RNA病毒檢驗創新技術開發

Development of Innovative Techniques for RNA Virus Diagnosis

黃榮淵(Rong-Yuan Huang)

指導教授 ： 張大慈

國立清華大學/生命科學院/分子與細胞生物研究所/博士(2009年)

https://doi.org/10.6843/NTHU.2009.00245

摘要

根據美國食品與藥物管理局(FDA)定義，體外診斷器材(In Vitro Diagnostic Device, IVD device)係用於診斷疾病或決定健康狀態，以治癒、緩和、處理或預防疾病或其後遺症之診斷用試劑、儀器與系統。相關器材主用於蒐集、準備及檢查取自於人體之樣本。本研究針對RNA病毒之體外檢測，開發新穎C型肝炎病毒(Hepatitis C virus, HCV)核酸檢測(Nucleic Acid Testing, NAT)及人類免疫缺陷病毒(Human immunodeficiency virus, HCV)免疫檢測(EIA, enzyme immunoassay)系統雛型。
本研究首先以生物資訊軟體Remus與MISA分析C型肝炎病毒之5’untranslated region序列，針對六種HCV基因型設計專一性的探針，開發新型的HCV genotyping PCR-ELOSA (Enzyme-Linked Oligonucleotide-Sorbent Assay)病毒核酸檢測方法。實際比較本項新型技術與市面上通用之HCV核酸檢測試劑之性能，發現於HCV血清標準品與100支HCV陽性檢體中，本技術之有效檢測率及靈敏度均較高，顯示本技術能提升HCV病毒檢驗之品質與成效。
本研究以電腦模擬篩選(in silico)與體外(in vitro)醣晶片分析，發現米根黴菌(Rhizopus oryzae)葡萄糖澱粉酶(glucoamylase, GA)的澱粉結合蛋白(starch-binding domain, SBD)之立體結構與人類單株抗體2G12相似，且與2G12一般可識別HIV表面醣蛋白抗原gp120。本研究設計直接三明治ELISA分析，證明RoSBD的確可利用蛋白質與多醣分子之交互作用與HIV1-gp120之特殊醣抗原結合。此外，亦發現麴菌(Aspergillus niger) 葡萄糖澱粉酶的澱粉結合蛋白(AnSBD)亦具相同作用。本研究首先發現SBD為新穎的HIV gp120結合蛋白，並利用其結構與功能特性開發創新的病毒偵測理論與試劑。
綜合言之，本論文發現並報導具新穎性之HCV病毒基因型與HIV病毒抗原檢測技術，並已開發出HCV genotyping PCR-ELOSA與HIV-SBD antigen ELISA檢測試劑雛型，未來將可實際應用於RNA病毒之分子檢測與改良，具體貢獻於生物醫學體外診斷器材領域。

關鍵字

RNA病毒

並列摘要

In vitro diagnostic device (IVD device) includes reagents, calibrators, sample collection device, control materials, and related instruments or apparatus according to the definition of U.S. Food and Drug Administration (USFDA). IVD device is now widely used for diagnostic, monitoring or preventive purposes. In this thesis investigation of in vitro diagnostic strategy for infectious RNA viruses leads to development of proto-type diagnosis including nucleic acid testing (NAT) system for Hepatitis C virus (HCV) and enzyme immunoassay (EIA) for Human immunodeficiency virus (HIV).
At first Remus and MISA bioinformatics software were employed to analyze the 5’ untranslated region sequences in several HCV strains. Then the oligonucleotide primers and probes specifically reacted to six different HCV genotypes were designed and tested in a new HCV genotyping Polymerase Chain Reaction Enzyme-Linked Oligonucleotide-Sorbent Assay (PCR-ELOSA). Screening of 100 clinical serum samples using our novel PCR-ELOSA system indicated higher sensitivity and specificity than commercial NAT diagnostic kits. . It demonstrated the quality and performance of this new technique.
In addition, in silico molecular modeling was carried out to investigate protein-glycan interaction. It was found that the structure of starch-binding domain (SBD) of Rhizopus oryzae glucoamylase (RoGA) was quite similar to the VH domain of human 2G12 monoclonal antibody, which recognized the HIV envelope glycoprotein. Here direct sandwich ELISA and in vitro glycan array analysis proved that RoSBD could bind to HIV1-gp120 glycoprotein through protein-polysaccharide interaction. Likewise, the SBD of Aspergillus niger glucoamylase (AnSBD) showed the same binding specificity. Hence we have firstly discovered and reported novel binding ability between RoSBD / AnSBD and a special glycan structure of HIV1 gp120 envelope glycoprotein, which leads to development of novel molecular diagnostic theory and reagents for detection of RNA virus, and may further contribute to the fields of biomedicine and biotechnology.

並列關鍵字

RNA virus

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