以大腸桿菌C41與C43菌株表現登革熱病毒蛋白NS1、NS2、NS4與E

Abstract

登革熱病毒屬黃熱病毒科(Flaviviridae)黃熱病毒屬(Flavivirus)。它會造成登革熱(dengue fever)、登革出血熱(dengue hemorrhagic fever)及登革休克症候群(dengue shock syndrome)等症狀。登革熱病毒產生蛋白質的過程中，會先產生單一個蛋白質轉譯區，再切割成10 個獨立的蛋白質。包含3 個結構性蛋白(structural proteins) 以及7 個非結構性蛋白(non-structuralproteins)，其中外膜蛋白(envelope protein)便屬於結構性蛋白，位於病毒雙層膜的外側，是一種穿膜的蛋白質，被認為可和受體結合，與感染宿主細胞有關。NS1 與NS2A、NS2B、NS4A、NS4B 是較小的非結構性蛋白，具有疏水的特性，皆是與膜有關的蛋白質。已知NS2B 會參與蛋白切割□切割反應，而NS1、NS2A、NS4A、NS4B 則與病毒的複製有關。為研究蛋白質的功能，首先必須建構質體並於大腸桿菌系統中表現蛋白質，本研究將針對NS1、NS2A、NS2B、NS4A、NS4B 與E 蛋白等蛋白質做探討，由於這些蛋白質皆具有疏水的特性，因此選用大腸桿菌C41（DE3）及C43（DE3）菌株以利其表現。將基因構築於修飾過pET-30(+)表現載體：pET?5T-HAHis上， 分別於基因末端接上融合蛋白HA-His(influenza Hemagglutinin-Hexahistidine protein)以利抗體偵測。將此建構質體送入大腸桿菌C41（DE3）或C43（DE3）菌株後，皆可採用西方點墨法(Western blot)偵測其表現，但以Comassie blue 染色則無法偵測到。

Dengue virus is a member of family Flaviviridae, genus of Flavivirus. Dengue viruses cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Dengue virus encodes 10 proteins in a single open reading frame, including 3 structural proteins and 7 non-structural proteins. Envelope protein is one of the structural proteins locates outside of the virus particle, which can bind to receptors on host cells and causes infection. NS2A, 2B, 4A and 4B are small non-structural proteins which are membrane-associated proteins exhibiting hydrophobic profiles. NS2B has been suggested to involve in protease activity. NS1, NS2A, NS4A and NS4B have been suggested to involve in virus replication. Individual expression plasmids were constructed and expressed in E.coli C41（DE3）and C43（DE3） strains due to their hydrophobic profiles. These genes were cloned individually into a modified pET-30(+) vector, pET?5T-HAHis. Their proteins were expressed as HA-His (influenza Hemagglutinin- Hexahistidine protein) fusion proteins for antibodies detection. Recombinant proteins fused with HA-His tag could be detected by Western blotting analysis, but not by Comassie blue staining.