复制型HBV重组腺病毒的制备

Construction of recombinant adenovirus with replicative HBV genome

目的 通过细菌体内同源重组方法，构建含1．3拷贝HBV DNA片段（HBV4．1）的HBV复制型重组腺病毒。方法从质粒pTh—HBV4．1上获取HBV4．1片段，插入腺病毒穿梭质粒载体pAdTrack—CMV中，构建含HBV4．1的重组穿梭质粒pAdTrack-CMV／HBV4．1。然后在大肠杆菌BJ5183内与pAdEasy—1进行同源重组。形成重组腺病毒质粒pAd—GFP／HBV4．1，经脂质体2000转染HEK293细胞进行包装和扩增，获得腺病毒Ad-GFP／HBV4．1。通过荧光显微镜下观察GFP绿色荧光蛋白的表达，检测腺病毒滴度和感染效率；采用PCR法检测腺病毒DNA上有无HBV基因组存在。结果经酶切鉴定确认含HBV穿梭质粒pAdTrack—CMV／HBV4．1扣重组腺病毒基因组质粒pad—GFP／HBV4．1构建成功；GFP荧光检测提示重组腺病毒Ad-GFP／HBV4．1已成功包装；PCR扩增表明重组腺病毒内携带有HBVDNA片段。在HEK293细胞中获得的病毒滴度可达（4．2—4．8）×10^7 efu／ml；当MOI为100时，感染HEK293细胞的效率〉90％。结论成功构建了携带1．3拷贝HBV基因组的重组腺病毒Ad-GFP／HBV4．1，为进一步建立高水平HBV复制型细胞和动物模型以用于HBV生物学特性的研究奠定了基础。

Objective To construct recombinant adenovirus containing 1.3 copies of HBV DNA fragments（ HBV4.1 ） through the bacteria in vivo homologous recombination methods. Methods The recombinant plasmid pAdTrack-CMV/HBV4.1 was constructed throuth subclone of a HBV 4 . 1 fragment from pTh - HBV 4 . 1 plasmid to adenovirus shuttle vector pAdTrack - CMV . pAdTrack - CMV /HBV4.1 was transformed into E. eoli BJ5183 which contained PAdEasy-I for homologous recombination. Plasmids pAd-GFP/HBV4.1 was identified with restriction endonuelease PaeI. It was digested with PaeI and sequently transfeeted into human embryo kidney 293 cells （HEK293）using Lipofeetamine 2000. Through the observation of green fluorescent protein expression, titers and infection efficiency of adenovirus was detected;the HBV DNA within the adnovirus geome was identified by PCR. Results By conffinning with restrictiong en- donuelease,plasmid pAdTraek-CMV/HBV4.1 and pAd-GFP/HBV4. 1 containing HBV4.1 fragment was successfully constructed; Re- combination adenovirus Ad-GFP/HBV4.1 earring HBV4.1 fragment was successfully obtained. The virus titer was up to （4.2- 4.8） × 10^7 efu/ml;When the MOI of 100, the efficieney for infecting of HEK293 cells was 90% . Conclusion The adenoviruses containing HBV4.1 fragment was constructed successfully. It with be very useful for the establishment of high level HBV replication cell and animal models,and therefore for the study of the biological characteristics of HBV in future.