HBV核心区基因，定向克隆于V-1012(V-HBc)。并人工合成硫代修饰的免疫刺激DNA寡核苷酸[CpG ODN]及非免疫刺激寡核苷酸[非CpG ODN]，分组免疫Balb/c小鼠，用ELISA检测血清抗-HBc抗体的转换，通过LDH释放法观察小鼠脾细胞对表达HBcAg的SP2/0细胞的特异性杀伤活性，并通过荷瘤试验观察小鼠注射表达HBcAg的SP2/0细胞后肿瘤生长情况以判断体内CTL活性。 3．用CpG ODN与HBV S区基因真核表达载体(V-HBs)联合免疫HBsAg转基因鼠，用ELISA观察小鼠血清HBsAg及抗体转换，通过免疫组化 [SP法]、病理HE染色及电镜观察小鼠肝组织HBsAg表达量的改变、肝组织炎症活动度及超微结构的变化。 结果：1．pRSC-HBV/HCV转染SP2/0细胞可见游离 HBcAg及HCV核蛋白的表达，5只免疫鼠中全部出现抗一HCV及抗一HBV抗体，小鼠脾细胞对转染pRSC-HBV/HCV的SPZ/0细胞的特异性细胞毒活性为sl％，明显高于空载对照组，荷瘤试验显示免疫组小鼠注射转染pRSC-HBV/HCV的驴/0细胞2周时肿瘤平均直径为7．25土 豆．15mm，明显小于空载对照组小鼠肿瘤直径门．85士0．25mm)，二者差异显著 (P＜0．05)。注射肿瘤细胞后3周，2只pRSC－HCV/HBV免疫组小鼠仍然存活，而对 照组则全部死亡。 2．免疫刺激DNA序列联合基因免疫实验显示，V-HBC＋CPG ODN组及V一 HBC/CpG组免疫鼠脾细胞对转染pRSC-HBV的SP2/0细胞的特异性细胞毒活性分别 为 18％、60％，均高于 V-HBC＋非 CpG ODN组及 V-HBC对照组。荷瘤试验显示 V 一mc联合 CpG ODN组及 V一HBc/CpG在免疫 2周时肿瘤直径分另为 5．8 f 0．l恤。 4．85士0．75mm，明显小于 V-HBC联合非 CPG ODN及 V-HBC对照组门．85土0．65、 19．05士0．75j，差异显著(P＜0．05)。注射肿瘤细胞后 3周，V一HBC联合 CPG ODN 组及 V-HBc/CpG免疫组小鼠仍然存活，而对照组全部死亡。 3．在HBV转基因鼠的抗病毒实验显示，V-HBS联合CpG ODN组6只免疫鼠中 有2只血清抗一ms抗体阳性，其平均效价为56．21士15．16mIWml，高于免疫保护 所需的最低水平(10ml/ml)，血清HBsAg浓度在免疫后第8周时明显降低，并 有2只转阴，而单用V-HBS组及V-1012对照组小鼠血清抗一HBS抗体均阴性、 HBsAg含量无明显降低。V－HBs＋CpG ODN组肝组织 HBsAg的表达量低于 V-HBs组及 对照组，差异显著(P＜0．05)。V-HBS联合CPG ODN组肝组织病理及电镜观察可见 大量炎细胞浸润，炎症组织活动度积分明显高于V-HBS组及对照组。 结论：PRSC一HBV/HCV在Balb ＊小鼠可同时诱导对HBV及HCV的体液免疫应答 及细胞免疫应答，提示真核双表达载体可用于构建联合基因疫苗，该研究为HBV/HCV 联合基因免疫探索了新的策略。CpG ODN联合V－HBc及V一HBc/CpG在Balb/c体内 可增强其特异性的细胞免疫应答，进一步在 HBV转基因鼠的实验研究显示，CPG ODN 联合 V－HBS可增强其免疫应答及抗病毒效应。该实验证实 CpG ODN联合基因兔疫可 增强机体的免疫应答及抗HBV效应，为打破慢性乙型肝炎免疫耐受及基因兔疫治疗 探索了新的方法。更多还原

【Abstract】 Aims: To develop a HCV combined HBV DNA-based therapeutic vaccine and tostudy the immune responses of HBV-based immunization with plasmid by appendingimmune stimulatory DNA sequence (ISS) to HBV core gene or by coinjecting CpG-oligodeoxynucleotides in Balb/c mice and in HBV transgenic mice.Methods:1. The HBV core gene and HCV core cDNA were inserted into the eukaryoticexpression vector with two multiple cloning sites mammalian expression vector underthe CMV promoter and RSC promoter respectively, named pRSC-HBV/HCV. Theexpression of SP2/0 cells transfected with pRSC-HBV/HCV was assessed by Westernblot and immunofluorescence studies. The Balb/c mice were immunized by multiplesites intramuscular injection with pRSC-HBV/HCV. The anti-HBc Ab and anti-HCV Abwere detected by ELISA. CTL activity of splenocytes against SP2/0 expressing HBcAgand HCV nucleocasid cells was determined in standard LDH release method. The tumorsize and animal survival were evaluated as an in vivo index of CTL activity generated byimmunization with the plasmid DNA construct after inoculated with SP2/0 cellsexpressing both the HBcAg and HCV core protein.2. The HBV core gene or HBV core gene appended ISS, obtained by ploymerasechain reaction, were inserted into the eukaryotic expression vector under the CMVpromoter respectively, named V-HBc and V-HBc/CpG. CpG-ODN and non CpG-ODNwere synthesized and phosphorothioate-modified. The Balb/c mice were immunizedby multiple sites intramuscular injection:①Group1, V-HBc+ non CpG ODN; ②GrouP2, V-HBc/CpG; ③Group3, V-HBc+CpG ODN; ④Group4, V-HBc. The anti-HBc Ab was measured by ELISA. CTL acivity of splenocytes against pRSC-HBV/HCVtransfected SP2/0 cells was determined in standard LDH release method. The tumor sizeand animal survival were evaluated as an in vivo index of CTL activity generated byimmunization with the plasmid DNA construct after inoculated with SP2/0 cellsexpressing HBcAg.3. The HBV transgenic mice were immunized by multiple sites intramuscularinjection with V-HBs and CpG ODN or V-HBs alone or V-1012 controls.The anti-HBsAb and HBsAg in serum from immunized mice was measured by ELISA. Theexpression of HBsAg in liver tissue was detected by inununohistochemistry method (SP).The histological activity index was calculated with Knodell’s method and theultrasttuctAfe ofhepatocytes was observed by electfomicroscopy.Rcsults:1. The HBcAg and HCV nucleocaPsid expressed simultaneously in SP2/0 cellstransfected with pRSC-HBVMCV Both anti-HBc and ani-HCV core Ab was detectedin all inununized mice with pRSC-HBVMCV. The specific CTL activity of splenocytesagainst SP2/0 cells expressing both HBV and HCV nucleocaPsid were 8l% in miceimmunized with pRSC-HBV/HCV, Which is significantly higher than that in inununizedmice with pRSC controls. The boor size after inoculated with SP2/0 cells expressingHBcAg and HCV nucleocaPsid in mice immunized with pRSC-HBVnsCV wereremarkably reduced compared with mice injected with pRSC.2. The ani-HBc Ab could be detected in every mice inununized with V-HBC+CpGODN, V-HBC/CpG, V-HBC+ Non CpG ODN as well as V-HBc contrOl grouPs. Thespecific CTL activity of splenocytes apainst SP2/0 cells expressing HBcAg were l8% inmice immunized with V-HBC+CpG ODN and 60% in mice injected with V-HBC/CpG,Which is significantly higher than that in immunized mice with V-HBC+ Non CpG ODNas well as V-HBc control grouPs. The tUmor size after inoculated with SP2/0 cellsexpressing HBcAg in mice inUnwhzed with V-HBC+CpG ODN and in mice immunizedWth V-HBC/CpG were remarkably reduced comPared with mice injected with V-HBC+Non CpG ODN as we1l as V-HBc cothel grouPs.3. The ani-HBs Ab in seru!n could be detected and HBsAg in serum and livertissues could not be measured in 2 out of 6 HBV transgenic mice iInmunized With V-HBs+CpG ODN. There were not significanly changes in the level of anti-HBs Ab andHBsAg in serum from HBV transgenic mice inununized with V更多还原