本文選題：乙肝病毒核心蛋白病毒樣顆粒(HBc-VLP)　切入點(diǎn)：病毒樣顆粒(VLP)　出處：《中南大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:乙肝病毒核心蛋白可自我組裝成病毒樣顆粒(VLP),HBc-VLP作為表位遞呈系統(tǒng)可用于攜帶外源表位。除了應(yīng)用于VLP疫苗和表位疫苗以外,VLP還可用于病毒顆粒的攝取,病毒藥物活性以及樹突狀細(xì)胞(DCs)的抗原遞呈功能等研究中。然而,我們發(fā)現(xiàn)一些商業(yè)化單克隆抗體并不能特異性識(shí)別HBc-VLP,同樣也無法應(yīng)用于細(xì)胞內(nèi)病毒樣顆粒的定位研究,于是我們不得不購(gòu)買大量單抗以找到一種合適的,這一過程無疑費(fèi)力又費(fèi)錢。因次,要制備出一個(gè)HBc-VLP特異性多功能單克隆抗體以滿足多種研究的不同需要。 方法:①構(gòu)建與表達(dá)HBV核心抗原:截短的HBc基因(aa1-144)通過PCR擴(kuò)增而來,PCR產(chǎn)物經(jīng)酶切后,轉(zhuǎn)入表達(dá)載體pET-28a。陽性質(zhì)粒轉(zhuǎn)染BL21(DE3)大腸桿菌菌株,用IPTG誘導(dǎo);②快速純化重組HBc蛋白:離心獲得濕重為9克的菌體,重懸加入溶菌酶后超聲破菌獲得包含體。將包含體加入10ml變性緩沖液(50mM Tris-HCL,pH 8.0和0.1M尿素),不能溶解的成分通過離心(30 min 12000g)去除。用復(fù)性緩沖液稀釋至2mg/ml的濃度。復(fù)性蛋白溶液用雙蒸水(pH6.0)透析以去除殘留的尿素。并用SDS-PAGE確定其純度;③電鏡分析所制備的VLP;④制備抗HBc-VLP單克隆抗體:用HBc-VLP(aa1-144)作為免疫原來免疫BALB/c雌鼠。經(jīng)過幾次免疫后,將抗體滴度高的BALB/c鼠的脾細(xì)胞與SP2/0骨髓瘤細(xì)胞融合。先用ELISA篩選,再進(jìn)行western印跡分析以進(jìn)一步篩選,獲得HBc-VLP特異性抗體;⑤鼠單核-巨噬細(xì)胞系RAW264.7的吞噬作用的檢測(cè);⑥利用共聚焦激光掃描顯微鏡對(duì)HBc-VLP表位疫苗的攝取過程進(jìn)行觀察;⑦HepG2.2.15細(xì)胞中活HBV的觀察:將HepG2.2.15細(xì)胞接種于玻片上并培養(yǎng),以觀察先前所篩選的陽性單抗是否能與自然病毒顆粒反應(yīng)。HepG2細(xì)胞作為陰性對(duì)照。 結(jié)果:①成功構(gòu)建了pET-28a-HBc(aa1-144),經(jīng)SDS-PAGE分析,其所表達(dá)的目的蛋白分子量為19kDa;②經(jīng)western印跡分析驗(yàn)證該目的蛋白的確具備HBc抗原的免疫原性;③0.1M尿素所溶解的HBc蛋白可形成病毒樣顆粒;經(jīng)超純水透析后的HBc-VLP的結(jié)構(gòu)更加規(guī)則統(tǒng)一;④用HBc-VLP作為免疫原來免疫BALB/c雌鼠通過ELISA和western印跡分析篩選到5個(gè)HBc-VLP特異性抗體;⑤篩選到3個(gè)可與被APC攝取的HBc-VLP相結(jié)合的特異性單抗。這三個(gè)單抗的編號(hào)分別為3H2D7-F6,1H7D7-D2和388E6-D3;⑥利用HepG2.2.15細(xì)胞系篩選到1個(gè)單抗能特異性結(jié)合自然狀態(tài)下的HBc顆粒,此單抗編號(hào)為3H2D7-F6。 結(jié)論:①以截短的HBc-VLP骨架作免疫原,以此表位遞呈系統(tǒng)為篩選平臺(tái),最終篩選出1個(gè)可用于鑒定HBc-VLP,嵌合體HBc-VLP疫苗和細(xì)胞內(nèi)乙肝病毒衣殼蛋白的抗體。該抗體具備多功能抗HBc-VLP作用,應(yīng)用此多功能單抗可節(jié)約大量經(jīng)費(fèi)。 ②構(gòu)建與表達(dá)的目的蛋白具有HBc抗原的免疫原性。 ③0.1M尿素所溶解的HBc蛋白可形成病毒樣顆粒。
[Abstract]:Objective: HBV core protein can be self-assembled into virus-like particles HBc-VLP as epitope presenting system can be used to carry exogenous epitopes. It can also be used for the uptake of viral particles in addition to VLP vaccine and epitope vaccine. However, we found that some commercial monoclonal antibodies can not specifically recognize HBc-VLP, nor can they be applied to the localization of virus-like particles in cells. Therefore, we have to purchase a large number of McAbs to find a suitable one, and this process is painstaking and costly. Therefore, we have to produce a HBc-VLP specific multifunctional monoclonal antibody to meet the different needs of many kinds of research. Methods HBV core antigen: truncated HBc gene was amplified by PCR. The product was digested into the expression vector pET-28a. the positive plasmid was transfected into E. coli strain BL21mDE3. The recombinant HBc protein was rapidly purified by IPTG induction. The recombinant HBc protein was obtained by centrifugation with a wet weight of 9 g. After adding lysozyme in heavy suspension, the inclusion was obtained by ultrasonic breaking. The inclusion body was added to 10ml denaturing buffer solution (50mm Tris-HCL, pH 8.0 and 0.1M urea). The insoluble components were removed by centrifugation (30 min 12000g). The refolding buffer was diluted to the concentration of 2mg / ml. Sex protein solution was dialyzed with double distilled water pH 6.0) to remove residual urea. Monoclonal antibody against HBc-VLP was prepared by using SDS-PAGE to determine its purity. Monoclonal antibody against HBc-VLP was prepared by using HBc-VLPna1-144). After several times of immunization, the female mice were immunized. The spleen cells of BALB/c mice with high antibody titer were fused with SP2/0 myeloma cells. The spleen cells were screened by ELISA and then analyzed by western blotting. Detection of phagocytosis of monocyte macrophage RAW264.7 derived from HBc-VLP specific antibody the uptake of HBc-VLP epitope vaccine in 7HepG2.2.15 cells was observed by confocal laser scanning microscopy (confocal laser scanning microscope). Planted on a glass and cultured, To observe whether the previously screened positive monoclonal antibody can react with natural virus particles. HepG2 cells were used as negative control. Results the pET-28a-HBcca1-144G was successfully constructed by 1: 1. The molecular weight of the expressed protein was 19kDaNi2 by SDS-PAGE analysis. The result of western blot analysis showed that the target protein had the immunogenicity of HBc antigen and the HBc protein dissolved by 30.1 M urea could form virus-like particles. After ultrapure water dialysis, the structure of HBc-VLP was more regular and uniform. HBc-VLP was used to immunize BALB/c female mice. Five HBc-VLP specific antibodies were screened by ELISA and western blotting analysis. Three of them could be combined with HBc-VLP uptake by APC. The numbers of the three McAbs were 3H2D7-F6H7D7-D2 and 388E6-D3O6, respectively, which were screened by HepG2.2.15 cell line to specifically bind to HBc particles in natural state. The McAb number is 3H _ 2D _ 7-F _ 6. Conclusion the truncated HBc-VLP skeleton was used as the immunogen by using the epitope presenting system as the screening platform. Finally, an antibody which can be used to identify HBc-VLP, chimeric HBc-VLP vaccine and hepatitis B virus capsid protein was screened. The antibody has multifunctional anti HBc-VLP effect, and the application of this multifunctional monoclonal antibody can save a lot of money. 2 the constructed and expressed target protein has immunogenicity of HBc antigen. HBc protein dissolved in 30.1 M urea can form virus-like particles.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前4條

1 邱愛東;四川地區(qū)宮頸癌患者組織中高危型人乳頭瘤病毒的分子流行病學(xué)研究[D];吉林大學(xué);2006年

2 聞曉波;新城疫病毒樣顆粒的構(gòu)建及其出芽機(jī)制的研究[D];中國(guó)農(nóng)業(yè)科學(xué)院;2006年

3 韓廣洲;人單純皰疹病毒Ⅱ型（HSV-2）轉(zhuǎn)化片段惡性轉(zhuǎn)化人乳頭瘤病毒16型（HPV-16）永生化人宮頸鱗狀上皮細(xì)胞的細(xì)胞生物學(xué)和分子生物學(xué)研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);1997年

4 李炯;口蹄疫病毒空衣殼蛋白的表達(dá)加工及其免疫原性研究[D];中國(guó)農(nóng)業(yè)科學(xué)院;2009年

相關(guān)碩士學(xué)位論文 前10條

1 劉春國(guó);新城疫病毒樣顆粒的研究[D];中國(guó)農(nóng)業(yè)科學(xué)院;2004年

2 喬彩霞;禽白血病勞斯肉瘤病毒衣殼蛋白p27基因的克隆、表達(dá)及免疫學(xué)診斷方法的建立[D];中國(guó)農(nóng)業(yè)科學(xué)院;2004年

3 季新成;豬瘟病毒E2基因的克隆及其在昆蟲細(xì)胞中的表達(dá)[D];西北農(nóng)林科技大學(xué);2004年

4 張秀華;HPV6型早期蛋白E7在尖銳濕疣組織中的表達(dá)[D];山東大學(xué);2005年

5 烏恩奇;四川廣元宮頸癌患者組織人乳頭瘤病毒的分子流行病學(xué)調(diào)查[D];吉林大學(xué);2006年

6 吳芬芳;新城疫病毒樣顆粒免疫效力的初步研究[D];新疆農(nóng)業(yè)大學(xué);2007年

7 高秀春;G9型豬輪狀病毒VP2/4/6/7蛋白真核表達(dá)及組裝病毒樣顆粒研究[D];東北農(nóng)業(yè)大學(xué);2007年

8 崔曉辰;A群輪狀病毒單克隆抗體及免疫膠體金試紙條制備[D];東北農(nóng)業(yè)大學(xué);2008年

9 胡秋炅;PPV SC-1株VP2蛋白Cys-402、214的定點(diǎn)突變及其對(duì)PPV VLPs影響的初步研究[D];四川農(nóng)業(yè)大學(xué);2008年

10 孔娜;H5亞型AIV HA1基因與雞IL-18成熟蛋白基因重組桿狀病毒表達(dá)載體的構(gòu)建及其表達(dá)[D];山東農(nóng)業(yè)大學(xué);2008年

，

本文編號(hào)：1623457