Prokaryotic expression and polyclonal antibody preparation of human adenovirus type 7 DNA binding protein.
10.3760/cma.j.cn112150-20211110-01043
- Author:
Yun ZHU
1
;
Lin Lin ZHANG
2
;
Ya Li DUAN
2
;
Zheng De XIE
1
Author Information
1. Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Research Unit of Critical Infection in Children, Chinese Academy of Medical Sciences, Beijing Children's Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children's Health, Beijing 100045, China.
2. Laboratory of Infection and Virology, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China.
- Publication Type:Journal Article
- MeSH:
Adenoviruses, Human/genetics*;
Animals;
Antibody Specificity;
Blotting, Western;
DNA-Binding Proteins/metabolism*;
Enzyme-Linked Immunosorbent Assay;
Escherichia coli/genetics*;
Immunoglobulin G;
Rabbits
- From:
Chinese Journal of Preventive Medicine
2022;56(2):171-177
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To express DNA-binding protein (DBP) of human adenovirus (HAdV) type 7 using the prokaryotic expression system, and product anti-HAdV-7 DBP rabbit polyclonal antibody. Methods: The HAdV-7 DBP gene was synthesized and cloned into prokaryotic expressing vector pET30a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein DBP was expressed by induced Isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with Ni-NTA affinity column. The titer of anti-DBP polyclonal antibody produced in immunized rabbit was measured by indirect ELISA, and the specificity of the antibody was identified by Western blotting and indirect immunofluorescence assay (IFA). In addition, purified rDBP was used as coating antigen for indirect ELISA assay to detect specific IgM and IgG antibodies against DBP in the serum of children infected with HAdV. Results: The HAdV-7 DBP plasmid was constructed successfully. The purified recombinant DBP was more than 95% after purification. The titer of polyclonal antibody was 1∶1 024 000. The polyclonal antibody showed high specificity in vitro using Western blotting and IFA. The positive rate of specific anti-DBP IgM and IgG antibody in acute-phase serum samples collected from children infected with HAdV were 50.0% (19/38) and 63.2% (24/38), respectively, using indirect ELISA. Conclusion: In summary, the HAdV-7 rDBP is expressed using prokaryotic expression system, and the recombinant HAdV-7 DBP protein and the anti-DBP rabbit polyclonal antibody with high titer are prepared.
