采用基于CDR3δ肽结合特性的免疫—生物化学技术策略鉴定TCRγδ所识别的新型配体-hMSH2蛋白

发布时间：2018-02-26 23:19

本文关键词： γδT细胞 CDR3δ 蛋白抗原 hMSH2 肿瘤昆虫病毒表达系统　出处：《中国协和医科大学》2008年博士论文　论文类型：学位论文

【摘要】： 尽管人类外周血γδT细胞在整个T细胞群体中所占比例较少,但其在机体对抗感染和肿瘤的免疫应答中的重要作用却深受关注。然而,至今为止,γδT细胞所识别的抗原则很少被鉴定。不了解T细胞受体(TCR)γδ所识别配体,也就不可能深入了解γδT细胞的生物学功能。因此,进一步发现和鉴定TCRγδ所识别配体则成为γδT细胞研究领域亟待解决的突出问题。如何通过新的技术策略,来发现TCRγδ所识别新型配体是本研究着重考虑的科学问题,这也是进一步了解TCRγδ抗原识别特异性和多样性机制,揭示γδT细胞的生物学功能的关键问题。 TCR的抗原结合部位是由六个抗原决定簇(CDR)形成的,这六个CDR在识别抗原时作用是不相同的,其CDR3区在抗原识别时占有主导地位。鉴于TCRδCDR3区(CDR3δ)与抗体重链的CDR3区在结构和功能上的相似性,因此,我们提出了CDR3δ的一级结构是决定TCRγδ识别抗原特异性的关键部位的学术假说。这一假说已为我们实验室前期工作所验证。我们根据卵巢上皮癌(OEC)组织浸润的γδT细胞(γδTIL)TCR的一个CDR3δ的基因序列(OT3),人工合成了OT3肽,并通过体外一系列OT3肽与靶细胞、靶组织或来源于靶细胞的蛋白的体外结合实验,验证OT3肽的结合特异性,所采用的方法包括OT3肽介导的生物传感器,免疫荧光技术,酶免疫技术和OT3肽竞争结合实验。同时,构建了用OT3序列替代抗体重链CDR3区序列的OT3移植抗体OT3Ab,也进行相应实验。本研究又重复了部分上述验证工作。结果显示,OT3肽和OT3Ab在对靶细胞、靶组织或来源靶细胞的蛋白的结合活性上享有相似的特异性。这就证明了CDR3δ确实在TCRγδ抗原识别特异性上起关键作用,也提示OT3肽能作为一个特异的探针,去筛选TCRγδ识别的抗原。在上述验证工作的基础上,本研究共采用了三种技术策略去寻找TCRγδ识别的蛋白抗原:一是以OT3肽为探针在噬菌体随机展示十二肽库中筛选与OT3肽结合的十二肽,通过序列比对获得相应蛋白;二是以OT3肽为探针在人卵巢癌cDNAλ噬菌体表达文库筛选与OT3肽结合的克隆,通过测序鉴定蛋白;三是通过OT3肽偶联的亲和层析,从OEC肿瘤细胞系(SKOV3细胞)的总蛋白中分离与OT3肽特异性结合的蛋白,再利用质谱技术鉴定蛋白的种类。应用第一种技术策略,我们获得了三个OT3肽特异结合的十二肽。结合和功能验证实验结果显示,这些十二肽不但能特异性地结合TCRγδ,还能在体外促进γδT细胞活化,提示这些十二肽具有TCRγδ的表位肽活性。然而,在BLAST比对中未发现与这些多肽在序列上相匹配的人类相关蛋白,提示这些十二肽可能为来源于其它种属的表位肽。筛选人卵巢癌cDNAλ噬菌体表达文库要求探针具有较高的特异性和亲和力,在这两方面OT3肽不如一般的抗体,因此第二种策略也失败了。通过第三种策略,我们成功鉴定了三个TCRγδ识别的肿瘤相关抗原,它们是丙酮酸激酶3,人mutS同源蛋白2(hMSH2)和热休克蛋白(HSP)60。由于已有报道HSP60能被TCRγδ所识别,故鉴定出HSP60表明我们的策略是可行的。而丙酮酸激酶3以及与DNA损伤修复有关的hMSH2可能是TCRγδ识别的新的蛋白抗原。本研究就hMSH2是否为TCRγδ配体这一问题进行了验证。RT-PCR结果显示,在卵巢癌细胞系SKOV3中,hMSH2 cDNA存在散在分布的点突变。SKOV3细胞培养上清中检测到分泌形式的hMSH2。用Western blotting在SKOV3细胞总蛋白中还发现一个60kD的hMSH2的缺失突变表达形式。而且,在包括SKOV3细胞在内的很多肿瘤细胞系细胞膜上均检测到hMSH2的表达。免疫组化结果证明,肿瘤组织中也有异位表达的hMSH2。另外,Vδ2γδT细胞对SKOV3细胞的细胞毒活性也能被抗hMSH2抗体部分封闭。同时,本研究还构建和表达了五种重组的hMSH2蛋白,包括hMSH2全长蛋白,分别含hMSH2 N端两个结构域和C端两个结构域的蛋白片段,以及SKOV3细胞表达的含有散在突变的两个分段蛋白。所有的这五个蛋白都能特异性与Vδ2γδT细胞结合,并刺激其活化增殖,分泌γ干扰素,并且促进Vδ2γδT细胞对靶细胞的杀伤活性。此外,本研究还利用昆虫病毒表达系统,得到了比原核大肠杆菌表达系统更具生物活性的hMSH2蛋白,这为hMSH2蛋白进一步结构与功能研究奠定了基础。综上所述,本研究取得了以下学术成果: 1、证明了CDR3δ在决定TCRγδ识别抗原特异性上的关键作用,TCRγδ识别抗原时仅仅依赖CDR3δ一级结构的特异性,因此人工合成的CDR3δ肽OT3是寻找TCRγδ抗原的很奏效的探针; 2、建立了一个采用基于CDR3δ肽结合特性的免疫-生物化学技术策略鉴定TCRγδ所识别的蛋白抗原的新的有效的方法; 3、首次发现在癌变的情况下,异位表达的hMSH2很可能是一个新的Vδ2γδT细胞所识别的肿瘤配体分子,其生物学意义在于对固有免疫系统的预警作用; 4、证明了昆虫病毒表达系统是一个稳定而有效的真核表达系统,在昆虫细胞中能完成很多翻译后的修饰,在使表达的外源蛋白保有天然蛋白的生物学活性上至关重要。在上述的四点成果中,第二项和第三项最具有创新性。第二项成果解决了一项长期困扰免疫学界的难题,为鉴定TCRγδ所识别新型配体提供了一个有效的关键技术。第三项成果揭示了γδT细胞免疫监视的新型作用方式,为全面阐明γδT细胞生物学功能提供了重要的线索,也为肿瘤的诊治提供了一个重要的靶标分子。
[Abstract]:Although the proportion of human peripheral blood T cells in the whole cell population in T, but its important role in protecting the body against infection and tumor immune response is concerned. However, so far, the identification of gamma delta T cell antigens are rarely identified. No solution of T cell body (the identification of gamma delta TCR) ligand, it is impossible to understand the biological function of gamma delta T cells. Therefore, the discovery and identification of TCR gamma delta ligand recognition has become the urgent problems of gamma delta T cell research. Through new technology and methods, to find the identification of novel ligands of gamma delta TCR this is a scientific problem considered, which is to further understand the TCR gamma delta antigen recognition specificity and diversity mechanism, the key problems to reveal the biological function of gamma delta T cells.
The antigen binding site of TCR is composed of six epitopes (CDR) formed by the six CDR role in antigen recognition is not the same, the CDR3 region plays a leading role in antigen recognition. In view of the TCR CDR3 Delta region (CDR3 delta) similarity, and the antibody heavy chain CDR3 in the region the structure and function of the result, we put forward a structure of CDR3 8 is the key part of TCR gamma delta antigen recognition specificity of the academic hypothesis. This hypothesis has been confirmed by previous work in our laboratory. We according to epithelial ovarian cancer (OEC) tissue infiltration of gamma delta T cells (gamma delta TIL) the gene sequence of a CDR3 Delta TCR (OT3), the synthetic peptide OT3 in vitro, and through a series of OT3 peptide and target cells, combined with experimental target tissue or derived from the target cell proteins in vitro, verify the binding specificity of OT3 peptide, the method including OT3 peptide mediated biosensor the sensor, immunofluorescence technique Operation, enzyme immunoassay and OT3 peptide competitive binding experiment. At the same time, OT3 was constructed with OT3 transplantation antibody OT3Ab antibody heavy chain sequence substitution sequence CDR3, corresponding experiments. This study also repeated part of the verification work. The results showed that OT3 peptide and OT3Ab on target cells, binding to the target tissue or source the target cell specificity of the protein similar to enjoy the activity. This proves that the CDR3 delta does play a key role in TCR gamma delta antigen recognition specificity, suggesting that OT3 peptide as a specific probe to screening, identification of gamma delta TCR antigen.
Based on the above verification work, this study adopted three kinds of technology strategy to search for protein antigen TCR gamma delta recognition: one is the screening of twelve peptide binding peptide OT3 in phage displayed twelve peptide library with OT3 peptide probe, to obtain the corresponding protein by sequence alignment; two is the cloning and expression of cDNA library screening OT3 peptide binding probe in human ovarian carcinoma cDNA phage to OT3 peptide was identified by sequencing the protein by affinity chromatography; three OT3 peptide coupling, from OEC tumor cells (SKOV3 cells) and OT3 peptide separated total protein specific binding protein by mass spectrometry identification of protein species application of the first technique strategy, we obtained twelve peptide binding three OT3 peptide specific binding and functional verification. The results of experiments show that these twelve peptides can not only bind to TCR gamma delta, can promote the in vitro activation of gamma delta T cells, These twelve peptides with TCR gamma delta epitope peptide activity. However, human proteins associated with these peptides matched in sequence were found in BLAST ratio, suggesting that these twelve peptides may be derived from other species. Screening the epitope peptide of human ovarian cancer cDNA phage expression library for lambda probe has high specificity and affinity of antibodies in the two aspects of the OT3 peptide is not as well as usual, so the second strategy failed. By the third strategy, we successfully identified three TCR gamma delta recognition of tumor associated antigens, they are pyruvate kinase 3, mutS homologous protein 2 (hMSH2) and heat shock protein (HSP) 60. because it has been reported that HSP60 can be identified by the TCR gamma delta, we identified HSP60 show that our method is feasible. The pyruvate kinase 3 and DNA damage repair related hMSH2 may be TCR gammadelta identification of new protein antigen.
The study on whether hMSH2 TCR gamma delta ligands for this validated.RT-PCR results showed that in human ovarian cancer cell line SKOV3, the hMSH2 cDNA mutation distribution of.SKOV3 cells in the presence of scattered points and cultured with Western blotting in total protein of SKOV3 cells also found a 60kD deletion mutation of hMSH2 expression detected by secretion the form of hMSH2. in the supernatant. Moreover, including SKOV3 cells, many tumor cell line hMSH2 was detected by immunohistochemistry. Results show that there are in the tumor tissue of the ectopic expression of hMSH2. in 2, V delta gamma delta T cells to the cytotoxic activity of SKOV3 cells can also be anti hMSH2 antibody partially closed.
At the same time, we also construct and expression of five recombinant hMSH2 proteins, including hMSH2 proteins, protein fragments containing hMSH2 N two terminal domain and C terminal two domains, and contains scattered in the two segment of mutant proteins expressed in SKOV3 cells. The five egg white all the specific combination of the 2 and V delta gamma Delta T cells, and stimulate the proliferation and secretion of interferon gamma, V delta 2 and promote the cytotoxic activity of gamma delta T cells to target cells.
In addition, this study also use the baculovirus expression system, the ratio of E.coli expression system has the biological activity of hMSH2 protein, which lays a foundation for further research of hMSH2 protein structure and function.
In summary, this research has made the following achievements:
1, proved the key role of TCR in determining CDR3 delta gamma delta antigen recognition specificity, specificity of TCR gamma delta CDR3 delta antigen recognition depends only on the primary structure, so the synthetic peptide OT3 is a CDR3 delta probe work for TCR gamma delta antigen;
2, set up a protein antigen recognized by immune CDR3 delta peptide binding characteristics of bio chemical technology strategy based on the identification of gamma delta TCR new effective method;
3, for the first time found in the case of carcinogenesis, ectopic expression of hMSH2 is probably a new identification 2 V delta gamma delta T cells tumor ligand and its biological significance in the early warning of the innate immune system;
4, prove the baculovirus expression system is a stable and efficient eukaryotic expression system can complete many of the post-translational modifications in insect cells, is of vital importance in the biological activity of the exogenous protein expression retains the natural protein.
In four the above results in second and third. Second of the most innovative achievement solves a long-standing problem in the field of immunology, as identified by the identification of TCR gamma delta ligand model provides a key technology. The third results reveal a new role of gamma delta T cell immunity. Seen, provides an important clue for the elucidation of gamma delta T cell biology function, but also provides an important molecular target for tumor diagnosis and treatment.

【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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