[Construction and prokaryotic expression of recombinant gene c-Abeta-c and the immunogenicity analysis of the fusion protein].
OBJECTIVE
To construct the recombinant prokaryotic expression plasmid pHGhis/c-Abeta-c and evaluate the immunogenicity of the fusion protein expressed in E.coli DH5 alpha.


METHODS
The gene fragments of HBc1-71, HBc88-144 and Abeta(1-42) were amplified by PCR. Then the Abeta(1-42) gene was inserted between HBc1-71 and HBc88-144, yielding the recombinant gene c-Abeta-c. c-Abeta-c gene was cloned into pGEMEX and then subcloned into pHGhis plasmids. c-Abeta-c fusion protein expression in transformed E.coli DH5alpha was induced at 42degrees Celsius. The expressed fusion protein was analyzed by SDS-PAGE. Six BALB/c mice recieved intraperitoneal injection (i.p.) of c-Abeta-c fusion protein purified by saturated ammonium sulfate. The anti-Abeta antibody was detected by indirect ELISA.


RESULTS
The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After temperature induction, fusion protein was expressed and existed in the sediment of the bacterial lysate. The expression level was 16% of all the proteins in the sediment. After 3 times of immunization, the titer of anti-Abeta antibody in sera of BALB/c mice reached up to 1:16,000, while the anti-HBc antibody was undetectable.


CONCLUSIONS
Recombinant c-Abeta-c gene can be expressed in E.coli DH5 alpha. The expressed protein exists in the sediment of the bacterial lysate and has a strong immunogenicity. This study lays the foundation for the experimental animal study of Alzheimer's disease genetic engineering vaccine.
Feng
Gai-feng
GF
Department of Human Anatomy, Medical College, Xi'an Jiaotong University, Xi'an 710061, China. fenggaifeng@163.com
Hu
Hai-tao
HT
Li
Yue-ying
YY
Han
Hua
H
Wang
Quan-ying
QY
Yang
Guang-xiao
GX
D016229:Amyloid beta-Peptides / Q000235:genetics*; D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006512:Hepatitis B Core Antigens / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D016229:Amyloid beta-Peptides; D006512:Hepatitis B Core Antigens; D011993:Recombinant Fusion Proteins