人類細胞株生產含多種亞型的 干擾素-a之蛋白質體學研究;Proteomics study of purified interferon-a mixture produced from human lymphoblastoid cell line

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題名:	人類細胞株生產含多種亞型的干擾素-a之蛋白質體學研究;Proteomics study of purified interferon-a mixture produced from human lymphoblastoid cell line
作者:	潘冠廷;Kuan-Ting Pan
貢獻者:	生命科學研究所
關鍵詞:	蛋白質體學;干擾素;interferon;proteomics
日期:	2004-07-08
上傳時間:	2009-09-22 10:17:46 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	干擾素屬於細胞激素的成員之一，具重要免疫調節性功能，會因病毒感染細胞而被誘發生成，並抑制人體內病毒的複製，是抵抗多種病毒感染的第一道防線。此外，干擾素還擁有如抑制細胞生長、以及廣泛的免疫調節等多種作用。由於以上特性，干擾素在醫療上已成功地用作多種病毒感染或是癌症的治療，其中包含B型肝炎與C型肝炎、白血病，淋巴瘤等疾病皆具有療效。然而，這些產品大部分是利用基因工程由大腸桿菌或中國倉鼠卵巢細胞所生產之重組的干擾素。重組干擾素的缺點在於大腸桿菌不具醣化機制，而中國倉鼠卵巢細胞的醣化和人類細胞不同，這樣的產品半衰期短、藥效低，且幾乎百分之百會引發副作用。因此，以人體自然生產的干擾素用作臨床治療一直是多年以來藥物開發的重要課題。人類干擾素-?是由13個胺基酸序列相同度約在78-95%的蛋白質所組成（即有13種亞型），其中只有?-2與?-14為醣蛋白，其他則無醣化結構。不同的亞型具有不同強度的抗病毒、抗細胞生長以及其他生物活性。在亞型組成比例上與人體自然生產之干擾素-?相似的產品，具有較高的藥效與較低的自體免疫反應。然而直到現在，干擾素-?在製備時的低濃度及其亞型組成的複雜度仍阻礙了天然干擾素-?藥物的發展。本研究則採用建教合作計畫所專利之人類淋巴纖維母細胞株，其可以生物反應器培養。本研究乃利用蛋白質體學之方法，建立分析模式，藉此分析溶液中濃度低（?g/ml），而又包含一群胺基酸序列極為相似的蛋白質。由賽德醫藥科技公司利用管柱層析所得之干擾素-?多種亞型混合物，經反相高效能液相層析儀分析，分成13-15個主要的波峰，而以硫酸十二酯鈉聚丙烯醯胺凝膠電泳分析其分子量分佈在19-24 kD之間，且分子量大於20 kD的部份應屬醣化之亞型粗估佔50-80%。而每一反相高效能液相層析之波峰透過聚丙烯醯胺凝膠電泳與醣結構偵測染劑進行分析，判斷其醣化亞型主要分佈在反相高效能液相層析中比重組干擾素-?2a滯留時間較早的波峰內，且佔全部波峰面積之57-60%。由切醣酵素分析顯示，該純化干擾素-?偵測不到N-linked的亞型，即無干擾素-?14之存在，且質譜分析中亦無此亞型。此外，經由電灑法四極棒飛行時間串聯式質譜儀的胺基酸序列分析，經各干擾素-?亞型經胰蛋白酶水解後之代表胜肽序列比對，確認有五種亞型存在(?1(13)、?2、?5、?8、?21)，並有多種亞型尚未確認(?4/10/14/17、?4/7/16/17/21) ，故純化干擾素-?中含有至少6種最多11種不同亞型。在質譜分析的前處理方面，胰蛋白酶水解條件的最適化過程中，發現還原反應的溫度由37?C提高至56?C時，鑑定亞型時的胺基酸序列覆蓋度由平均10%提高至平均47%，而若在還原反應前以95?C水浴5分鐘，更可達58-66%的序列覆蓋度。本研究提供一有效且敏感之方法探討自然生產之干擾素-?中的亞型，將可有效幫助天然之混合性蛋白質藥物的發展。 Interferon (IFN) with important immunomodulatory function. is one of the members of cytokines They are produced in response to viruses and they inhibit virus replication. Besides antiviral activities, they also show antiproliferative and immunomodulatory activities. The pleiotropic properties of IFNs have been successfully exploited in therapeutic application in several types of cancer and viral infection diseases, including leukemia, lymphoma, hepatitis B, hepatitis C…etc. However, most of the products currently on the market are recombinant IFNs expressed from Escherichia coli (E. coli) or Chinese Hamster Ovary (CHO) cell. Due to the absent or different glycosylation of such recombinant proteins, they exhibit certain shortage, including shorter half-life, less potency and almost 100% side effect. The need of natural human IFNs for clinical use is on the rise in the last several years. The human interferon-a (IFN-a) family is comprised of 13 homologous subtypes with 78-95% identity at amino acid sequence level. Only IFN-?2 and IFN-?14 are glycosylated subtypes. Previous studies have demonstrated that each of the IFN-? subtypes showed quantitatively distinct patterns in the antiviral, growth inhibitory, and other biologic activities. And IFN-a with natural structural conformation and similar composition of subtypes has higher efficacy and is less immunogenic. Until now, the minute quantities and the complexity of subtypes composition of native IFN-a produced from human cells still hindered the development of naturally derived products. In this study, the patented human lymphoblastoid cell line was cultured in bioreactor. Our goal is to establish an analytic methodology by proteomics approach to analyze a group of proteins at low concentration (?g/ml) with high identity in amino acid sequence. The purified IFN-a by a 3-step column chromatography was prepared by CytoPharm Inc. Taiwan. The IFN-? mixture was further separated into 11-15 major peaks by reversed-phase high performance liquid chromatography. The molecular weight of this preparation is 19-24 kD with 50-80% higher than 20 kD, which indicating the glycosylated subtypes. From the analysis of SDS-PAGE and stained by glyco-staining, the glycosylated subtypes were located in the peaks with earlier retention time than recombinant IFN-?2a when separated by RP-HPLC and contained 57-60% of total peak area. And there are no detectable N-linked subtypes from the deglycosylation analysis indicating there were no IFN-?14 in purified IFN-? that also confirmed by MS/MS analysis. Based on the peptide sequences analysis by electrospray quadrupole-time-of-flight tandem mass spectrometry, confirmed the present of 5 subtypes (?1(13)、?2、?5、?8、?21) and several subtypes (?4/10/14/17、?4/7/16/17/21) were not completely identified. Therefore, there were at least 6 and most 11 subtypes in purified IFN-?. For the sample pretreatment for MS analysis, changing the temperature of reduction from 37?C to 56?C raised the peptide coverage from 10% to 47% when analyzed by tandem mass spectrometry. And 58-66% amino acid sequence coverage for the identified subtypes was obtained when cooking the sample for 5 minutes before reduction. Our study provides an effective and sensitive approach to characterize the subtype composition of an IFN mixture that may efficiently accelerate the development of natural IFN drug.
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