發(fā)布時(shí)間：2018-03-09 01:22

  本文選題：碳納米管　切入點(diǎn)：豬瘟病毒　出處：《河南科技大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：碳納米管屬于石墨烯家族,具有較強(qiáng)的跨細(xì)胞膜能力,經(jīng)過功能化的碳納米管能夠攜帶多種有機(jī)或無機(jī)粒子,如蛋白質(zhì)、核酸和化學(xué)藥物等活性分子進(jìn)入細(xì)胞內(nèi)部,激活先天性免疫細(xì)胞,如單核細(xì)胞、巨噬細(xì)胞和樹突狀細(xì)胞等。目前國(guó)內(nèi)外學(xué)者對(duì)碳納米管的研究主要集中在針對(duì)于腫瘤的診斷和治療方面,隨著對(duì)碳納米管的深入研究,越來越多的學(xué)者開始關(guān)注碳納米管在疫苗載體方面的應(yīng)用。豬瘟(classical swine fever,CSF)是由豬瘟病毒(classical swine fever virus,CSFV)感染豬所引起的一種高致病性接觸性傳染病,在養(yǎng)豬業(yè)中危害非常嚴(yán)重。E2蛋白是豬瘟病毒的主要保護(hù)性抗原,含有豬瘟病毒高度保守的抗原決定簇,能誘導(dǎo)機(jī)體產(chǎn)生豬瘟病毒的中和抗體。為探究碳納米管作為疫苗載體對(duì)動(dòng)物免疫的影響,本實(shí)驗(yàn)通過制備碳納米管-豬瘟病毒E2蛋白抗原,免疫小鼠后,檢測(cè)抗體效價(jià),以評(píng)估碳納米管顆粒疫苗對(duì)免疫的影響。本實(shí)驗(yàn)主要包括三個(gè)部分:1)用大腸桿菌進(jìn)行誘導(dǎo)表達(dá)CSFV-E2蛋白,SDS-PAGE鑒定重組蛋白E2的表達(dá)形式,采用His標(biāo)簽鎳柱親和層析法進(jìn)行純化,并經(jīng)Western Blot鑒定表達(dá)蛋白的抗原特異性。2)用EDC/NHS作偶聯(lián)劑,將純化后的重組蛋白E2與羧基化多壁碳納米管偶聯(lián),制備成碳納米管-E2蛋白抗原,并通過考馬斯亮藍(lán)法檢測(cè)連接蛋白量。3)以碳納米管-E2蛋白、E2蛋白、弗氏佐劑-E2蛋白免疫小鼠,兩次免疫,間隔兩周,于二免后第10天,使用間接ELISA檢測(cè)抗體效價(jià)。結(jié)果:經(jīng)考馬斯亮藍(lán)法檢測(cè)后,測(cè)得1mg羧基化多壁碳納米管連接蛋白量為480μg。E2蛋白組抗體效價(jià)最高達(dá)到1:3200,碳納米管-E2蛋白組抗體效價(jià)最高達(dá)到1:12800,弗氏佐劑-E2蛋白組抗體效價(jià)最高達(dá)到1:51200。結(jié)論:本研究通過構(gòu)建碳納米管-E2蛋白抗原,并對(duì)小鼠進(jìn)行免疫,通過ELISA檢測(cè),證明碳納米管能夠增強(qiáng)免疫反應(yīng),促進(jìn)機(jī)體特異性抗體的產(chǎn)生。
[Abstract]:Carbon nanotubes (CNTs) belong to the graphene family and have strong transmembrane capability. Functionalized CNTs can carry a variety of organic or inorganic particles, such as proteins, nucleic acids and chemical drugs, into the cells. Activation of congenital immune cells, such as monocytes, macrophages and dendritic cells. More and more scholars are beginning to pay attention to the application of carbon nanotubes in vaccine carriers. Classical swine virus is a highly pathogenic contact infectious disease caused by classical swine fever virus (CSFV) infection in pigs. E2 protein is the main protective antigen of CSFV and contains highly conserved antigenic determinant of CSFV. In order to investigate the effect of carbon nanotubes (CNTs) as vaccine carrier on animal immunity, we prepared E2 protein antigen of CSFV and immunized mice with antibody titers. In order to evaluate the effect of CNT particle vaccine on immunity, this experiment mainly includes three parts: 1) expression of recombinant protein E2 was identified by SDS-PAGE of CSFV-E2 protein induced by Escherichia coli, and purified by His label nickel column affinity chromatography. The antigen specificity of the expressed protein was identified by Western Blot. 2) using EDC/NHS as coupling agent, the purified recombinant protein E2 was coupled with carboxylated multiwalled carbon nanotubes to prepare a carbon nanotube protein antigen. The mice were immunized with carbon nanotube-E2 protein E2 protein and Freund's adjuvant -E2 protein by Coomassie brilliant blue method. The mice were immunized twice for two weeks, 10 days after the second immunization. Indirect ELISA was used to detect antibody titer. Results: after Coomassie brilliant blue assay, The highest titer of antibody in carboxylated multiwalled carbon nanotubes (480ug / E2) protein group was 1: 3200, the highest antibody titer of carbon nanotube E2 protein group was 1: 12800, and that of Freund's adjuvant-E2 protein group was 1: 51200.Conclusion:. By constructing carbon nanotube-E2 protein antigens, The results of ELISA test showed that carbon nanotubes could enhance the immune response and promote the production of specific antibodies.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3

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相關(guān)期刊論文 前2條

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本文編號(hào)：1586397