title: Expression, purification and production of antisera against recombinant truncated VP22 protein.
abstract: Cell-penetrating peptides (CPPs) are non-invasive vectors that can efficiently transport bioactive cargo across the cell membrane. Naturally occurring CPPs, such as the tegument protein VP22 of the Herpes simplex virus type 1, can potentiate protein-drug delivery into living cells. The aim of the present study was to construct anti-VP22 antibodies that can be used to detect VP22-fusion drugs. Therefore, 60- and 45-amino acid peptides corresponding to the N-terminus and C-terminus of VP22, respectively, were cloned, expressed and purified. Subsequently, polyclonal antisera against them were generated. The DNA sequence, cloned into the pGEX-5X-1 vector, was transformed into E. coli BL21 (DE3). After inducing expression with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 25°C for 4 h, the recombinant VP22 proteins were purified by electroelution. The high titers of polyclonal antisera obtained subsequent to immunization of mice with the purified recombinant truncated VP22 was confirmed by ELISA. Western blot and immunofluorescence analysis showed that the antisera detected both the truncated and full-length VP22 protein. Therefore, the polyclonal antisera against VP22 may be used in the detection of the intracellular location of VP22-fusion protein drugs.
authors.affiliation: Department of Pharmacy, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, P.R. China.
authors.forename: Xian
authors.initials: X
authors.lastname: Yu
authors.affiliation: Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China.
authors.forename: Jun
authors.initials: J
authors.lastname: Lei
authors.affiliation: Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China.
authors.forename: Zhengmin
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan 637007, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
keywords: VP22; antisera; expression; purification