【摘要】：脂蛋白相關(guān)磷脂酶A2(lipoprotein associated phospholipase A2,LP-PLA2)屬于VIIA型磷脂酶A2,它可以預(yù)測(cè)與冠脈事件有關(guān)的動(dòng)脈粥樣硬化和中風(fēng)危險(xiǎn)。目前,在國(guó)內(nèi),LP-PLA2臨床診斷試劑主要依賴于從國(guó)外進(jìn)口,所需費(fèi)用昂貴,限制了該檢測(cè)項(xiàng)目在我國(guó)的推廣及應(yīng)用,因此,研發(fā)LP-PLA2體外免疫診斷試劑尤為重要。 目的本研究旨在獲取人脂蛋白相關(guān)磷脂酶A2蛋白,制備相應(yīng)抗體,同時(shí)驗(yàn)證經(jīng)生物信息學(xué)預(yù)測(cè)的表位肽的抗原性,為L(zhǎng)P-PLA2體外免疫診斷試劑的研發(fā)奠定基礎(chǔ)。 方法采用Trizol法從分化的THP-1細(xì)胞中提取總RNA,經(jīng)RT-PCR擴(kuò)增目的片段LP-PLA2;利用分子克隆技術(shù)構(gòu)建原核表達(dá)質(zhì)粒;轉(zhuǎn)化入大腸桿菌BL21(DE3),優(yōu)化誘導(dǎo)表達(dá)條件,進(jìn)行多步驟純化并除去標(biāo)簽,獲得目的蛋白,免疫新西蘭大白兔,制備抗LP-PLA2的多克隆抗體;運(yùn)用多種生物信息學(xué)方法對(duì)LP-PLA2抗原表位進(jìn)行預(yù)測(cè),并用純化的LP-PLA2多克隆抗體鑒定其抗原性。 結(jié)果 1.成功構(gòu)建了PET-28a(+)-LP-PLA2,PGEX-4T-2-LP-PLA2,pCold TF-LP-PLA2等重組表達(dá)質(zhì)粒。 2.經(jīng)IPTG誘導(dǎo)表達(dá)后,SDS-PAGE電泳顯示:LP-PLA2在PET-28a(+)-LP-PLA2表達(dá)載體中不表達(dá);在PGEX-4T-2-LP-PLA2表達(dá)載體中少量表達(dá),且主要以包涵體形式存在;在pCold TF-LP-PLA2表達(dá)載體中,獲得表達(dá)量較高的融合蛋白,主要以上清形式存在。 3.將pCold TF-LP-PLA2誘導(dǎo)表達(dá)上清經(jīng)鎳柱親和層析純化后,獲得TF-LP-PLA2重組蛋白,將該重組蛋白用HRV-3C prolease酶切后,用藍(lán)膠分離純化,獲得了純度約為90%、分子量約為45kD的蛋白,與預(yù)期相符,且該蛋白能夠被市售的LP-PLA2多克隆抗體識(shí)別,證明獲得了純度較高的LP-PLA2目的蛋白。 4.制備了抗LP-PLA2的多克隆抗體,抗體效價(jià)1:5.12×106 ,Western blot結(jié)果表明,該抗體特異性較高。 5.運(yùn)用生物信息學(xué)軟件綜合分析預(yù)測(cè)了LP-PLA2的三段表位肽,體外抗原抗體免疫反應(yīng)實(shí)驗(yàn)證實(shí)了三段表位肽均具有良好的抗原性。 結(jié)論成功表達(dá)并純化獲得了高純度的LP-PLA2蛋白;制備了效價(jià)和純度均較高的LP-PLA2多克隆抗體;運(yùn)用生物信息學(xué)分析方法預(yù)測(cè)了LP-PLA2的三段表位肽,并證實(shí)了其具有良好的抗原性,可用于進(jìn)一步制備多克隆和單克隆抗體。本研究為L(zhǎng)P-PLA2體外診斷試劑的研發(fā)奠定了基礎(chǔ)。
[Abstract]:Lipoprotein associated phospholipase A 2 (lipoprotein associated phospholipase A 2) belongs to VIIA type phospholipase A 2, which can predict the risk of atherosclerosis and stroke associated with coronary events. At present, the LP-PLA2 clinical diagnostic reagent mainly depends on imported from abroad, and the cost is expensive, which limits the application and popularization of the test project in China. Therefore, it is particularly important to develop LP-PLA2 in vitro immunodiagnostic reagent. Objective to obtain human lipoprotein associated phospholipase A2 protein and prepare the corresponding antibody, and to verify the antigenicity of the epitope peptide predicted by bioinformatics, so as to lay a foundation for the development of LP-PLA2 immunological diagnostic reagent in vitro. Methods the total RNA, was extracted from differentiated THP-1 cells by Trizol method and the target fragment LP-PLA2; was amplified by RT-PCR. The prokaryotic expression plasmid was constructed by molecular cloning technique. E. coli BL21 (DE3) was transformed into Escherichia coli (DE3), and the expression conditions were optimized. The target protein was purified and the label was removed. The target protein was immunized against New Zealand white rabbits, and the polyclonal antibody against LP-PLA2 was prepared. LP-PLA2 epitopes were predicted by various bioinformatics methods and their antigenicity was identified with purified LP-PLA2 polyclonal antibodies. Result 1. PET-28a ()-LP-PLA2,PGEX-4T-2-LP-PLA2,pCold TF-LP-PLA2 and other recombinant expression plasmids were successfully constructed. 2. After IPTG induced expression, SDS-PAGE electrophoresis showed that LP-PLA2 was not expressed in PET-28a ()-LP-PLA2 expression vector, but was expressed in a small amount in PGEX-4T-2-LP-PLA2 expression vector, and mainly in the form of inclusion body. In the pCold TF-LP-PLA2 expression vector, the fusion protein with high expression level was obtained, and the main supernatant was found. 3. The recombinant protein of pCold TF-LP-PLA2 was purified by nickel column affinity chromatography. The recombinant protein was digested with HRV-3C prolease and purified with blue gel. The purity of the recombinant protein was about 90%. The protein with a molecular weight of about 45kD was in line with the expectation, and the protein could be recognized by the LP-PLA2 polyclonal antibody on the market. It was proved that the purified LP-PLA2 target protein was obtained. 4. The polyclonal antibody against LP-PLA2 was prepared. The titer of the antibody was 1: 5.12 脳 10 ~ 6, Western blot. The specificity of the antibody was high. 5. The three-segment epitope peptides of LP-PLA2 were predicted by bioinformatics software. The results of antigen-antibody immunoreactivity in vitro confirmed that all three epitope peptides had good antigenicity. Conclusion High purity LP-PLA2 protein was successfully expressed and purified, and LP-PLA2 polyclonal antibody with high titer and purity was prepared. The three-segment epitope peptide of LP-PLA2 was predicted by bioinformatics, and its antigenicity was confirmed. It can be used to prepare polyclonal and monoclonal antibodies. This study laid a foundation for the development of LP-PLA2 in vitro diagnostic reagent.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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