Construction And Rescue Of The Infectious Clone Of Bovine Adenovirus Type 1

At present, human adenovirus type 5(HAdV5) has been widely used as one of vehicles for gene therapy and vaccine study. However, its application is hampered by the pre-exciting anti-HAdV5 immunity in majority of people. To circumvent the bottleneck, two strategies have been taken. One is to modify the capsids of HAd V5, or replace HAdV5 with other human rare serotype HAdVs or non-human adenoviruses. The other is to deliver HAdV5 through mucosal immune system. For the first strategy, animal adenoviruses can be developed as an ideal vectors instead of HAdV5 because there is no cross-immunity between animal adenoviruses and human adenovirus. For the second strategy, as the reported neurotoxicity for intranasal immunization, oral mucosal immunity is more convenient, time-saving highly secure, and widely application prospect.Bovine adenovirus type 1(BAdV1) was isolated from healthy cattle intestine, which had no cross-immunity with human adenovirus and was highly stable in bovine intestine. Thus, BAdV1 could match the both needs for HAdV5 alternative. In this work, we tried to construct a novel adenovirus vector system — the infectious clone of BAdV1 based on bovine adenovirus type 1 which has great potentials for the gene function study of BAdV1 as well as for gene therapy and oral vaccine study based on BAdV1 vector. This research mainly divided into 2 parts:1. Construction of the recombinant plasmid containing full-length BAdV1 by homologous recombination in bacteriaFirst, LITR and RITR of BAdV1 genome containing two I-Sce Ⅰ endonuclesase recognition site were synthesized. And the left and right homologous arms(LITR+E1 and RITR+E4) of BAdV1 were obtained respectively by overlap PCR between LITR and amplified E1, or RITR and amplified E4, which were cloned into pUC18 to construct shuttle plasmid pUC18-E1-E4. Secondly, the linear genome of BAdV1 was isolated from wtBAdV1 infected VIDO DT1 cells. And then the infectious plasmid pUCBAdV1 flanked by I-SceⅠendonuclesase recognition site was constructed successfully by homologous recombination in E. coli BJ5183 between the Afl II- linearized pUC18-E1-E4 plasmid and the isolated linear BAdV1 genome, which has been identified by further restriction enzyme analysis.2. Rescue of the recombinant BAdV1 in VIDO DT1Recombinant BAdV1 was successfully packaged in VIDO DT1 cells expressing the endonuclease I-SceⅠtransfected by the circle genome pUCBAdV1. This proves that VIDO DT1 cells can be used to rescue and propagate BAd V1 at high viral titer. What’s more, there were no much difference between the growth tendency of recombinant BAdV1 and wtBAdV1. This suggests that viral vector based on BAdV1 make an important foundation for BAdV1 vector vaccine based on BAdV1.