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Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

World Journal of Gastroenterology 2010年第24期16卷 3078-3082页

作者： Ma, Chun-Ling Wang, Gui-Bin Gu, Run-Guo Wang, Fang Shandong Med Coll Immunol Lab Linyi 276002 Shandong Peoples R China Renmin Hosp Linyi City Imaging Div Linyi 276003 Shandong Peoples R China Nanjing Med Univ Affiliated Hosp 1 Dept Lab Med Nanjing 210029 Jiangsu Prov Peoples R China

AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic ***: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion 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 recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western ***: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg 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 ex-pressed 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 vi-rus gene therapy.

关键词： Calreticulin Hepatitis B virus Hepatitis B surface antigen adenovirus expression vector Fusion protein Therapeutic vaccine

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