关键词: HBV preS2/S, 重组腺病毒载体, 复制型腺病毒

Abstract:

Objective   To assess the biosafty of HBV preS2/S-AD5，a recombinant adenovirus.    Methods   The recombinant plasmid HBV preS2/S-AD5 was constructed with the first generation adenovirus vector (clontech). After digestion into linearity with PacI, preS2/S-AD5 was transfected four times into HEK-293 cells with liposome.  Four strains of replicationdefective preS2/S-AD5, named as N1, N2, N3 and N4 were obtained. HBV preS2/S-AD5 was passaged and titered in HEK-293 before inoculation of Hep-2，Hela and A549 cells and specific-pathogen-free (SPF) BALB/C mice through the nasal mucous membrane. Then the cytopathic effect (CPE) of infected cells and the general health condition of inoculated mice were observed everyday. After 10 days the important organs of the mice were taken and fixed by formalin for histopathological tests.   Results    No CPE was observed on cells which were inoculated with the first generation of N1, the 4th generation of N2, the 6th generation of N3 and the 8th generation of N4. There were no abnormities in the general health condition of the mice and no pathological changes in histological sections were observed. But there was obvious CPE on the cells inoculated with the 11th generation of N1, and mice inoculated with the 11th generation of N1 showed severe diarrhea with pathological changes in the intestines and lungs.   Conclusion   The [KG*3]recombinant [KG*3]adenovirus HBV [KG*3]preS2/S-AD5 constructed [KG*3]in our lab displays some phenomena as if it has reversely mutated to replication-competent Ads (RCAs), which can produce diarrhea and slight pneumonia in inoculated mice.