人B7-H3两种异构体基因转染细胞株的构建及鼠抗人B7-H3单克隆抗体的研制

发布时间：2018-05-20 11:54

本文选题：2IgB7-H3 + 4IgB7-H3　；参考：《苏州大学》2010年硕士论文

【摘要】： T细胞的有效活化需要两条独立的但是互补的信号途径。第一信号是抗原递呈细胞(APC)递呈的抗原肽-主要组织相容性复合物(MHC),与特殊的T细胞抗原受体复合物TCR/CD3识别,并将信号传递给T细胞。第二信号是不可缺少的共刺激信号,它由T细胞和APC表面的共刺激分子相互作用所激发。共刺激分子并不仅仅单独激发T细胞的活化,它们还协同TCR/CD3复合物提供增强信号。T细胞上的CD28和B7(B7-1和B7-2)相互作用产生的信号,是公认的最经典的共刺激信号。共刺激信号有B7和CD28两大超家族成员,它们在调节T细胞的活化和耐受中发挥着关键作用,有望用于临床治疗。这些通路不仅提供促进和维持T细胞应答的正性第二信号,它们同样能够下调T细胞应答,提供必要的负性第二信号。负性共刺激信号在限制、中断和减弱T细胞应答中发挥效应,这对于调节T细胞耐受和自身免疫非常重要。人B7-H3由于mRNA剪接的不同存在着2IgB7-H3和4IgB7-H3两种异构体。鉴于B7-H3两种异构体的功能差异尚不清楚,因此值得进一步深入探讨。鉴此,本课题制备了2IgB7-H3和4IgB7-H3两种异构体的转基因细胞株,比较分析了B7-H3两种异构体对T细胞的免疫调控作用。此外,本研究还研制了1株抗人B7-H3单克隆抗体,为进一步研究B7-H3分子的生物学功能奠定基础。一、人B7-H3两种异构体基因转染细胞株的建立及对T细胞刺激作用差异比较目的:建立2IgB7-H3基因转染细胞株和4IgB7-H3基因转染细胞株,探讨B7-H3两种异构体在介导T细胞免疫应答中是否存在差异。方法:RT-PCR法从诱导成熟的DC细胞中克隆人B7-H3两种异构体基因2IgB7-H3和4IgB7-H3的编码区,经EcoR I和BamH I双酶切后插入pIRES2-EGFP真核表达载体构建pIRES2-EGFP/2IgB7-H3和pIRES2-EGFP/4IgB7-H3重组子,采用脂质体法转染脑胶质瘤细胞株SHG44。经G418抗性筛选,采用免疫荧光标记和流式细胞术分析2IgB7-H3和4IgB7-H3在SHG44细胞上的表达。继而,利用MTT法和酶联免疫吸附测定法(ELISA)分析两种异构体基因转染细胞株对T细胞体外增殖和细胞因子分泌的影响。结果:成功构建了可以稳定表达2IgB7-H3和4IgB7-H3的基因转染细胞株。体外生物学功能分析表明,与转染空载体的SHG44/mock细胞相比,SHG44/2IgB7-H3和SHG44/4IgB7-H3均能有效抑制T细胞增殖以及对IFN-γ的分泌,且两者的协同抑制作用不存在显著差异。结论:B7-H3两种异构体分子均能负性调控T细胞介导的免疫应答,但两者的生物学功能并不存在显著的差异。二、鼠抗人B7-H3单克隆抗体的制备及鉴定目的:研制鼠抗人B7-H3单克隆抗体,并对其生物学特性进行鉴定。方法:以高表达人2IgB7-H3和4IgB7-H3的293T细胞为免疫原,常规免疫小鼠、细胞融合和筛选。SHG44/2IgB7-H3和SHG44/4IgB7-H3基因转染细胞为抗体筛选阳性细胞,SHG44/mock基因转染细胞为抗体筛选阴性细胞,经间接免疫荧光标记和流式细胞术对杂交瘤细胞进行反复筛选和多次亚克隆化,流式细胞术分析抗体对B7家族不同成员基因转染细胞的识别。流式细胞术分析B7-H3在各类细胞株上的表达特性。结果:获得了一株持续稳定分泌鼠抗人B7-H3单克隆抗体的杂交瘤细胞株13F8,该单克隆抗体(克隆号13F8)对B7-H3两种异构体均具有较好的识别特异性。结论:以获得的抗人B7-H3单克隆抗体13F8作为检测手段,发现B7-H3的表达谱较为广泛,该抗体的获得为进一步研究B7-H3的生物学功能与临床运用奠定了基础。
[Abstract]:There are two independent but complementary signaling pathways for T cell activation . The first signal is antigen peptide - major Histocompatibility Complex ( MHC ) presented by antigen presenting cell ( APC ) . It is excited by co - stimulatory molecule interaction of T cells and APC surfaces . The co - stimulatory molecules play a key role in regulating the activation and tolerance of T cells . These pathways not only provide positive second signals for the promotion and maintenance of T cell responses , but also provide the necessary negative second signals . Negative co - stimulatory signals exert an effect in limiting , interrupting , and reducing T cell responses , which are important for regulating T cell tolerance and autoimmune diseases .

There are two isoforms of B7 - H3 and 4IgB7 - H3 in human B7 - H3 . In view of the unclear functional differences between the two isoforms of B7 - H3 , the immunoregulatory effects of the two isoforms of B7 - H3 on T cells are discussed .

Establishment of a human B7 - H3 gene transfected cell line and comparison of T cell stimulating effect

Objective : To establish a 2 IgB7 - H3 gene - transfected cell line and a 4IgB7 - H3 gene - transfected cell line , and investigate whether the two isoforms of B7 - H3 are different in mediating T cell immune response .

Methods : The eukaryotic expression vector pIRES2 - EGFP / 2IgB7 - H3 and pIRES2 - EGFP / 4IgB7 - H3 were constructed by RT - PCR from mature DC cells . The pIRES2 - EGFP / 2IgB7 - H3 and pIRES2 - EGFP / 4IgB7 - H3 were transfected into the eukaryotic expression vector pIRES2 - EGFP / 4IgB7 - H3 .

Results : In vitro biological function analysis showed that SHG44 / 2IgB7 - H3 and SHG44 / 4IgB7 - H3 effectively inhibited T cell proliferation and IFN - 纬 secretion compared with SHG44 / mock cells transfected with empty vector .

Conclusion : B7 - H3 can regulate T cell mediated immune response negatively , but there is no significant difference in the biological function of B7 - H3 .

Preparation and identification of mouse anti - human B7 - H3 monoclonal antibody

Objective : To develop a mouse anti - human B7 - H3 monoclonal antibody and to identify its biological characteristics .

Methods : Human 2IgB7 - H3 and 4IgB7 - H3 were used as immunogen , and the cells were fused and screened . SHG44 / 2IgB7 - H3 and SHG44 / 4IgB7 - H3 gene were used to screen the positive cells . SHG44 / 2 IgB7 - H3 and SHG44 / 4IgB7 - H3 gene were used to screen negative cells .

Results : A hybridoma cell line 13F8 stably secreting anti - human B7 - H3 monoclonal antibody against human B7 - H3 was obtained .

Conclusion : The anti - human B7 - H3 monoclonal antibody 13F8 is used as the detection means , and the expression profile of B7 - H3 is found to be more extensive , which lays a foundation for further research on the biological function and clinical application of B7 - H3 .
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】