【作者】 陈开旭；

【导师】 马正海；

【作者基本信息】 新疆大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 腺病毒载体是基因治疗中最为常用的一种载体,具有以下优点:安全性高,经人工改造的减毒腺病毒对人体无明显毒副作用;腺病毒DNA不会整合入宿主基因组,避免了对宿主基因的干扰;腺病毒可增强MHC分子和共刺激分子的表达,从而促进抗原递呈;腺病毒载体容量大,最大可容纳约37 kb的外源DNA;可高效感染哺乳动物细胞,能够获得高滴度的病毒。高危型人乳头瘤病毒(HPV)的持续感染是诱发宫颈癌的主要原因,其中以HPV-16和HPV-18最为常见。新疆南部维吾尔聚居区是我国宫颈癌高发区,该地区妇女宫颈癌的发病率高,发病年龄较其它民族低,其发生与HPV-16的感染密切相关。HPV主要衣壳蛋白L1结构保守,能够自组装形成病毒样颗粒,是宫颈癌预防性疫苗的理想靶抗原。哺乳动物卵透明带(zona pellucida, ZP)是由卵母细胞分泌并覆盖于卵母细胞外的一层糖蛋白基质,在种属特异性精卵识别、结合,诱导精子与透明带的结合、顶体反应、避免多精受精和胚胎植入前的保护等方面发挥着重要作用。ZP主要由3种糖蛋白(ZP1、ZP2和ZP3)组成,ZP3在受精过程中具有十分重要的作用,能够与精子专一性地结合并诱导精子的顶体反应。大量研究证实:抗ZP3抗体能够阻断精卵结合。因此,ZP3可作为免疫不育的理想靶抗原。本研究将携带新疆株人乳头状瘤病毒16型(HPV-16)主要衣壳蛋白L1基因和鼠卵透明带3 ( murine zona pellucida 3)基因的腺病毒重组DNA转染HEK 293A细胞,使其在HEK 293A细胞中进行包装;继而扩增重组腺病毒Adeno-mZP3和Adeno-L1,利用TCID50(半数组织培养感染剂量)法分别测定滴度, Adeno-L1滴度为:1.2×1010 PFU/mL,Adeno-mZP3滴度为:2.5×1011 PFU/mL, Adenovirus滴度为:2.4×1011 PFU/mL。将获得的重组腺病毒Adeno-mZP3和Adeno-L1分别以滴鼻和灌胃两种途径免疫BALB/c小鼠。实验组每只小鼠接种1×108 PFU重组腺病毒,对照组每只小鼠接种1×108 PFU野生型腺病毒,每隔两周免疫1次,免疫3次,免疫后第14天、第28天和第28天采集小鼠血清,ELISA法检测重组腺病毒诱导的体液免疫反应,并比较两种免疫途径诱导免疫反应的差异。接种重组腺病毒Adeno-mZP3的雌性小鼠在第3次免疫后7天与已验证有生殖能力的雄鼠合笼,观察雌鼠受孕情况,判断重组腺病毒Adeno-mZP3所诱导的小鼠免疫不育效果。免疫检测结果显示:重组腺病毒Adeno-mZP3和Adeno-L1通过滴鼻和灌胃免疫小鼠后第14d、第28d、第42d,小鼠血清中相应抗体水平均比对照组小鼠高,二者存在极显著性差异(P<0.001);说明2种重组腺病毒通过滴鼻和灌胃免疫小鼠均能激发较强的体液免疫反应,且在3次免疫后,滴鼻免疫组小鼠血清中抗体水平高于灌胃免疫组。重组腺病毒Adeno-mZP3免疫组合笼实验的结果表明,滴鼻免疫小鼠的生育率和平均窝崽数均低于对照组,实验组与对照组的平均窝崽数存在显著性差异(P=0.021<0.05);灌胃免疫小鼠的生育率和平均窝崽数与对照组相比没有明显降低,实验组与对照组的平均窝崽数差异不显著(P=0.102>0.05)。以上结果说明腺病毒载体疫苗滴鼻免疫能诱导产生较好的免疫效果。更多还原

【Abstract】 In recent years, Adenovirus vectors (AdVs) have been increasingly considered as gene therapy vectors for treating human diseases for the following features: they have been proven safe and effective after being used as live vaccines for immunizing with wild-type (wt) live AdVs showed no significant side effects; they do not integrate their viral DNA into host chromosomes, thereby avoiding the possibility of disturbing vital cellular genes or inducing cancer as has been the case with retrovirus vectors; they can modulate dendritic cell maturation by increasing the expression of major histocompatibility complex (MHC) and costimulating molecules; they can accommodate a large size of foreign DNA of up to 37 kb, and they can be easily produced in large quantities.A growing number of evidence indicates that persistent infection of high-risk HPV, particularly type 16 and 18, is an important initiating agent of cervical carcinogenesis. The south of Xinjiang where Uygur nationality inhabits is a region with a high incidence of cervical cancer, and the women in this region usually suffer cervical cancer much younger than other nationalities. This phenomena is considered as a closely relationship with the infection of HPV-16. The major capsid protein L1 has a conservative protein structure, and can assemble into virus-like particles (VLPs) automatically. So L1 is an ideal target antigen of prophylactic vaccines against cervical cancer.An acellular glycoproteinaceous matrix termed zona pellucida (ZP) surrounds the mammalian oocyte and plays a pivotal role in species-specific sperm–egg recognition and binding, induction of acrosomal exocytosis in ZP-bound spermatozoa, avoidance of polyspermy, and protection of the embryo prior to implantation. The mouse ZP is composed of three biochemically distinct glycoproteins designated as ZP1, ZP2, and ZP3. ZP3, the putative primary sperm receptor, binds to the head region of acrosome-intact spermatozoa and induces acrosomal exocytosis. There is considerable evidence implicate that anti-ZP3 antibodies can block sperm-egg binding, which can be used as an ideal target antigen for immunocontraception.In this study, adenovirus recombinant DNA which carry murine zona pellucida 3 gene and the L1 of HPV-16 from Xinjiang were transfected into HEK 293A cells to package viral particles respectively, the recombinant adenovirus named as Adeno-mZP3 and Adeno-L1. Then Adeno-mZP3, Adeno-L1 and wild type adenovirus were amplified in HEK 293A cells and TCID50 (50% tissue culture infectious doses) assay was employed to detect the titers of viruses. The titer of Adeno-mZP3 is 2.5×1011 PFU/mL, the titer of Adeno-L1 is 1.2×1010 PFU/mL and the titer of wild type Adenovirus is 2.4×1011 PFU/mL. BALB/c mice were immunized three times (interval 14d) with 1×108 PFU Adeno-L1, Adeno-mZP3 and wild type Adenovirus via intragastric route and intranasal route respectively, and the mice immunized with wild type Adenovirus as control. The sera of the mice were collected at 14 d, 28 d and 42 d respectively, and then ELISA tested the level of IgG against L1 and mZP3 respectively. The immunological effects of difeent recombinant adenovirus vaccines and the different immunization routes were analyzed by Two-way ANOVA. The female mice inoculated three times with Adeno-mZP3 were coupled with male mice possessing reproductive capacity to observe the pregnant situation, and then the immunocontraceptive efficacy induced by Adeno-mZP3 was evaluated.The results of ELISA showed that the level of antibody against L1 (or mZP3) from the mice immunized with Adeno-L1 (or Adeno-mZP3) via intranasal (or intragastric route) is extremely higher (P<0.001) than the serumal antibody level of mice in control group at 14th d, 28th d and 42nd d. These results suggested that both Adeno-L1 and Adeno-mZP3 can stimulate humoral immune response via intranasal and intragastric route. However, the serumal antibody of mice immunized via intranasal route was higher than that of mice immunized via intragastric route. Meanwhile antifertility assay indicate that the fertility and mean litter size of mice inoculated with Adeno-mZP3 via intranasal route were reduced significantly, and the mean litter size of mice immunized with Adeno-mZP3 were significantly lower than that of control group (P=0.021<0.05). But the reduction of the fertility and mean litter size of mice inoculated with Adeno-mZP3 via intragastric route was not significant, and the mean litter size of mice immunized with Adeno-mZP3 were not between treatment group and control group were not significantly lower than that of control group (P=0.102>0.05). So it is more effective to immunize mice with recombinant adenovirus via intranasal immunization than intragastric immunization in our study.更多还原