本文選題：協(xié)同刺激分子 + PD-L2�。� 參考：《蘇州大學(xué)》2011年碩士論文

【摘要】：協(xié)同刺激分子在免疫調(diào)節(jié)中起著重要的作用,PD-1(CD279)所介導(dǎo)的負(fù)性信號(hào)能導(dǎo)致T細(xì)胞的凋亡和功能衰竭,PD-L2(B7-DC,CD273)作為PD-1的第二配體,與PD-1作用抑制T細(xì)胞活化和IFN-γ的分泌。PD-L2為B7超家族成員,在蛋白結(jié)構(gòu)上包含有IgV樣區(qū)、IgC樣區(qū)、跨膜區(qū)和一個(gè)短而保守的胞漿區(qū)尾部。最近研究表明,PD-L2與血吸蟲(chóng)病、肺結(jié)核和過(guò)敏性哮喘的發(fā)病密切相關(guān)。PD-L2在同種異體反應(yīng)、自身免疫性疾病中也發(fā)揮重要作用。研究表明,樹(shù)突狀細(xì)胞表面PD-L2與活化T細(xì)胞上PD-1相互作用后可顯著抑制效應(yīng)性T細(xì)胞的生物學(xué)功能和IL-2等細(xì)胞因子的產(chǎn)生,PD-L2分子的缺失導(dǎo)致PD-1/PD-L2抑制途徑發(fā)生異常,進(jìn)而引發(fā)機(jī)體產(chǎn)生自身免疫性疾病。 許多協(xié)同刺激分子能分別以細(xì)胞膜型和可溶性兩種形式存在,包括OX40L、CD40L、B7-H3和PD-1等�？扇苄苑肿涌梢酝ㄟ^(guò)蛋白水解酶裂解細(xì)胞上的膜型分子而形成,也可以由免疫細(xì)胞直接分泌。許多可溶性協(xié)同刺激分子具有臨床診斷價(jià)值,然而,可溶性PD-L2(sPD-L2)分子在機(jī)體中扮演何種角色,在疾病的發(fā)生發(fā)展中發(fā)揮何種生物學(xué)作用,目前尚缺乏深入研究。 本課題研究獲得了一株新型鼠抗人PD-L2功能性單克隆抗體,并對(duì)其生物學(xué)特性進(jìn)行了初步研究;在此基礎(chǔ)上建立了特異性檢測(cè)人sPD-L2的ELISA方法,并對(duì)該ELISA檢測(cè)體系的穩(wěn)定性和精確性等進(jìn)行分析、評(píng)價(jià);利用sPD-L2的ELISA方法對(duì)健康人和支氣管哮喘患者外周血中的sPD-L2表達(dá)水平及特性進(jìn)行分析。 一、鼠抗人PD-L2單克隆抗體的制備及生物學(xué)特性的研究 【目的】研制特異性識(shí)別人PD-L2(B7-DC)的鼠單克隆抗體,并對(duì)其生物學(xué)特性和PD-L2分子的表達(dá)特性進(jìn)行初步分析 【方法】以高表達(dá)人PD-L2分子的基因轉(zhuǎn)染細(xì)胞L929/PD-L2作為免疫原,常規(guī)免疫BALB/c小鼠,采用B淋巴細(xì)胞雜交瘤技術(shù)進(jìn)行細(xì)胞融合,以L929/PD-L2作為抗體篩選細(xì)胞,L929/mock為對(duì)照細(xì)胞,經(jīng)間接免疫熒光標(biāo)記和流式細(xì)胞術(shù)分析、反復(fù)篩選和多次克隆化培養(yǎng),篩選出分泌特異性鼠抗人PD-L2單克隆抗體的雜交瘤細(xì)胞株;采用Western blot、Ig亞型快速定性試紙法、間接免疫熒光法和競(jìng)爭(zhēng)結(jié)合抑制實(shí)驗(yàn)對(duì)單抗進(jìn)行生物學(xué)特性的分析,繼而利用該單抗進(jìn)行免疫熒光標(biāo)記和流式細(xì)胞術(shù)檢測(cè)PD-L2在腫瘤細(xì)胞株和免疫細(xì)胞上的表達(dá)特性。 【結(jié)果】通過(guò)多次融合和反復(fù)篩選,成功獲得一株特異性鼠抗人PD-L2的雜交瘤,該雜交瘤分泌的單克隆抗體能特異識(shí)別人PD-L2分子。繼而利用上述研制獲得的單克隆抗體8F2進(jìn)行免疫熒光標(biāo)記和流式細(xì)胞檢測(cè)發(fā)現(xiàn),PD-L2表達(dá)在THP-1、SHI-1、U937等單核來(lái)源的腫瘤細(xì)胞株,以及上調(diào)性表達(dá)在成熟的樹(shù)突狀細(xì)胞和調(diào)節(jié)性T細(xì)胞上。 【結(jié)論】成功地獲得一株特異性鼠抗人PD-L2單克隆抗體,并對(duì)其生物學(xué)特性和表達(dá)譜進(jìn)行初步分析,證明其識(shí)別抗原表位不同于商品化抗體,是一株新型鼠抗人PD-L2單抗,這為進(jìn)一步研究PD-1/PD-L2信號(hào)通路在免疫應(yīng)答中的生物學(xué)作用提供了有價(jià)值的物質(zhì)基礎(chǔ)。 二、特異性檢測(cè)人可溶性PD-L2蛋白ELISA試劑盒的研制及哮喘患者外周血sPD-L2表達(dá)水平的檢測(cè)分析 【目的】研制特異性檢測(cè)人sPD-L2的ELISA試劑盒,為定量分析不同來(lái)源的臨床標(biāo)本和研究sPD-L2的功能提供有效的檢測(cè)手段。利用該ELISA試劑盒檢測(cè)健康人和哮喘患者外周血sPD-L2蛋白的表達(dá),分析sPD-L2在哮喘發(fā)生發(fā)展過(guò)程中的作用機(jī)制。 【方法】利用anti-PD-L2 mAb(8F2)作包被抗體在碳酸鹽緩沖液中預(yù)包被ELISA板,biotin-anti-PD-L2 mAb(10D6)作為檢測(cè)抗體識(shí)別與8F2相結(jié)合的抗原,再與Streptavidin-HRP反應(yīng),最后用TMB底物進(jìn)行顯色反應(yīng),建立雙抗體夾心法檢測(cè)sPD-L2的酶聯(lián)檢測(cè)試劑盒,并對(duì)該試劑盒的特異性進(jìn)行分析。利用上述建立的ELISA試劑盒檢測(cè)了80例健康人和110例哮喘患者外周血中sPD-L2的表達(dá)。 【結(jié)果】成功研制出檢測(cè)人sPD-L2的ELISA方法,該方法能特異性測(cè)定人sPD-L2蛋白,而與其他蛋白無(wú)交叉反應(yīng)。試劑盒檢測(cè)的標(biāo)準(zhǔn)曲線在抗原濃度為1.56-100ng/ml范圍內(nèi)有良好的線性關(guān)系,并具有良好的穩(wěn)定性、準(zhǔn)確性和特異性。利用該sPD-L2 ELISA方法對(duì)哮喘患者及健康人的外周血中該因子的表達(dá)水平進(jìn)行定量分析顯示,哮喘患者血清中sPD-L2的含量顯著升高。 【結(jié)論】特異性檢測(cè)人sPD-L2 ELISA試劑盒的建立為定量分析sPD-L2蛋白因子提供了有效的檢測(cè)手段。與健康對(duì)照組相比,哮喘患者外周血sPD-L2的表達(dá)水平明顯升高。 綜上所述,本課題成功研制出能穩(wěn)定分泌特異性鼠抗人PD-L2單克隆抗體,在此基礎(chǔ)上研制出檢測(cè)人sPD-L2的ELISA試劑盒,并且利用該試劑盒檢測(cè)了哮喘病人外周血sPD-L2的表達(dá)。
[Abstract]:PD - L1 plays an important role in immune regulation . PD - 1 ( CD279 ) - mediated negative signal can induce apoptosis and function failure of T cells . PD - L2 ( B7 - DC , CD273 ) plays an important role in inhibiting T cell activation and IFN - 緯 secretion . PD - L2 is a member of B7 superfamily . PD - L2 plays an important role in allogeneic and autoimmune diseases .

Soluble molecules can be formed by proteolytic enzyme lysis of membrane - type molecules on cells and can also be secreted directly by immune cells . Many soluble co - stimulatory molecules have clinical diagnostic value , however , what role the soluble PD - L2 ( sPD - L2 ) molecules play in the organism , and what biological effect is played in the development of the disease , there is a lack of further study .

In this study , a novel anti - human PD - L2 functional monoclonal antibody against human PD - L2 was obtained and its biological characteristics were studied . On the basis of this , an ELISA method for detecting human sPD - L2 was established , and the stability and accuracy of the ELISA system were analyzed and evaluated . The sPD - L2 expression level and characteristics in peripheral blood of healthy and bronchial asthma patients were analyzed by ELISA .

Preparation and Biological Characteristics of Monoclonal Antibodies Against Human PD - L2

Objective To develop a murine monoclonal antibody specifically recognizing human PD - L2 ( B7 - DC ) , and to analyze its biological characteristics and the expression of PD - L2 molecules .

The hybridoma cell lines secreting specific mouse anti - human PD - L2 monoclonal antibodies were screened by indirect immunofluorescence and flow cytometry . Western blot , Ig subtype rapid qualitative test paper , indirect immunofluorescence assay and competitive binding inhibition assay were used to analyze the biological characteristics of monoclonal antibodies against human PD - L2 .

The hybridoma secreting monoclonal antibody can specifically recognize the human PD - L2 molecule . The monoclonal antibody 8F2 obtained by the above - mentioned development can be used for immunofluorescence labeling and flow cytometry . PD - L2 is expressed in the tumor cell line of THP - 1 , SHI - 1 , and cell line , and the supernatant is expressed on mature dendritic cells and regulatory T cells .

Conclusion The specific anti - human PD - L2 monoclonal antibody against human PD - L2 has been successfully obtained , and its biological characteristics and expression profiles are analyzed . It is shown that the recognition epitope is different from commercialized antibody , which is a new mouse anti - human PD - L2 monoclonal antibody , which provides valuable material basis for further studying the biological function of PD - 1 / PD - L2 signaling pathway in immune response .

Development of ELISA Kits for Specific Detection of Human Soluble PD - L2 Protein and Detection of sPD - L2 Expression Level in Peripheral Blood of Patients with Asthma

Objective To develop an ELISA kit for specific detection of sPD - L2 , and to provide an effective means for quantitative analysis of clinical specimens from different sources and to study the function of sPD - L2 . The expression of sPD - L2 protein in peripheral blood of healthy people and asthma patients was detected by ELISA kit . The mechanism of sPD - L2 in the development of asthma was analyzed .

The ELISA plate was pre - coated with anti - PD - L2 mAb ( 8F2 ) in carbonate buffer . The biotin - anti - PD - L2 mAb ( 10D6 ) was used as the detection antibody to identify the antigen combined with 8F2 . Then , the ELISA kit was used to detect sPD - L2 .

The results showed that the ELISA method for detecting human sPD - L2 was successfully developed . The method can specifically determine the human sPD - L2 protein without cross reaction with other proteins . The standard curve detected by the kit has a good linear relationship in the range of 1 . 56 - 100 ng / ml , and has good stability , accuracy and specificity . The quantitative analysis of the expression level of the factor in the peripheral blood of patients with asthma and healthy people by using the sPD - L2 ELISA method shows that the content of sPD - L2 in the serum of asthma patients is significantly increased .

Conclusion The sPD - L2 ELISA kit has been established to provide an effective means for quantitative analysis of sPD - L2 protein factor . Compared with the healthy control group , the expression level of sPD - L2 in peripheral blood of patients with asthma is significantly increased .

In conclusion , this study successfully developed an anti - human PD - L2 monoclonal antibody which can stably secrete specific murine PD - L2 . On the basis of this , an ELISA kit for detecting human sPD - L2 was developed , and the expression of sPD - L2 in peripheral blood of asthmatic patients was detected by using the kit .
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392

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相關(guān)期刊論文 前3條

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本文編號(hào)：2071948