ArticleWorld journal of gastroenterology2010
Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector.
Article in World journal of gastroenterology, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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4 authors at 1 institution in 2 countries.
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Abstract
aimTo generate recombinant adenoviral vector containing calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.
methodsCRT 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.
resultsThe 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.
conclusionCRT/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.
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