Brief Article
Copyright ©2010 Baishideng. All rights reserved
World J Gastroenterol. Jun 28, 2010; 16(24): 3078-3082
Published online Jun 28, 2010. doi: 10.3748/wjg.v16.i24.3078
Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector
Chun-Ling Ma, Gui-Bin Wang, Run-Guo Gu, Fang Wang
Chun-Ling Ma, Run-Guo Gu, Immunology Laboratory of Shandong Medical College, Linyi 276002, Shandong Province, China
Gui-Bin Wang, Imaging Division, Renmin Hospital of Linyi City, Linyi 276003, Shandong Province, China
Fang Wang, Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Author contributions: Ma CL, Wang GB and Wang F performed the majority of experiments; Gu RG provided vital agents and was involved in editing the manuscript; Ma CL designed the study and wrote the manuscript.
Supported by Grants from National Natural Science Foundation of China, No. 30901344
Correspondence to: Chun-Ling Ma, Professor and Chief, Immunology Laboratory of Shandong Medical College, Linyi 276002, Shandong Province, China.
Telephone: +86-539-8075592 Fax: +86-539-8075592
Received: February 23, 2010
Revised: April 23, 2010
Accepted: April 30, 2010
Published online: June 28, 2010

AIM: 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 × 1011 pfu/mL. The CRT-HBsAg fusion protein was expressed by HEK 293A cells correctly.

CONCLUSION: 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.

Keywords: Calreticulin, Hepatitis B virus, Hepatitis B surface antigen, Adenovirus expression vector, Fusion protein, Therapeutic vaccine