Screening and Production of Anti HBsAg_a Determinant ScFv

• Main Authors: Widjajana, Moonika Sari, 陳美花
• Other Authors: Li,Yaw-Kuen
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/6y984b

碩士 === 國立交通大學 === 跨領域分子科學國際碩士學位學程 === 104 === 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.