Prokaryotic expression of a recombinant protein of adeno-associated virus capsid conserved regions and preparation of its polyclonal antibody.
10.12122/j.issn.1673-4254.2022.06.20
- Author:
Shu Yue LI
1
;
Chun Yu CAO
1
;
Hao ZHANG
1
;
Yu Ling LI
1
;
Xiong Zhou ZHANG
1
;
Zi Can YANG
1
;
Yan XIA
2
;
Lei WANG
1
;
Ya Feng LÜ
1
Author Information
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.
- Publication Type:Journal Article
- Keywords:
adeno-associated virus;
polyclonal antibodies;
polyvalent antigenic peptide;
protein purification
- MeSH:
Animals;
Antibodies;
Capsid;
Capsid Proteins/genetics*;
Dependovirus/genetics*;
Prokaryotic Cells;
Rabbits;
Recombinant Proteins/genetics*
- From:
Journal of Southern Medical University
2022;42(6):944-948
- CountryChina
- Language:Chinese
-
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.
