A Single Chain Fv Specific Against Western Equine Encephalitis Virus

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Authors
  1. Xu, B.
  2. Kriangkum, J.
  3. Nagata, L.P.
  4. Fulton, R.E.
  5. Suresh, M.R.
Corporate Authors
Defence R&D Canada - Suffield, Ralston ALTA (CAN)
Abstract
A recombinant single chain Fv (scFv) specific against Western equine encephalitis virus (WEE) was developed and characterized. The scFv was generated from 11D2 hybridoma producing anti-WEE antibody reactive to E1 component of viral envelope glycoprotein. V sub L and V sub H gene segments of 11D2 scFv were generated and joined together with a (gly sub 4 ser) sub 3 linker by polymerase chain reaction (PCR). The resulting scFv was successfully expressed in P. pastoris expression system. Fifteen individual plasmids were tested and six of them were shown to drive scFv expression. DNA sequence analysis from three productive plasmids showed that they all carried the same V sub L and V sub H gene segments with a few base differences. Comparison of 11D2 scFv DNA sequence to the Kabat database showed that V sub H of 11D2 antibody belonged to subgroup IIID and sub-family XIV, while V sub L domain did not belong to any known subgroup or subfamily. Western blot analysis of 11D2 scFv using anti-c-myc antibody for detection showed different band pattern among clones derived from different plasmids. This was thought to be due to the different glycosylation where amino acid substitution occurred. Successful purification of 11D2 scFv could be done by immobilized metal affinity chromatography with an unoptimized yield of 700 Mu g/L. Functional studies showed that 11D2 could bind to its respective WEE antigen as demonstrated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA).
Keywords
WEEV (Western Equine Encephalitis Virus);Sequencing;Genetic engineering;Recombinant antibody technology;Genetic analysis;ELISA (Enzyme Linked Immunosorben);Nucleic acid sequencing
Report Number
DRDC-SUFFIELD-SL-2003-091 — Reprint
Date of publication
14 May 2003
Number of Pages
10
Reprinted from
Ybridoma, vol 18, no 4, 1999, p 315-323
DSTKIM No
CA022368
CANDIS No
519234
Format(s):
Hardcopy;Document Image stored on Optical Disk

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