B 型肝炎表面抗原a 決定位之單鍊抗體篩選與製造

Abstract

B 型肝炎病毒是一個造成世界性公共衛生問題的病原體；和其他國家相比，台灣被認為是一個高度受B 型肝炎病毒所感染的國家。在一般的檢測中，常利用B型肝炎病毒表面抗原(HBsAg)當作主要的檢測標記，來檢測B 型肝炎；此外，我們以表面抗原a 決定位(HBsAg_a)來篩選M13 噬菌體，可以使我們獲得對於HBsAg 具有專一性的單鍊抗體(scFv)。

首先，具有攜帶HBsAg_a 序列的大腸桿菌BL-21 的方法已經從先前的研究得到，此蛋白質表現後利用Ni-NTA 管柱進行純化；下一步，利用西方點墨法來檢查抗原(HBsAg_a)和抗體(anti-HBsAg)標準品之間的親和力並取得陽性的結果。

利用噬菌體展示技術，HBsAg_a 藉由M13 噬菌體反覆篩選數次，接著再以酵素結合免疫吸附分析(ELISA)來進行親和力測定。然後，在高親和性的篩選當中進一步篩選出單一菌落，用來製作單株噬菌體抗體，選擇高靈敏度(1Am)及高選擇性(1Fm)的兩株來製作目標scFv。

下一步，將scFv 基因定序後可知序列具有抗體重鍊與輕鍊之變異區(VH,VL)，將其剪接於pET-22b 載體上。進一步，將這兩組克隆先轉型至大腸桿菌DH-5α，進行序列的確認與放大後再以BL-21 表現。

最後，製作scFv 且純化，scFv 與HBsAg_a 之間的親和力藉由西方點墨法來測定並獲得陽性的結果。此外，以ELISA 和微尺度熱泳動 (MST)測量解離常數，兩組方法所測得的scFv 解離常數：1Fm ~ 3 nM 和1Am ~15 nM；在未來的工作可藉由兩組anti-HBsAg_a scFv，應用於偵測肝炎病毒的試劑，或在肝炎療程中，當作一個藥物載體。

Hepatitis B Virus (HBV) is a human pathogen substance that poses a worldwide health problem. In Taiwan, the prevalence of this infection can be determined as highly prevalent country as compared to other countries. HBV contains surface antigens (HBsAg) which is used as primary marker for identification of HBV infection in routine diagnosis. HBsAg_a determinant, a part of HBsAg, has been employed in this research. This protein serves as a target for being screened by M13 bacteriophages to produce scFv specific for anti-HBsAg_a determinant protein.

First of all, E. coli BL-21 coded specific gene for HBsAg_a determinant was obtained from previous study. This protein then was expressed and purified. Next, western blot analysis was performed, and gave positive affinity to standard anti-HBsAg.

By employing phage display technology, HBsAg_a determinant was being screened by M13 bacteriophages, then specific gene was amplified in E. coli XL-1 blue, and titers were determined by using ELISA. Furthermore, single colony was isolated for producing monoclonal phage antibody. Two clones which given high sensitivity and high specificity were chosen to produce target scFv.

Next, E. coli XL-1 Blue coded specific gene for anti-HBsAg_a determinant was constructed in pET-22b vector. This gene was amplified and implied the complete size product of scFv, VH and VL. These 2 clones were transformed into E. coli DH-5α and BL-21, respectively, to produce scFv anti-HBsAg_a determinant protein.

Finally, scFv specific for anti-HBsAg_a determinant protein was being produced and purified. Affinity binding of this scFv was performed by using western blot analysis and gave positive result. Impressively, by performing ELISA and MST (Micro Scale Thermophoresis) method, these 2 scFvs dissociation constant was around ~ 3 nM and ~15 nM. Based on these results, in future work, these 2 specific scFvs for anti-HBsAg_a determinant protein will be used either as an agent for HBV diagnosis or as drug carrier in HBV treatment.