心臟型脂肪酸結(jié)合蛋白（H-FABP）的原核表達(dá)、純化及其單克
發(fā)布時(shí)間：2018-11-24 09:16
【摘要】：通過RT-PCR擴(kuò)增人心臟型脂肪酸結(jié)合蛋白H-FABP的基因片斷，將目的基因與原核表達(dá)載體pET28a連接，構(gòu)建原核表達(dá)重組質(zhì)粒pET28a-H-FABP。將重組質(zhì)粒轉(zhuǎn)化到大腸桿菌BL21中，通過IPTG誘導(dǎo)表達(dá)人心臟型脂肪酸結(jié)合蛋白。利用pET28a構(gòu)建的H-FABP原核表達(dá)質(zhì)粒在BL21實(shí)現(xiàn)了高效表達(dá)，經(jīng)SDS-PAGE電泳分析顯示，表達(dá)的重組蛋白分子質(zhì)量約為15kD。采用離子交換層析柱純化H-FABP重組蛋白，通過Q-HP柱（pH11.5）純化后得到高純度H-FABP重組蛋白，含量約為0.48mg/mL。Western blotting鑒定純化后重組蛋白特異性結(jié)果表明，所得重組蛋白與商品化的H-FABP單抗呈特異性反應(yīng)。 利用B細(xì)胞表位在線預(yù)測軟件，綜合分析蛋白結(jié)構(gòu)β-轉(zhuǎn)角，蛋白抗原表面可及性，蛋白柔韌性，蛋白抗原性，疏水性及線性抗原表位，我們選擇了具有較強(qiáng)抗原特異性的H-FABP區(qū)段作為免疫原與重組H-FABP一起免疫動(dòng)物。通過改良免疫方法將H-FABP全長抗原及表位肽免疫新西蘭大白兔獲得兔抗人H-FABP抗血清，應(yīng)用蛋白A柱將抗血清中IgG組分純化分離出來，應(yīng)用親和層析技術(shù)將抗體中針對(duì)重組蛋白標(biāo)簽的抗體去除，得到高特異針對(duì)H-FABP的兔抗人多克隆抗體。通過ELISA，Western blotting等方法證實(shí)了該抗體的高效價(jià)和高特異性。 使用重組H-FABP全長抗原及表位肽免疫Balb/c小鼠，應(yīng)用改良的雜交瘤技術(shù)將免疫小鼠的脾細(xì)胞與Balb/c小鼠骨髓瘤細(xì)胞Sp2/0在聚乙二醇作用下融合，，HAT選擇性培養(yǎng)，間接酶聯(lián)免疫吸附方法對(duì)融合細(xì)胞培養(yǎng)上清進(jìn)行篩選，選擇抗H-FABP單克隆抗體陽性的細(xì)胞株采用有限稀釋法連續(xù)進(jìn)行亞克隆，建立穩(wěn)定分泌抗H-FABP單克隆抗體的雜交瘤細(xì)胞株。采用小鼠腹腔內(nèi)誘生法擴(kuò)大培養(yǎng)陽性單克隆細(xì)胞株后，腹腔注射石蠟處理過的小鼠，制備腹水。腹水經(jīng)HiTrap IgG PurificationHP親和層析柱純化，所得的單克隆抗體進(jìn)行效價(jià)測定，親和力測定。經(jīng)篩選得到6株穩(wěn)定分泌抗H-FABP單克隆抗體的雜交瘤細(xì)胞株，經(jīng)亞類鑒定均為IgG型，6株單抗效價(jià)均在1：256000以上，親和力最高可達(dá)9.02×10~(-9)。Western blotting鑒定表明6株單克隆抗體均具有較高的特異性。成功制備的這6株抗H-FABP特異性的單克隆抗體，可應(yīng)用于建立一種新型H-FABP免疫檢測方法。 利用實(shí)驗(yàn)室已制備的抗H-FABP抗體作為捕獲抗體和檢測抗體，以H-FABP重組蛋白作為檢測抗原,進(jìn)行抗體配對(duì)試驗(yàn)，經(jīng)過優(yōu)化ELISA反應(yīng)條件，分別檢測26例臨床AMI血清及34例正常血清標(biāo)本中的H-FABP，并進(jìn)行結(jié)果比較分析，結(jié)果成功得到了能檢測天然H-FABP的配對(duì)抗體3-H5—1-F10，并以此成功建立了檢測H-FABP的雙抗體夾心ELISA方法。因此，所獲得的高特異性抗體3-H5—1-F10將會(huì)為今后H-FABP的檢測以及新型免疫檢測試劑盒的研發(fā)奠定重要的基礎(chǔ)。
[Abstract]:The gene fragment of human heart fatty acid binding protein (H-FABP) was amplified by RT-PCR. The target gene was ligated with prokaryotic expression vector pET28a to construct the prokaryotic expression plasmid pET28a-H-FABP.. The recombinant plasmid was transformed into Escherichia coli BL21 and induced by IPTG to express human cardiac fatty acid binding protein. The prokaryotic expression plasmid of H-FABP constructed by pET28a was highly expressed in BL21. The molecular weight of the expressed recombinant protein was about 15kD by SDS-PAGE electrophoresis. The recombinant H-FABP protein was purified by ion exchange chromatography and purified by Q-HP column (pH11.5). The high purity H-FABP recombinant protein was obtained. The specific content of the recombinant protein was identified by 0.48mg/mL.Western blotting. The recombinant protein reacted specifically with the commercial H-FABP monoclonal antibody. B cell epitope on-line prediction software was used to analyze the 尾 -rotation angle of protein structure, surface accessibility of protein antigen, protein flexibility, protein antigenicity, hydrophobicity and linear antigen epitope. We selected H-FABP region with strong antigen specificity as immunogen to immunize animals with recombinant H-FABP. Rabbit anti-human H-FABP antiserum was obtained by immunizing New Zealand white rabbits with H-FABP full-length antigen and epitope peptide by modified immune method. The IgG component in the antiserum was purified by protein A column. Rabbit anti-human polyclonal antibodies with high specificity to H-FABP were obtained by using affinity chromatography to remove the antibodies against recombinant protein labels. The high titer and specificity of the antibody were confirmed by ELISA,Western blotting and other methods. Balb/c mice were immunized with recombinant H-FABP full-length antigen and epitope peptide. The spleen cells of the immunized mice were fused with Balb/c mouse myeloma cells Sp2/0 under the action of polyethylene glycol and HAT was selectively cultured by modified hybridoma technique. Indirect enzyme-linked immunosorbent assay (Elisa) was used to screen the supernatant of the fusion cell culture. Subclones of the cell lines that were positive for H-FABP monoclonal antibody were subcloned by limited dilution method. To establish a hybridoma cell line secreting monoclonal antibody against H-FABP stably. Ascites were prepared by intraperitoneal injection of paraffin-treated mice. Ascites were purified by HiTrap IgG PurificationHP affinity chromatography. Six hybridoma cell lines stably secreting monoclonal antibodies against H-FABP were obtained. All of them were identified as IgG type by subclass identification, and the titers of 6 McAbs were above 1: 256000. The highest affinity of 9. 02 脳 10 ~ (-9). Western blotting showed that all of the 6 McAbs had high specificity. The six monoclonal antibodies against H-FABP can be used to establish a new H-FABP immunoassay. The anti H-FABP antibody prepared in the laboratory was used as the capture antibody and the detection antibody, and the recombinant H-FABP protein was used as the detection antigen. The antibody pairing test was carried out, and the conditions of ELISA reaction were optimized. H-FABPs were detected in 26 clinical AMI sera and 34 normal serum samples, and the results were compared and analyzed. The results showed that the paired antibodies 3-H5-1-F10, which could detect natural H-FABP, were successfully obtained. A double antibody sandwich ELISA method for the detection of H-FABP was successfully established. Therefore, the obtained high specific antibody 3-H5-1-F10 will lay an important foundation for the detection of H-FABP and the development of new immunoassay kit in the future.
【學(xué)位授予單位】：重慶師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1
本文編號(hào)：2353108
[Abstract]:The gene fragment of human heart fatty acid binding protein (H-FABP) was amplified by RT-PCR. The target gene was ligated with prokaryotic expression vector pET28a to construct the prokaryotic expression plasmid pET28a-H-FABP.. The recombinant plasmid was transformed into Escherichia coli BL21 and induced by IPTG to express human cardiac fatty acid binding protein. The prokaryotic expression plasmid of H-FABP constructed by pET28a was highly expressed in BL21. The molecular weight of the expressed recombinant protein was about 15kD by SDS-PAGE electrophoresis. The recombinant H-FABP protein was purified by ion exchange chromatography and purified by Q-HP column (pH11.5). The high purity H-FABP recombinant protein was obtained. The specific content of the recombinant protein was identified by 0.48mg/mL.Western blotting. The recombinant protein reacted specifically with the commercial H-FABP monoclonal antibody. B cell epitope on-line prediction software was used to analyze the 尾 -rotation angle of protein structure, surface accessibility of protein antigen, protein flexibility, protein antigenicity, hydrophobicity and linear antigen epitope. We selected H-FABP region with strong antigen specificity as immunogen to immunize animals with recombinant H-FABP. Rabbit anti-human H-FABP antiserum was obtained by immunizing New Zealand white rabbits with H-FABP full-length antigen and epitope peptide by modified immune method. The IgG component in the antiserum was purified by protein A column. Rabbit anti-human polyclonal antibodies with high specificity to H-FABP were obtained by using affinity chromatography to remove the antibodies against recombinant protein labels. The high titer and specificity of the antibody were confirmed by ELISA,Western blotting and other methods. Balb/c mice were immunized with recombinant H-FABP full-length antigen and epitope peptide. The spleen cells of the immunized mice were fused with Balb/c mouse myeloma cells Sp2/0 under the action of polyethylene glycol and HAT was selectively cultured by modified hybridoma technique. Indirect enzyme-linked immunosorbent assay (Elisa) was used to screen the supernatant of the fusion cell culture. Subclones of the cell lines that were positive for H-FABP monoclonal antibody were subcloned by limited dilution method. To establish a hybridoma cell line secreting monoclonal antibody against H-FABP stably. Ascites were prepared by intraperitoneal injection of paraffin-treated mice. Ascites were purified by HiTrap IgG PurificationHP affinity chromatography. Six hybridoma cell lines stably secreting monoclonal antibodies against H-FABP were obtained. All of them were identified as IgG type by subclass identification, and the titers of 6 McAbs were above 1: 256000. The highest affinity of 9. 02 脳 10 ~ (-9). Western blotting showed that all of the 6 McAbs had high specificity. The six monoclonal antibodies against H-FABP can be used to establish a new H-FABP immunoassay. The anti H-FABP antibody prepared in the laboratory was used as the capture antibody and the detection antibody, and the recombinant H-FABP protein was used as the detection antigen. The antibody pairing test was carried out, and the conditions of ELISA reaction were optimized. H-FABPs were detected in 26 clinical AMI sera and 34 normal serum samples, and the results were compared and analyzed. The results showed that the paired antibodies 3-H5-1-F10, which could detect natural H-FABP, were successfully obtained. A double antibody sandwich ELISA method for the detection of H-FABP was successfully established. Therefore, the obtained high specific antibody 3-H5-1-F10 will lay an important foundation for the detection of H-FABP and the development of new immunoassay kit in the future.
【學(xué)位授予單位】：重慶師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1
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