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

【摘要】： 畢赤酵母是近年來發(fā)展迅速,應用廣泛的真核表達系統(tǒng),能對蛋白進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外源蛋白,由于其自身胞外分泌的蛋白量很低,與胞內表達外源蛋白的方法相比,沒有細胞破碎過程中的細胞內溶物的釋放以及所帶來的細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。 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,為病毒主要結構蛋白。經研究發(fā)現,ORF2aa423-438在介導病毒與宿主細胞的結合中起重要作用,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,在原核和真核表達系統(tǒng)中均能自我組裝成顆粒。當外源表位插入其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的基因導入畢赤酵母中,構建工程菌株,進行分泌表達,優(yōu)化了重組菌株的培養(yǎng)條件,并對純化后的蛋白進行性質分析,驗證了融合蛋白上的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受體相關表位12A10的HBcAg基因克隆到畢赤酵母分泌表達載體pPIC9k,構建重組表達質粒pPIC9k-HBc149-12A10,用內切酶SacI將其線性化后電轉入畢赤酵母菌株GS115中。用G418篩選得到高抗性的轉化子經過培養(yǎng)和甲醇誘導,培養(yǎng)上清進行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。 其次,用MGY/MM、BMG/BMM和BMGY/BMMY三組培養(yǎng)基進行重組菌株的培養(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,進行誘導溫度、誘導甲醇濃度和誘導初始pH值的單因子實驗。在以上實驗的基礎上,通過正交試驗進一步優(yōu)化培養(yǎng)條件,得到最佳誘導條件:初始pH值為7.0,甲醇濃度為1.0%,誘導溫度為25℃。培養(yǎng)上清液通過切向流濃縮、更換緩沖液后,進行疏水層析純化。 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。小鼠免疫實驗表明,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受體相關的非免疫優(yōu)勢表位。 在本研究中,首次構建了能夠分泌表達攜帶有表位多肽的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,為克服酵母胞內表達出現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，

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