本文關(guān)鍵詞： γδT細(xì)胞 CDR3δ 蛋白抗原 hMSH2 腫瘤 昆蟲(chóng)病毒表達(dá)系統(tǒng)　出處：《中國(guó)協(xié)和醫(yī)科大學(xué)》2008年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 盡管人類(lèi)外周血γδT細(xì)胞在整個(gè)T細(xì)胞群體中所占比例較少,但其在機(jī)體對(duì)抗感染和腫瘤的免疫應(yīng)答中的重要作用卻深受關(guān)注。然而,至今為止,γδT細(xì)胞所識(shí)別的抗原則很少被鑒定。不了解T細(xì)胞受體(TCR)γδ所識(shí)別配體,也就不可能深入了解γδT細(xì)胞的生物學(xué)功能。因此,進(jìn)一步發(fā)現(xiàn)和鑒定TCRγδ所識(shí)別配體則成為γδT細(xì)胞研究領(lǐng)域亟待解決的突出問(wèn)題。如何通過(guò)新的技術(shù)策略,來(lái)發(fā)現(xiàn)TCRγδ所識(shí)別新型配體是本研究著重考慮的科學(xué)問(wèn)題,這也是進(jìn)一步了解TCRγδ抗原識(shí)別特異性和多樣性機(jī)制,揭示γδT細(xì)胞的生物學(xué)功能的關(guān)鍵問(wèn)題。 TCR的抗原結(jié)合部位是由六個(gè)抗原決定簇(CDR)形成的,這六個(gè)CDR在識(shí)別抗原時(shí)作用是不相同的,其CDR3區(qū)在抗原識(shí)別時(shí)占有主導(dǎo)地位。鑒于TCRδCDR3區(qū)(CDR3δ)與抗體重鏈的CDR3區(qū)在結(jié)構(gòu)和功能上的相似性,因此,我們提出了CDR3δ的一級(jí)結(jié)構(gòu)是決定TCRγδ識(shí)別抗原特異性的關(guān)鍵部位的學(xué)術(shù)假說(shuō)。這一假說(shuō)已為我們實(shí)驗(yàn)室前期工作所驗(yàn)證。我們根據(jù)卵巢上皮癌(OEC)組織浸潤(rùn)的γδT細(xì)胞(γδTIL)TCR的一個(gè)CDR3δ的基因序列(OT3),人工合成了OT3肽,并通過(guò)體外一系列OT3肽與靶細(xì)胞、靶組織或來(lái)源于靶細(xì)胞的蛋白的體外結(jié)合實(shí)驗(yàn),驗(yàn)證OT3肽的結(jié)合特異性,所采用的方法包括OT3肽介導(dǎo)的生物傳感器,免疫熒光技術(shù),酶免疫技術(shù)和OT3肽競(jìng)爭(zhēng)結(jié)合實(shí)驗(yàn)。同時(shí),構(gòu)建了用OT3序列替代抗體重鏈CDR3區(qū)序列的OT3移植抗體OT3Ab,也進(jìn)行相應(yīng)實(shí)驗(yàn)。本研究又重復(fù)了部分上述驗(yàn)證工作。結(jié)果顯示,OT3肽和OT3Ab在對(duì)靶細(xì)胞、靶組織或來(lái)源靶細(xì)胞的蛋白的結(jié)合活性上享有相似的特異性。這就證明了CDR3δ確實(shí)在TCRγδ抗原識(shí)別特異性上起關(guān)鍵作用,也提示OT3肽能作為一個(gè)特異的探針,去篩選TCRγδ識(shí)別的抗原。 在上述驗(yàn)證工作的基礎(chǔ)上,本研究共采用了三種技術(shù)策略去尋找TCRγδ識(shí)別的蛋白抗原:一是以O(shè)T3肽為探針在噬菌體隨機(jī)展示十二肽庫(kù)中篩選與OT3肽結(jié)合的十二肽,通過(guò)序列比對(duì)獲得相應(yīng)蛋白;二是以O(shè)T3肽為探針在人卵巢癌cDNAλ噬菌體表達(dá)文庫(kù)篩選與OT3肽結(jié)合的克隆,通過(guò)測(cè)序鑒定蛋白;三是通過(guò)OT3肽偶聯(lián)的親和層析,從OEC腫瘤細(xì)胞系(SKOV3細(xì)胞)的總蛋白中分離與OT3肽特異性結(jié)合的蛋白,再利用質(zhì)譜技術(shù)鑒定蛋白的種類(lèi)。應(yīng)用第一種技術(shù)策略,我們獲得了三個(gè)OT3肽特異結(jié)合的十二肽。結(jié)合和功能驗(yàn)證實(shí)驗(yàn)結(jié)果顯示,這些十二肽不但能特異性地結(jié)合TCRγδ,還能在體外促進(jìn)γδT細(xì)胞活化,提示這些十二肽具有TCRγδ的表位肽活性。然而,在BLAST比對(duì)中未發(fā)現(xiàn)與這些多肽在序列上相匹配的人類(lèi)相關(guān)蛋白,提示這些十二肽可能為來(lái)源于其它種屬的表位肽。篩選人卵巢癌cDNAλ噬菌體表達(dá)文庫(kù)要求探針具有較高的特異性和親和力,在這兩方面OT3肽不如一般的抗體,因此第二種策略也失敗了。通過(guò)第三種策略,我們成功鑒定了三個(gè)TCRγδ識(shí)別的腫瘤相關(guān)抗原,它們是丙酮酸激酶3,人mutS同源蛋白2(hMSH2)和熱休克蛋白(HSP)60。由于已有報(bào)道HSP60能被TCRγδ所識(shí)別,故鑒定出HSP60表明我們的策略是可行的。而丙酮酸激酶3以及與DNA損傷修復(fù)有關(guān)的hMSH2可能是TCRγδ識(shí)別的新的蛋白抗原。 本研究就hMSH2是否為T(mén)CRγδ配體這一問(wèn)題進(jìn)行了驗(yàn)證。RT-PCR結(jié)果顯示,在卵巢癌細(xì)胞系SKOV3中,hMSH2 cDNA存在散在分布的點(diǎn)突變。SKOV3細(xì)胞培養(yǎng)上清中檢測(cè)到分泌形式的hMSH2。用Western blotting在SKOV3細(xì)胞總蛋白中還發(fā)現(xiàn)一個(gè)60kD的hMSH2的缺失突變表達(dá)形式。而且,在包括SKOV3細(xì)胞在內(nèi)的很多腫瘤細(xì)胞系細(xì)胞膜上均檢測(cè)到hMSH2的表達(dá)。免疫組化結(jié)果證明,腫瘤組織中也有異位表達(dá)的hMSH2。另外,Vδ2γδT細(xì)胞對(duì)SKOV3細(xì)胞的細(xì)胞毒活性也能被抗hMSH2抗體部分封閉。 同時(shí),本研究還構(gòu)建和表達(dá)了五種重組的hMSH2蛋白,包括hMSH2全長(zhǎng)蛋白,分別含hMSH2 N端兩個(gè)結(jié)構(gòu)域和C端兩個(gè)結(jié)構(gòu)域的蛋白片段,以及SKOV3細(xì)胞表達(dá)的含有散在突變的兩個(gè)分段蛋白。所有的這五個(gè)蛋白都能特異性與Vδ2γδT細(xì)胞結(jié)合,并刺激其活化增殖,分泌γ干擾素,并且促進(jìn)Vδ2γδT細(xì)胞對(duì)靶細(xì)胞的殺傷活性。 此外,本研究還利用昆蟲(chóng)病毒表達(dá)系統(tǒng),得到了比原核大腸桿菌表達(dá)系統(tǒng)更具生物活性的hMSH2蛋白,這為hMSH2蛋白進(jìn)一步結(jié)構(gòu)與功能研究奠定了基礎(chǔ)。 綜上所述,本研究取得了以下學(xué)術(shù)成果: 1、證明了CDR3δ在決定TCRγδ識(shí)別抗原特異性上的關(guān)鍵作用,TCRγδ識(shí)別抗原時(shí)僅僅依賴(lài)CDR3δ一級(jí)結(jié)構(gòu)的特異性,因此人工合成的CDR3δ肽OT3是尋找TCRγδ抗原的很奏效的探針; 2、建立了一個(gè)采用基于CDR3δ肽結(jié)合特性的免疫-生物化學(xué)技術(shù)策略鑒定TCRγδ所識(shí)別的蛋白抗原的新的有效的方法; 3、首次發(fā)現(xiàn)在癌變的情況下,異位表達(dá)的hMSH2很可能是一個(gè)新的Vδ2γδT細(xì)胞所識(shí)別的腫瘤配體分子,其生物學(xué)意義在于對(duì)固有免疫系統(tǒng)的預(yù)警作用; 4、證明了昆蟲(chóng)病毒表達(dá)系統(tǒng)是一個(gè)穩(wěn)定而有效的真核表達(dá)系統(tǒng),在昆蟲(chóng)細(xì)胞中能完成很多翻譯后的修飾,在使表達(dá)的外源蛋白保有天然蛋白的生物學(xué)活性上至關(guān)重要。 在上述的四點(diǎn)成果中,第二項(xiàng)和第三項(xiàng)最具有創(chuàng)新性。第二項(xiàng)成果解決了一項(xiàng)長(zhǎng)期困擾免疫學(xué)界的難題,為鑒定TCRγδ所識(shí)別新型配體提供了一個(gè)有效的關(guān)鍵技術(shù)。第三項(xiàng)成果揭示了γδT細(xì)胞免疫監(jiān)視的新型作用方式,為全面闡明γδT細(xì)胞生物學(xué)功能提供了重要的線(xiàn)索,也為腫瘤的診治提供了一個(gè)重要的靶標(biāo)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.

【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

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本文編號(hào)：1540117