[Construction of replication-deficient recombinant adenoviral vector carrying HBsAg and HSP70 chimeric gene and its expression in vitro].
OBJECTIVE
To construct a recombinant adenoviral vector carrying HBcAg-HSP70 chimeric gene by homologous recombination in bacteria and to detect its expression in vitro.


METHODS
Heat shock protein 70 gene from Mycobacterium tuberculosis were amplified by PCR and were cloned to adenoviral shuttle plasmid pAdTrack-CMV-HBsAg. Then the resultant pAdTrack-CMV-HBsAg-HSP70 was cotransfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying HBsAg-HSP70 gene (pAd-HBsAg-HSP70) was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 cells. Several kinds of mammal cells (293 cells and Vero cells) were infected with adenoviruses and the expression of HBsAg-HSP70 was detected by RT-PCR and ELISA in vitro.


RESULTS
The adenoviral plasmids pAd-HBsAg-HSP70 were obtained by selection for kanamycin resistance and confirmed by restriction endonuclease Pac analyses. The recombinant adenoviruses Ad-HBsAg-HSP70 were packaged successfully in 293 cells. The titer of Ad-HBsAg-HSP70 was up to 2 x 10(12) pfu/L after the second passage of proliferation in 293 cells. HBsAg and HSP70 were expressed efficiently in mammal cells after infection.


CONCLUSIONS
The recombinant adenoviruses expressing HBsAg and HSP70 were constructed successfully which can be used further in study of gene therapy for HBV.
The Eighth Hospital of Guangzhou City, Guangzhou, China.
Chun-liang
CL
Lei
Cheng-hui
CH
Huang
Zhan
Z
Yang
Xiao-ping
XP
Tang
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003673:Defective Viruses / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014709:Vero Cells; D014779:Virus Replication / Q000235:genetics
D018840:HSP70 Heat-Shock Proteins; D006514:Hepatitis B Surface Antigens; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins