S Li1, C Cao1, H Zhang1, Y Li1, X Zhang1, Z Yang1, Y Xia2, L Wang1, Y Lü1. 1. Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, Three Gorges University Medical College, Yichang 443000, China. 2. Hubei Provincial Key Laboratory of Occurrence and Intervention of Rheumatic Diseases, Affiliated Hospital of Hubei University for Nationalities, Enshi 445000, China.
Abstract
OBJECTIVE: To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody. METHODS: The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay. RESULTS: The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10. CONCLUSION: We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.
OBJECTIVE: To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody. METHODS: The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay. RESULTS: The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10. CONCLUSION: We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.
Entities:
Keywords:
adeno-associated virus; polyclonal antibodies; polyvalent antigenic peptide; protein purification
Authors: Toloo Taghian; Miklos G Marosfoi; Ajit S Puri; Oguz I Cataltepe; Robert M King; Elise B Diffie; Anne S Maguire; Douglas R Martin; Deborah Fernau; Ana Rita Batista; Tim Kuchel; Chris Christou; Raj Perumal; Sundeep Chandra; Paul D Gamlin; Stephanie G Bertrand; Terence R Flotte; Diane McKenna-Yasek; Phillip W L Tai; Neil Aronin; Matthew J Gounis; Miguel Sena-Esteves; Heather L Gray-Edwards Journal: Mol Ther Date: 2019-11-16 Impact factor: 11.454
Authors: Markus Riessland; Anna Kaczmarek; Svenja Schneider; Kathryn J Swoboda; Heiko Löhr; Cathleen Bradler; Vanessa Grysko; Maria Dimitriadi; Seyyedmohsen Hosseinibarkooie; Laura Torres-Benito; Miriam Peters; Aaradhita Upadhyay; Nasim Biglari; Sandra Kröber; Irmgard Hölker; Lutz Garbes; Christian Gilissen; Alexander Hoischen; Gudrun Nürnberg; Peter Nürnberg; Michael Walter; Frank Rigo; C Frank Bennett; Min Jeong Kye; Anne C Hart; Matthias Hammerschmidt; Peter Kloppenburg; Brunhilde Wirth Journal: Am J Hum Genet Date: 2017-01-26 Impact factor: 11.025
Authors: Jerry R Mendell; Samiah Al-Zaidy; Richard Shell; W Dave Arnold; Louise R Rodino-Klapac; Thomas W Prior; Linda Lowes; Lindsay Alfano; Katherine Berry; Kathleen Church; John T Kissel; Sukumar Nagendran; James L'Italien; Douglas M Sproule; Courtney Wells; Jessica A Cardenas; Marjet D Heitzer; Allan Kaspar; Sarah Corcoran; Lyndsey Braun; Shibi Likhite; Carlos Miranda; Kathrin Meyer; K D Foust; Arthur H M Burghes; Brian K Kaspar Journal: N Engl J Med Date: 2017-11-02 Impact factor: 91.245
Authors: Marti Cabanes-Creus; Samantha L Ginn; Anais K Amaya; Sophia H Y Liao; Adrian Westhaus; Claus V Hallwirth; Patrick Wilmott; Jason Ward; Kimberley L Dilworth; Giorgia Santilli; Arkadiusz Rybicki; Hiroyuki Nakai; Adrian J Thrasher; Adrian C Filip; Ian E Alexander; Leszek Lisowski Journal: Mol Ther Methods Clin Dev Date: 2018-11-01 Impact factor: 6.698
Authors: Xiangjun He; Brian Anugerah Urip; Zhenjie Zhang; Chun Christopher Ngan; Bo Feng Journal: J Mol Med (Berl) Date: 2021-02-16 Impact factor: 4.599