本文選題：乙肝病毒核心抗原 + 融合蛋白　； 參考：《廈門大學(xué)》2008年碩士論文

【摘要】： 畢赤酵母是近年來發(fā)展迅速,應(yīng)用廣泛的真核表達(dá)系統(tǒng),能對蛋白進(jìn)行良好的翻譯后修飾,如信號肽加工、蛋白折疊、二硫鍵形成、部分脂基化和O-連接和N-連接糖基化修飾等。此外,畢赤酵母可以分泌表達(dá)外源蛋白,由于其自身胞外分泌的蛋白量很低,與胞內(nèi)表達(dá)外源蛋白的方法相比,沒有細(xì)胞破碎過程中的細(xì)胞內(nèi)溶物的釋放以及所帶來的細(xì)胞碎片的分離等問題,具有易于分離純化、下游工藝簡單等優(yōu)點(diǎn)。 戊型肝炎是由戊型肝炎病毒(HEV)引起的一種世界性的危害嚴(yán)重的傳染病,主要流行于亞洲、非洲和墨西哥等地區(qū)。HEV基因組含3個開放讀碼框架(ORF),其中ORF2編碼660個氨基酸的肽鏈,為病毒主要結(jié)構(gòu)蛋白。經(jīng)研究發(fā)現(xiàn),ORF2aa423-438在介導(dǎo)病毒與宿主細(xì)胞的結(jié)合中起重要作用,是12A10單克隆抗體的識別表位。 乙型肝炎病毒核心抗原(HBcAg)是一種良好的免疫載體,在原核和真核表達(dá)系統(tǒng)中均能自我組裝成顆粒。當(dāng)外源表位插入其MIR區(qū)時,仍可以形成顆粒,并且外源表位暴露在顆粒的表面,得到充分的展示。本研究將融合12A10表位肽的HBcAg的基因?qū)氘叧嘟湍钢?構(gòu)建工程菌株,進(jìn)行分泌表達(dá),優(yōu)化了重組菌株的培養(yǎng)條件,并對純化后的蛋白進(jìn)行性質(zhì)分析,驗(yàn)證了融合蛋白上的12A10表位有良好的免疫原性,探討了HEV表位疫苗的可行性,為酵母分泌表達(dá)蛋白顆粒提供了一個很好的范例。 首先,將嵌合有HEV受體相關(guān)表位12A10的HBcAg基因克隆到畢赤酵母分泌表達(dá)載體pPIC9k,構(gòu)建重組表達(dá)質(zhì)粒pPIC9k-HBc149-12A10,用內(nèi)切酶SacI將其線性化后電轉(zhuǎn)入畢赤酵母菌株GS115中。用G418篩選得到高抗性的轉(zhuǎn)化子經(jīng)過培養(yǎng)和甲醇誘導(dǎo),培養(yǎng)上清進(jìn)行SDS-PAGE電泳,與對照GS115/pPIC9k比較,在22kD處有一條差異蛋白帶;通過Western Blot鑒定,該條帶與單抗12A10有特異性反應(yīng)。 其次,用MGY/MM、BMG/BMM和BMGY/BMMY三組培養(yǎng)基進(jìn)行重組菌株的培養(yǎng),間接法ELISA表明,BMGY/BMMY明顯優(yōu)于其他兩組培養(yǎng)基。以BMGY/BMMY培養(yǎng)基為基礎(chǔ),進(jìn)行誘導(dǎo)溫度、誘導(dǎo)甲醇濃度和誘導(dǎo)初始pH值的單因子實(shí)驗(yàn)。在以上實(shí)驗(yàn)的基礎(chǔ)上,通過正交試驗(yàn)進(jìn)一步優(yōu)化培養(yǎng)條件,得到最佳誘導(dǎo)條件:初始pH值為7.0,甲醇濃度為1.0%,誘導(dǎo)溫度為25℃。培養(yǎng)上清液通過切向流濃縮、更換緩沖液后,進(jìn)行疏水層析純化。 最后,CsCl等密度梯度離心測得分泌出來的重組蛋白的密度為1.32g/ml。透射電鏡觀察顯示,純化的重組蛋白為均一的直徑30nm左右的空心顆粒。小鼠免疫實(shí)驗(yàn)表明,純化顆粒免疫8周后鼠血清中的特異性12A10抗體滴度可達(dá)到1.6×10~5。與含有12A10表位的p239重組顆粒的免疫原性相比,產(chǎn)生12A10特異性抗體水平有明顯的提高,提示重組顆粒較好地呈遞了HEV受體相關(guān)的非免疫優(yōu)勢表位。 在本研究中,首次構(gòu)建了能夠分泌表達(dá)攜帶有表位多肽的HBcAg蛋白顆粒的畢赤酵母工程菌,為畢赤酵母胞外分泌表達(dá)其它大尺度的重組病毒顆粒提供了參考,為研究攜帶表位多肽的顆粒載體的疫苗提供了范例,為克服酵母胞內(nèi)表達(dá)出現(xiàn)的難以純化的難題提供了新的途徑。
[Abstract]:Pichia pastoris is a kind of eukaryotic expression system which has been developed rapidly in recent years . It can be used for post - translational modification of the protein , such as signal peptide processing , protein folding , disulfide bond formation , partial glycosylation , O - connection and N - linked glycosylation modification .



HEV genome contains three open reading frames ( ORFs ) which encode 660 amino acid peptide chains , which are the main structural proteins of the virus . The results show that ORF2aa423 - 438 plays an important role in mediating the binding of virus to host cell , and is the recognition epitope of monoclonal antibody 12A10 .



The core antigen of hepatitis B virus ( HBV ) is a kind of good immune carrier , which can be self - assembled into particles in both prokaryotic and eukaryotic expression systems . When the exogenous epitope is inserted into its MIR region , it can still form particles , and the exogenous epitope is exposed on the surface of the particles to obtain a sufficient display . The present study provides a good example of the feasibility of the epitope vaccine , and provides a good example for yeast secretion expressing protein particles .



First of all , we cloned into Pichia pastoris secretion expression vector Z9k , and then transformed into Pichia pastoris GS115 by restriction enzyme SacI . The high - resistance transformants were cultured and methanol - induced to culture supernatant for SDS - PAGE electrophoresis . The band was identified by Western Blot .



The results showed that BMGY / BMMY was better than that of other two groups . The optimum conditions were obtained by orthogonal test . The optimal conditions were as follows : initial pH value was 7.0 , methanol concentration was 1.0 % , induction temperature was 25 鈩，

本文編號：1798740