抗 H7N9 流感病毒血球凝集素之單鏈抗體的製備與生化性質分析

Abstract

H7N9流感病毒自2013年爆發後，至2018年為止共有6波流感疫情，平均死亡率約為40%，且病毒株已突變為高病原性，並逐漸適應人群，對人類健康的潛在威脅更高，因此開發H7N9流感病毒的快速檢驗平台與治療性抗體日益重要。本實驗室已製備出抗H7N9流感病毒血球凝集素之鼠源單株抗體F3-2，此論文進一步選殖出F3-2之VH與VL基因，並透過大腸桿菌表現出可溶形式的F3-2單鏈抗體，且其具有結合H7N9 virion的能力。每1 L之大腸桿菌約可表現及純化出2 mg之F3-2單鏈抗體。為了使單鏈抗體在原態時能形成雙聚體，使其在結合能力上更接近單株抗體，本研究亦改變了單鏈抗體中連結VL與VH間之peptide linker的長度。當peptide linker少於5個胺基酸時，可以形成二價的diScFv，並且仍保有結合H7N9 virion的能力。每1 L之大腸桿菌約可表現及純化出1.63 mg之F3-2 diScFv。F3-2 diScFv與ScFv對H7HA之解離常數介於12.9–88.9 nM之間。本論文亦於F3-2單鏈抗體之C端接上抗病毒胜肽 (F3-2 ScFv-AVP)，希望可以增加其抑制H7N9流感病毒之功效。然而，F3-2 ScFv-AVP雖保有結合H7N9 virion的能力，且對H7HA的解離常數介於5.7–54.4 nM之間，但仍無法有效抑制H7N9流感病毒感染MDCK細胞。

After the first outbreak of the H7N9 influenza virus in 2013, there were six waves of H7N9 influenza epidemics until 2018, with an average mortality rate of about 40%. The virus strain has been mutated to high pathogenicity and gradually adapted to the population, posing a potential threat to human health. The preparedness of rapid-test kits and neutralizing antibodies against the novel H7N9 virus is becoming more and more important. F3-2 is a mouse monoclonal antibody developed in our laboratory that specifically targets H7N9 HA. The present study further cloned the VH and VL genes of F3-2 and expressed the recombinant F3-2 ScFv (single chain variable fragments) in E. coli as the soluble form, which retained the ability for binding to H7N9 virion. 2 mg of F3-2 ScFv can be purified from 1 L of E. coli culture. In order to make ScFvs form a dimer in the native condition to mimic the antigen-binding capability of a monoclonal antibody, a peptide linker with different length was also inserted between VL and VH domains. Data showed that when the peptide length was shorter than five amino acid residues (e.g. GGS, GGGS, or GGGGS), the F3-2 ScFv mainly formed as a diScFv, which also contained the ability for binding to H7N9 virion. 1.63 mg of F3-2 diScFv can be purified from 1 L of E. coli culture. The dissociation constants (KD) of F3-2 ScFv and F3-2 diScFv for binding to H7HA were in the range of 12.9-88.9 nM. To increase the capability of F3-2 ScFv and F3-2 diScFv for neutralizing H7N9 influenza virus, the antiviral peptide (AVP) was added to the C-terminus of the F3-2 ScFvs. The F3-2 ScFv-AVP performed the ability for binding to H7N9 virion, and had KD in the range of 5.7-54.4 nM for binding to H7HA. However, F3-2 ScFv-AVP couldn’t neutralize H7N9 virus replication in MDCK cells.