重组腺病毒HBV preS2/S-AD5的构建及生物安全性初步研究

Initial evaluation of the biosafty of recombinant HBV preS2/S-AD5

目的构建重组腺病毒HBVpreS2／S-AD5，并对其生物安全性进行初步评价。方法应用clontech公司的第一代腺病毒载体构建重组腺病毒质粒HBVpreS2／S-AD5，酶切线性化后分4次用脂质体方法转染HEK293细胞包装成复制缺陷型腺病毒HBYpreS2／S-AD5，分别命名为N1～4。扩增并滴定毒力后，接种Hep2、Hela和A549细胞，观察HBVpreS2／S-AD5在非容许细胞的增殖情况；同时，用N1～4分别鼻腔接种SPF级BALB／C小鼠，每日观察小鼠一般情况，第10d取小鼠重要组织并用福尔马林固定，做病理学检查。结果4株重组腺病毒HBVpreS2／S-AD5第1代N1、第4代N2、第6代N3、第8代N4株接种的细胞未见病变，接种的小鼠无一般状况改变，组织病理也未见任何改变；而第11代N1株重组腺病毒能在非容许细胞增殖并致细胞病变，接种的小鼠出现严重腹泻，并且小鼠肠、肺组织有明显的病理改变。结论本实验室构建的重组腺病毒HBVpreS2／S-AD5有突变为复制型腺病毒的现象，并可导致小鼠出现腹泻和轻型肺部炎症。

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 ceils inoculated with the llth 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 recombinant adenovirus HBV preS2/S-AD5 constructed 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.