Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector.
OBJECTIVE
To generate recombinant adenoviral vector containing calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.


METHODS
CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease digestion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5' end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinantion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombinant adenoviral vector to package and amplify recombinant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expression of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.


RESULTS
The CRT-HBsAg fusion gene was characterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HBsAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 x 10(11) pfu/mL. The CRT-HBsAg fusion protein was expressed by HEK 293A cells correctly.


CONCLUSIONS
CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B virus gene therapy.
Ma
Chun-Ling
CL
Immunology Laboratory of Shandong Medical College, Linyi 276002, Shandong Province, China. machl2003@163.com
Wang
Gui-Bin
GB
Gu
Run-Guo
RG
Wang
Fang
F
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D037282:Calreticulin / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006509:Hepatitis B / Q000235:genetics / Q000628:therapy; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism
D037282:Calreticulin; D006514:Hepatitis B Surface Antigens; D011993:Recombinant Fusion Proteins