Prokaryotic expression and immunogenicity of the chimeric HBcAg containing Aβ(1-15).
10.3969/j.issn.1672-7347.2012.03.014
- Author:
Gaifeng FENG
1
;
Hui JIN
;
Weixi WANG
;
Yihua QIAN
;
Quanying WANG
;
Guangxiao YANG
Author Information
1. Department of Human Anatomy and Histo/Embryology, School of Medicine, Xi'an Jiaotong University, Xi'an 710061,China.
- Publication Type:Journal Article
- MeSH:
Alzheimer Disease;
prevention & control;
Amyloid beta-Peptides;
genetics;
Animals;
Base Sequence;
Genetic Vectors;
genetics;
Hepatitis B Core Antigens;
genetics;
immunology;
Humans;
Mice;
Molecular Sequence Data;
Peptide Fragments;
genetics;
Recombinant Fusion Proteins;
genetics;
immunology;
metabolism;
Vaccines, Virus-Like Particle;
genetics;
immunology;
metabolism
- From:
Journal of Central South University(Medical Sciences)
2012;37(3):290-295
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To construct a recombinant prokaryoticexpression plasmid pET/ c-Aβ(15)-c, and evaluate the immunogenicity of its encoded fusion protein as expressed in E.coli.
METHODS:The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The synthetic, double-strand Aβ(1-15) gene was inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-71- Aβ(15) was spliced to HBc88-144, yielding the recombinant gene c-Aβ(15)-c; that gene was subcloned into pET-28a(+). The fusion protein (CA15C) expressed in the transformed E.coli BL21 was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and analyzed by SDS-PAGE. The virus-like particle (VLP) formed by fusion protein CA15C was observed with transmission electric microscope (TEM). Four Kunming (KM) mice were given intraperitoneal injections of CA15C, and the anti-Aβ antibody elicited was detected by indirect ELISA.
RESULTS:The sequence of the recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, the fusion protein was expressed, mainly in the sediment from the bacterial lysate. The expression level was 40% of total protein in the sediment. The CA15C could form VLP. After 5 rounds of immunization, the titer of anti-Aβ antibody in the sera of KM mice reached 1:10000, while the anti-HBc antibody was undetectable.
CONCLUSION:Recombinant c-Aβ(15)-c gene can be expressed in E.coli. The expressed protein can form VLPs and has a strong immunogenicity.
