小儿肾母细胞瘤新型疫苗的研制

发布时间：2018-07-27 13:05

【摘要】： 目的:对小儿肾母细胞瘤新型疫苗进行初步探索、研究,为该肿瘤的治疗以及替代手术后的放、化疗提供新的切入点。方法:首先对必需的实验材料进行制备和验证,包括抗体的制备和肿瘤细胞模型的建立。针对小儿肾母细胞瘤WT1基因,选取两段抗原表位肽HLA-A*0201、HLA-A*2402采用构建DNA疫苗的方式,运用Balb/c小鼠免疫检测的方法筛选最佳的抗原表位肽,为小儿肾母细胞瘤新型疫苗的研究奠定基础。用筛选得到的表位肽HLA-A*2402构建DNA疫苗,为最终新型疫苗的研制做对照。采用基因重组技术分别克隆编码小儿肾母细胞瘤WT1表位肽HLA-A*2402融合到乙型肝炎核蛋白基因片段(HBc)上,运用杆状病毒表达系统对融合蛋白进行表达,通过电镜观察得到的融合蛋白呈病毒样颗粒(VLP)。继而采用WB技术对表达的蛋白进行鉴定。鉴定好的VLP蛋白和DNA疫苗采用细胞模型进行研究,然后分别免疫Balb/c小鼠,采用ELISA、FCM、MTS等免疫学实验技术分别检测,初步判断基于WT1表位肽HLA-A*2402构建的以VLP为呈现载体的重组蛋白,作为该肿瘤治疗以及替代手术后的放、化疗的可行性。结果: (1)制备了合格的实验材料:对小儿肾母细胞瘤细胞进行了成功的原代培养,建立了细胞系用于实验研究;克隆了WT1基因部分片段的原核表达质粒,表达并纯化了蛋白,免疫家兔,采集血清制备了针对WT1的多克隆抗体。 (2) MTS及FCM初步证明,短肽HLA-A*2402在刺激淋巴细胞增殖方面优于HLA-A*0201,而前者无显著性差异(p0.05)。因此,HLA-A*2402为我们选择深入研究的对象。 (3)构建了含有HLA-A*2402的DNA疫苗,双酶切和测序结果证明构建成功。 (4)构建了HBc与HLA-A*2402偶联(couple)的以VLP为呈现载体的新型疫苗,双酶切和测序结果证明构建成功。运用杆状病毒表达系统对偶联的蛋白进行表达,WB和电镜检测证明目的蛋白的表达。 (5)运用以VLP为呈现载体的新型疫苗进行Balb/c小鼠的免疫,并以构建的DNA疫苗作为对照,结果表明以VLP为呈现载体的新型疫苗免疫小鼠产生了有效的特异性体液免疫和细胞免疫。抗体亚型分类表明特异性IgG1/IgG2a小于1,说明产生了Th1型为主的免疫应答。T淋巴细胞增殖试验证实免疫小鼠脾脏T淋巴细胞对ConA诱导的增殖反应增强;ELISPOT分析表明疫苗免疫组的特异性活化的可分泌IFN-γ的CD8+T细胞数显著多于对照组;FCM检测表明,疫苗免疫组的CD4+/CD8+比值显著低于对照组。这些结果提示以VLP为呈现载体的新型疫苗诱导了特异性CD8+T淋巴细胞应答。其激发的细胞免疫和体液免疫均明显优于DNA疫苗对照组。 (6)研究证实HLA-A*2402在小儿肾母细胞瘤的免疫治疗过程中扮演着重要的角色,而以VLP为呈现载体的新型疫苗为肿瘤的免疫治疗提供了新的思路。结论:所制备的小儿肾母细胞瘤以VLP为呈现载体的新型疫苗在小鼠体内可同时激发体液免疫和细胞免疫,优于相应的DNA疫苗,为肿瘤的免疫治疗提供新的方法和思路。更为重要的是将病毒有关成分引入到肿瘤的免疫治疗中,其良好的实验结果有待于肿瘤动物模型的进一步验证。
[Abstract]:Objective: To explore a new vaccine for Wilms tumor in children, and to provide a new breakthrough point for the treatment of this tumor and for postoperative radiotherapy and chemotherapy.
Methods: first, the necessary experimental materials were prepared and verified, including the preparation of the antibody and the establishment of the tumor cell model. According to the WT1 gene of the nephroblastoma in children, the two segment epitope peptide HLA-A*0201 was selected, and HLA-A*2402 was used to construct the DNA vaccine, and the best epitope peptide was screened by the method of immune detection in Balb/c mice. For the study of a new type of vaccine for nephroblastoma, the DNA vaccine was constructed with the selected epitope peptide HLA-A*2402, which was used as the control for the development of the final new vaccine. The gene recombination technique was used to clone the WT1 epitope peptide HLA-A*2402 of the children's nephroblastoma to the hepatitis B nucleoprotein gene fragment (HBc) and use the pole shape. The fusion protein was expressed by the virus expression system. The fusion protein obtained by electron microscopy was virus like particles (VLP). Then WB technology was used to identify the expressed protein. The identified VLP protein and DNA vaccine were studied by cell model, and then immune Balb/ c mice were immunized with ELISA, FCM, MTS and other immunoassay techniques. The preliminary determination of the recombinant protein based on the WT1 epitope peptide HLA-A*2402, based on the VLP as the carrier, was used as the tumor therapy and the possibility of chemotherapy after the replacement of the tumor.
Result:
(1) the qualified experimental materials were prepared: the primary culture of the children's nephroblastoma cells was successfully cultured, the cell line was established for experimental study, the prokaryotic expression plasmid of the partial fragment of WT1 gene was cloned, the protein was expressed and purified, the rabbit was immunized, and the polyclonal antibody against WT1 was prepared by the collection of serum.
(2) MTS and FCM preliminarily proved that the short peptide HLA-A*2402 is superior to HLA-A*0201 in stimulating lymphocyte proliferation, but the former has no significant difference (P0.05). Therefore, HLA-A*2402 is the object of our study.
(3) a DNA vaccine containing HLA-A*2402 was constructed. Double enzyme digestion and sequencing showed that the recombinant DNA vaccine was successfully constructed.
(4) a new vaccine was constructed with HBc and HLA-A*2402 coupling (couple) with VLP as the carrier. The results of double enzyme digestion and sequencing proved that the construction was successful. The protein expression of the coupling was expressed by the baculovirus expression system. The expression of the target protein was proved by WB and electron microscopy.
(5) the immunization of Balb/c mice was carried out with the new vaccine of VLP as the carrier, and the constructed DNA vaccine was used as the control. The results showed that the new vaccine immunized with VLP as the carrier produced effective specific humoral immunity and cell immunity. The specific IgG1/IgG2a of the antibody subtype was less than 1, indicating that the Th1 type was produced. The immune response.T lymphocyte proliferation test proved that the proliferation response of spleen T lymphocyte in immunized mice was enhanced by ConA, and ELISPOT analysis showed that the number of CD8+T cells that secreted IFN- gamma in the vaccine immunization group was significantly more than that of the control group; FCM detection showed that the CD4+/CD8+ ratio of the immunization group was significantly lower than that of the control group. These results suggest that a new type of vaccine with VLP as the carrier induces specific CD8+T lymphocyte response. Both the stimulated cell immunity and the humoral immunity are obviously superior to that of the DNA vaccine control group.
(6) the study confirms that HLA-A*2402 plays an important role in the immunotherapy of children with nephroblastoma, and a new vaccine with VLP as the carrier provides a new way of thinking for the immunotherapy of tumor.
Conclusion: the new vaccine with VLP as the carrier can stimulate both humoral and cellular immunity in mice. It is superior to the corresponding DNA vaccine and provides new methods and ideas for the immunotherapy of tumor. The results need to be further validated by animal models of tumor.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【共引文献】