| Summary: | 碩士 === 中原大學 === 生物科技研究所 === 102 === Influenza A H5N1 virus also known as highly pathogenic influenza viruses（HPAIV）are mainly spread among poultry and birds with severe symptoms or even death. The first case of human infection H5N1 from poultry is reported in Hong Kong in 1997. According to World Health Organization statistics , H5N1 have caused 375 deaths and up to 60 percent mortality rate from 2003 to June 2013. Comparing to other influenza A virus, mortality rate caused by H5N1 is out of 10-100 fold. Therefore, the influenza virus is a global public health problem which requires a high degree of attention. In this study, we express the single-chain variable fragment（scFv）generated from coding gene of H5N1 monoclonal antibody by baculovirus expression vector system（BEVS）. The scFv is a fusion protein of the variable regions of heavy chain（VH）and light chains（VL）of immunoglobulins, which connected with a short linker peptide of 10 to 25 amino acids. ScFv can be useful for diagnosis, therapy and research. In addition, our previous studies indicated that co-expression of the chaperon β-synuclein（β-syn）or a translation initiation factor eIF4E can enhance secreted protein productions in BEVS. Thus, a further combination of eIF4E and b-syn with scFv raise the possibility to increase the production of scFv in BEVS. We planed to express secreted scfv against HA protein of H5N1 in BEVS. After successfully cloned the scfv into expression vectors and generated the purified recombinant viruses, we could detect the scFv in infected cell lysate and medium by western blot analysis. Furthermore, we found that β-syn and eIF4E could enhance scFv protein secretion compared to the control. We can use BEVS to express and purify H5N1 scFv antibodies successfully by Ni-NTA column and we also validated that co-expression of β-syn and eIF4E together can prolongs cell viability. In the future, the function of purified ScFv such as competitive ELISA or neutralizing ability need to be further confirmed. We hope that it could expand the application in diagnostic or therapeutic for H5N1 infection.
|
|---|
