本文選題：馬IFN-γ + 單克隆抗體　； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2008年博士論文

【摘要】： γ-干擾素(IFN-γ)是機(jī)體內(nèi)一種具有抗病毒、抗腫瘤和免疫調(diào)節(jié)功能的重要細(xì)胞因子。研究表明,內(nèi)源性IFN-γ水平的高低在很大程度上可以反映機(jī)體的細(xì)胞免疫狀態(tài),因此對(duì)樣本中的IFN-γ進(jìn)行定量分析對(duì)免疫機(jī)制研究、免疫功能分析、疫苗免疫效果評(píng)估、器官移植、過敏反應(yīng)以及多種胞內(nèi)病原感染的診斷等方面都具有極其重要的理論意義和應(yīng)用價(jià)值。然而,迄今沒有應(yīng)用于檢測(cè)馬IFN-γ的單克隆抗體,用以建立相應(yīng)的檢測(cè)方法。本研究旨在建立檢測(cè)馬IFN-γ(Equine interferon-γ, EIFN-γ)抗原捕獲ELISA、細(xì)胞內(nèi)細(xì)胞因子染色(ICS)和酶聯(lián)免疫斑點(diǎn)試驗(yàn)(ELISPOT))等方法,從而為監(jiān)測(cè)馬機(jī)體免疫狀態(tài)、研究機(jī)體感染或免疫過程中的免疫效應(yīng)及探討相關(guān)免疫機(jī)制等提供技術(shù)平臺(tái)。 為此,本研究在克隆表達(dá)EIFN-γ之后將其免疫小鼠制備了馬IFN-γ單克隆抗體,然后分別對(duì)其進(jìn)行生物素和熒光素(FITC)標(biāo)記制備了生物素標(biāo)記的馬IFN-γ單克隆抗體和FITC標(biāo)記的馬IFN-γ單克隆抗體,在此基礎(chǔ)上成功建立了檢測(cè)EIFN-γ的相關(guān)免疫學(xué)方法。 首先,從ConA刺激的馬外周血單核細(xì)胞(PBMC)中采用RT-PCR擴(kuò)增獲得含信號(hào)肽的馬IFN-γ的編碼序列,將其克隆至載體pCR2.1-TOPO中。然后以從重組質(zhì)粒pCR-EIFN-γ為模版擴(kuò)增獲得編碼馬IFN-γ成熟蛋白(mature EIFN-γ, mEIFN-γ)的基因,并將其亞克隆至原核表達(dá)載體pET-28a(+)。經(jīng)PCR、酶切及序列測(cè)定后所獲得的重組子轉(zhuǎn)化至大腸桿菌感受態(tài)細(xì)胞BL21(DE3),經(jīng)IPTG誘導(dǎo),將其產(chǎn)物進(jìn)行SDS-PAGE分析和Western blot鑒定。利用表達(dá)綠色熒光蛋白(GFP)的重組水泡性口炎病毒(VSV*GFP)作為指示病毒對(duì)所表達(dá)的EIFN-γ進(jìn)行抗病毒活性測(cè)定。結(jié)果表明,mEIFN-γ開放閱讀框全長(zhǎng)為441bp,編碼146個(gè)氨基酸;其在大腸桿菌表達(dá)系統(tǒng)的表達(dá)產(chǎn)物以可溶性和包涵體兩種形式存在,重組蛋白相對(duì)分子量約為18 ku,且具有免疫反應(yīng)活性。采用Ni+親和層析對(duì)大腸桿菌表達(dá)系統(tǒng)所表達(dá)的mEIFN-γ在非變性和變性條件下分別進(jìn)行純化,均獲得了高純度的重組馬IFN-γ,且該重組蛋白具有抗病毒活性,其活性單位為1×103 AU/mL。 將原核表達(dá)的重組EIFN-γ免疫BALB/c小鼠(Mus musculus),經(jīng)4次免疫后,取脾細(xì)胞與SP2/0骨髓瘤細(xì)胞進(jìn)行融合,以昆蟲桿狀病毒表達(dá)系統(tǒng)獲得的重組馬IFN-γ作為檢測(cè)抗原進(jìn)行間接ELISA檢測(cè)篩選,最終獲得12株能穩(wěn)定分泌抗體的單細(xì)胞克隆株。利用間接免疫熒光(IFA)實(shí)驗(yàn)對(duì)12株單克隆抗體進(jìn)行鑒定,結(jié)果表明,所獲得的12株細(xì)胞分泌的抗體均為針對(duì)EIFN-γ的特異性抗體。單克隆抗體亞型鑒定結(jié)果顯示,其中4株為IgG1、2株為IgG2a、3株為IgG2b和其余3株為IgM,而且所有12株單克隆抗體的輕鏈均為κ鏈。將此12株單克隆抗體分別與兩個(gè)截短表達(dá)的重組馬IFN-γ蛋白(GST-mEIFN-γ(1-84)和GST-mEIFN-γ(80-146))進(jìn)行特異性ELISA檢測(cè),結(jié)果表明,其中有7株單克隆抗體識(shí)別GST-mEIFN-γ(1-84),而另5株單克隆抗體與GST-mEIFN-γ(80-146)發(fā)生反應(yīng),說明所獲得的12株單克隆抗體至少針對(duì)兩個(gè)或兩個(gè)以上不同的抗原表位。而且,其中一株單抗(SB10)具有抑制重組EIFN-γ抗病毒活性作用。 將兩株識(shí)別不同表位的抗馬IFN-γ單克隆抗體腹水(A5和SB10)純化后,對(duì)其中一株純化后的單抗(SB10)進(jìn)行生物素標(biāo)記,通過對(duì)重組EIFN-γ進(jìn)行ELISA檢測(cè)來建立馬γ-干擾素雙抗體夾心ELISA。經(jīng)過方陣滴定實(shí)驗(yàn)確定捕獲抗體的最佳濃度為1μg/mL,檢測(cè)抗體的工作效價(jià)為1: 1000。利用建立的方法對(duì)不同稀釋度的重組馬IFN-γ和絲裂原刺激的馬外周血單核細(xì)胞培養(yǎng)上清進(jìn)行檢測(cè)。結(jié)果表明,此方法檢測(cè)敏感度達(dá)到1ng/mL,特異性良好。 利用熒光素FITC對(duì)純化后的抗馬IFN-γ單克隆抗體(SB10)進(jìn)行標(biāo)記,制備FITC標(biāo)記的抗EIFN-γ單克隆抗體,然后采用該抗體對(duì)馬外周血單核細(xì)胞進(jìn)行胞內(nèi)EIFN-γ單染色,之后進(jìn)行流式細(xì)胞術(shù)檢測(cè),并且與商品化的FITC標(biāo)記的抗牛IFN-γ單克隆抗體(CC302)進(jìn)行對(duì)比試驗(yàn)。結(jié)果表明,FITC標(biāo)記的抗EIFN-γ單抗能夠有效地應(yīng)用于刺激后的馬外周血單核細(xì)胞的胞內(nèi)染色檢測(cè)。 分別將識(shí)別EIFN-γ不同表位的配對(duì)單抗作為捕獲抗體和檢測(cè)抗體,對(duì)經(jīng)刺激的馬PBMC所分泌的IFN-γ進(jìn)行ELISPOT檢測(cè),從而建立檢測(cè)馬γ-干擾素ELISPOT方法。經(jīng)過方陣滴定實(shí)驗(yàn)確定捕獲抗體的包被濃度為1μg/mL,檢測(cè)抗體的工作濃度為1: 1000。 本研究所初步建立的檢測(cè)EIFN-γ的抗原捕獲ELISA、細(xì)胞內(nèi)細(xì)胞因子染色及ELISPOT方法,可為監(jiān)測(cè)馬機(jī)體免疫狀態(tài)、研究機(jī)體感染或免疫過程中的免疫效應(yīng)及探討相關(guān)免疫機(jī)制等提供技術(shù)平臺(tái)。
[Abstract]:Interferon gamma (IFN- gamma) is an important cytokine with antiviral, anti-tumor and immunomodulatory functions in the body. The study shows that the level of endogenous IFN- gamma can reflect the cellular immune state of the body to a large extent. Therefore, the quantitative analysis of IFN- gamma in the sample is used to study the immune mechanism, the immune function analysis, and the vaccine. Immunological evaluation, organ transplantation, anaphylaxis and diagnosis of multiple intracellular pathogens are of great theoretical and application value. However, no monoclonal antibodies used to detect IFN- gamma have been used so far to establish a corresponding detection method. The aim of this study is to establish the detection of horse IFN- gamma (Equine interferon- gamma, EIFN- gamma) antigen capture ELISA, intracellular cytokine staining (ICS) and enzyme linked immunosorbent assay (ELISPOT), which provide a technical platform for monitoring the immune state of the body, studying the immune effect in the body infection or immune process and exploring the related immune mechanism.
In this study, the monoclonal antibody of horse IFN- gamma was prepared after cloning and expression of EIFN- gamma. Then, biotin and fluorescein (FITC) were used to produce biotin labeled McAbs of Ma IFN- gamma and McAb labeled by FITC. On this basis, the relationship between EIFN- gamma and EIFN- gamma was successfully established. Immunological methods.
First, the encoding sequence of horse IFN- gamma containing signal peptides was amplified by RT-PCR amplification from ConA stimulated peripheral blood mononuclear cells (PBMC), and was cloned into the carrier pCR2.1-TOPO. Then, the gene encoding Ma IFN- gamma mature protein (mature EIFN- gamma, mEIFN-) was amplified from the recombinant plasmid pCR-EIFN- gamma, and was subcloned to the original. Nuclear expression vector pET-28a (+). The recombinant protein obtained by PCR, enzyme digestion and sequencing was converted to BL21 (DE3) of Escherichia coli cells, and was induced by IPTG, and its products were identified by SDS-PAGE and Western blot. Recombinant vesicular stomatitis virus (VSV*GFP) expressed by the expression of green fluorescent protein (GFP) was used as a indicator of the expression of the virus. The antiviral activity of EIFN- gamma was measured. The results showed that the total length of the mEIFN- gamma open reading frame was 441bp and encoded 146 amino acids, and the expression product of the Escherichia coli expression system existed in two forms of soluble and inclusion bodies. The relative molecular weight of the recombinant protein was about 18 Ku, and it had the activity of immunoreaction. Ni+ affinity chromatography was used for the Escherichia coli. MEIFN- gamma expressed in the expression system was purified under the conditions of non denaturation and denaturation respectively, and high purity recombinant horse IFN- gamma was obtained. The recombinant protein had antiviral activity and its active unit was 1 x 103 AU/mL..
The recombinant EIFN- gamma immunized BALB/c mice (Mus musculus) were immunized with the prokaryotic expression. After 4 times immunization, the spleen cells were fused with the SP2/0 myeloma cells, and the recombinant horse IFN- gamma obtained by the insect baculovirus expression system was used as the detection antigen for indirect ELISA detection. Finally, 12 single cell clones capable of stabilizing the antibody were obtained. 12 monoclonal antibodies were identified by indirect immunofluorescence (IFA) test. The results showed that the antibodies secreted by 12 cells were specific antibodies against EIFN- gamma. The results of monoclonal antibody subtype identification showed that 4 of them were IgG1,2 and 3 were IgG2b and the other 3 strains were IgM, and all 12 monoclonal antibodies were light. The chain was kappa. The 12 monoclonal antibodies and two truncated recombinant horse IFN- gamma protein (GST-mEIFN- gamma (1-84) and GST-mEIFN- gamma (80-146)) were detected by specific ELISA. The results showed that 7 monoclonal antibodies identified GST-mEIFN- gamma (1-84), and the other 5 monoclonal antibodies responded to GST-mEIFN- gamma (80-146). 12 monoclonal antibodies were obtained for at least two or more than two different epitopes. Moreover, one of the monoclonal antibodies (SB10) could inhibit the antiviral activity of recombinant EIFN- gamma.
After purification of two different epitopes of anti horse IFN- gamma monoclonal antibody ascites (A5 and SB10), one of the purified monoclonal antibodies (SB10) was labeled with biotin. By ELISA detection of recombinant EIFN- gamma, the optimum concentration of the captured antibody was determined to be 1 u g/mL by a square matrix titration experiment. The work titer of detection antibody was 1: 1000.. The method was used to detect the supernatant of horse peripheral blood mononuclear cell culture of recombinant horse IFN- gamma and mitogen stimulated by different dilution degrees. The results showed that the sensitivity of this method was 1ng/mL and the specificity was good.
The purified anti horse IFN- gamma monoclonal antibody (SB10) was labeled with fluorescein FITC, and the anti EIFN- gamma monoclonal antibody labeled with FITC was prepared. Then the antibody was used to carry out intracellular EIFN- gamma staining of the horse peripheral blood mononuclear cells, then the flow cytometry was performed, and the anti bovine IFN- gamma monoclonal antibody (C) labeled with commercialized FITC (C) was used. C302) comparative test. The results showed that FITC labeled anti EIFN- gamma monoclonal antibody could be effectively applied to detect intracellular staining of peripheral blood mononuclear cells after stimulation.
The matched McAbs of different epitopes of EIFN- gamma were used to capture and detect antibodies, respectively, to detect the ELISPOT of IFN- gamma secreted by the stimulated horse PBMC, and to establish the detection method of IFN- gamma interferon ELISPOT. The concentration of the captured antibody was determined by the square matrix titration experiment and the concentration of the antibody was 1 mu g/mL, and the working concentration of the antibody was 1: 1000..
The antigen capture ELISA of EIFN- gamma, intracellular cytokine staining and ELISPOT method initially established in this study can provide a technical platform for monitoring the immune status of the horse body, studying the immune effect in the body infection or immune process and exploring the related immune mechanism.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 單鋒麗;牛γ干擾素單克隆抗體的制備與鑒定[D];揚(yáng)州大學(xué);2012年

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本文編號(hào)：2108620