Appl Microbiol Biotechnol (2010) 86:1067–1075 DOI 10.1007/s00253-009-2379-8 BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Production and characterization of a recombinant single-chain antibody against Hantaan virus envelop glycoprotein Jie Yang & Rui Chen & Junxia Wei & Fanglin Zhang & Yong Zhang & Lintao Jia & Yan Yan & Wen Luo & Yunxin Cao & Libo Yao & Jifeng Sun & Zhikai Xu & Angang Yang Received: 22 July 2009 / Revised: 18 November 2009 / Accepted: 19 November 2009 / Published online: 10 December 2009 # The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Hantaan virus (HTNV) is the type of Hantavirus causing hemorrhagic fever with renal syndrome, for which no specific therapeutics are available so far. Cell type-specific internalizing antibodies can be used to deliver therapeutics intracellularly to target cell and thus, have potential applica- tion in anti-HTNV infection. To achieve intracellular delivery of therapeutics, it is necessary to obtain antibodies that demonstrate sufficient cell type-specific binding, internalizing, and desired cellular trafficking. Here, we describe the prokaryotic expression, affinity purification, and functional testing of a single-chain Fv antibody fragment (scFv) against HTNV envelop glycoprotein (GP), an HTNV-specific antigen normally located on the membranes of HTNV-infected cells. Jie Yang, Rui Chen, and Junxia Wei contributed equally to this work. J. Yang : J. Sun (*) Department of Nephrology, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710038, People’s Republic of China e-mail: jifeng-sun@medmail.com.cn J. Yang : R. Chen : J. Wei : L. Jia : L. Yao State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi’an, Shaanxi 710032, People’s Republic of China F. Zhang : Y. Yan : W. Luo : Z. Xu (*) Department of Microbiology, Fourth Military Medical University, Xi’an, Shaanxi 710032, People’s Republic of China e-mail: zhikaixu@fmmu.edu.cn Y. Zhang : Y. Cao : A. Yang (*) Department of Immunology, Fourth Military Medical University, Xi’an, Shaanxi 710032, People’s Republic of China e-mail: agyang@fmmu.edu.cn This HTNV GP-targeting antibody, scFv3G1, was produced in the cytoplasm of Escherichia coli cells as a soluble protein and was purified by immobilized metal affinity chromato- graphy. The purified scFv possessed a high specific antigen- binding activity to HTNV GP and HTNV-infected Vero E6 cells and could be internalized into HTNV-infected cells probably through the clathrin-dependent endocytosis pathways similar to that observed with transferrin. Our results showed that the E. coli-produced scFv had potential applications in targeted and intracellular delivery of therapeutics against HTNV infections. Keywords Hantavirus . Hantavirus envelop glycoprotein . Single-chain Fv antibody fragment . Intracellular delivery Introduction Hantaviruses, which belong to the family of Bunyaviridae, are composed of a spherical lipid envelop, four viral proteins, and three single-stranded negative-sensed RNA segments (Schmaljohn and Dalrymple 1983). While the small (S [∼2.0 kb]) and medium segments (M [∼3.6 kb]) encode the nucleocapsid protein (NP) and the two envelop glycoproteins (GPs) G1 and G2, respectively, the large segment (L [∼6.0 kb]) contains the coding sequence for the viral RNA polymerase (Plyusnin et al. 1996). Hantaviruses can infect humans when virus-contaminated excreta from persistently but asymptomatically infected rodents are inhaled (Schmaljohn and Hjelle 1997). Within the genus Hantavirus, two groups have been identified: New World and Old World Hantaviruses. They not only differ in their geographical distribution but also vary regarding the