Viral Protein Expression of Human Immunodeficiency Virus Type I (HIV-1) from Taiwan

• Main Authors: Lee, Ming-Chang, 李明城
• Other Authors: Lee Chun-Nan
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/06501931836979682720

碩士 === 國立臺灣大學 === 醫事技術學系 === 86 === 1983年首次分離出第一型人類免疫缺乏病毒(HIV-1)後至今，目前全球已有超 過三千萬人受到感染。在尚無有效治療方法發明前，全面進行血液篩檢以控制病 毒擴散更是當務之急。目前國內 HIV-1的檢驗試劑主要仰賴國外進口，故期望能 發展出本土 HIV-1檢驗試劑，以降低檢驗成本。HIV-1的檢驗一般以偵測 HIV-1 抗體之應用為最普遍。而可利用來偵測抗體之抗原種類大致可分為三種：(1)完 整病毒之分解物；(2)重組蛋白質；(3)人工合成蛋白質片段。本研究選擇採用較 經濟、安全的重組蛋白質方式來製造HIV-1抗原(如：gp120、gp41、p31及 p24 等)。其用來表現重組蛋白質之基因序列是來自於台灣地區感染 HIV-1者身上之 病毒株。 本研究首先分析臺灣地區 HIV-1感染者體內病毒 gp41、p31及 p24蛋白質上之抗 原決定位與外國期刊曾發表此三種蛋白質上有效抗原決定位之氨基酸序列之差異 性，結果發現在 p31和 p24蛋白質上所分析之抗原決定位台灣的病毒株與國外的 病毒參考株彼此氨基酸序列十分相似。而在本研究中所分析之兩個 gp41蛋白質 的抗原決定位，台灣病毒株與國外病毒參考株之差異較大。 了解病毒株之間抗原決定位氨基酸序列之相似性後，以聚合酵素連鎖反應增幅之 基因構築在一蛋白質表現質體中，並送入大腸桿菌內，再利用 IPTG來誘發重組 蛋白質表現，最後用含有 Co2+的樹脂純化出重組蛋白質。本研究中總共表現了 八種重組蛋白質，包括了 B亞型全長和 C端短缺之外膜蛋白質 gp120、B和 E亞 型之透膜蛋白質 gp41、B和 E亞型之嵌入酵素蛋白質 p31、 E亞型之核殼蛋白質 p24和 B亞型 C端短缺之 p24等。而這些病毒蛋白質都是容易在感染者體內產生 抗體之蛋白質。將這些蛋白質與購得之 gp120、 gp41、p24單株抗體反應，結果 具有完整全長的重組蛋白質皆可與單株抗體反應，而在 gp120和 p24蛋白質 C端 有短缺的重組蛋白質 GP120-B.T及 P24-B.T則無法與購得之單株抗體反應。在這 八個重組蛋白質與 HIV-1感染者血漿的血清免疫反應結果發現，此八個蛋白質皆 可與 HIV-1 感染者之血漿有反應發生，唯獨 P24-B.T無論與 B或 E亞型病毒感 染者之血漿反應皆不明顯，推測 p24蛋白質的 C端可能具有一很有效的抗原決定 位，或是此部份蛋白質片段的缺失會改變整個蛋白質的結構，而影響了 p24的抗 原性。 由以上免疫反應可證實本研究已成功地利用較低成本培養細菌的方法，製造出 HIV-1之蛋白質抗原；期望所製造之重組蛋白質未來可應用於檢驗試劑的開發及 許多學術與臨床之研究上。 Since human immunodeficiency virus type 1 (HIV-1) was discovered in 1983, more than 30 millions people have been infected by HIV-1 in the world. Before the development of an efficient AIDS therapy, it is most important to screen donated blood and plasma by HIV-1 diagnostic kit to control the epidemic of HIV infection. The HIV-1 diagnostic kits used in Taiwan mostly are imported. In order to decrease the price of HIV-1 diagnosis, it is essential to develop a local HIV-1 diagnostic kit. For laboratory diagnosis of HIV-1, detection of HIV-1 specific antibodies is the most commonly used method. There are three different sources of antigen for antibody detection: (1) whole viral lysate, (2) recombinant protein, and (3) synthetic peptide. In this study, recombinant protein method was selected to produce antigen, because it is more economical and safe. The recombinant proteins gp120, gp41, p31 and p24 were expressed from the HIV-1 are prevalent in Taiwan. The amino acid sequences of some of the epitopes on the gp41, p31 and p24 proteins of Taiwanese HIV-1 were analyzed and compared with those of the reference HIV-1 strains. The analysis of the amino acid sequences had shown that the epitopes on the p31 and p24 were more conserved than the epitopes on the gp41 . The gp120, gp41, p31 and p24 genes were amplified by PCR, and cloned into the protein expression vector. Then the HIV-1 gene containing expression vectors were transformed into E.coli.. Protein expression was induced by adding IPTG. In this study, 8 recombinant proteins were generated, including the full length and C-terminal truncated gp120 of subtype B HIV-1, the transmenbrane portion deleted gp41 of subtype B and E HIV-1, the full length p31 of subtype B and E HIV-1, the full length p24 of subtype E HIV-1, and C-terminal truncated p24 of subtype B HIV-1. The recombinant proteins of gp120, gp41 and p24 were strongly reactive with monoclonal antibodies anti-gp120, anti-gp41 and anti-p24 , except the C-terminal truncated subtype B gp120 and p24 recombinant proteins. In the western blotting assay with HIV(+) plasma, all of the recombinant proteins had strong immunoreactivity, except the C-terminal truncated p24 recombinant protein which was much weaker than the others. According to the results of immunoreactivity, the gp120, gp41, p31, and p24 recombinant proteins of Taiwanese HIV-1 were successfully produced. We hope that these recombinant proteins could be applied for developing diagnostic kits and used in various studies.