Provided are a human type 55 replication defective adenovirus vector, a method for preparing the same and uses thereof. The human type 55 replication defective adenovirus vector is prepared by the following method: knocking out E1 and E3 genes from Ad55, substituting the open reading frame 6 or the open reading frames 2, 3, 4, 6 and 6/7 of E4 gene in Ad55 genome with the corresponding open reading frames of Ad5 genome. In addition, an exogenous gene expression cassette may also be integrated into the E1 gene region of Ad55. Said human type 55 replication defective adenovirus vector is able to be produced in large scale in 293, PerC6 and other helper cell lines, and is able to be concentrated and purified by density gradient centrifugation. The human type 55 replication defective adenovirus vector is unable to replicate in normal human cells, thus has an attenuated phenotype, and the vector can express exogenous genes in target cells with high efficiency, thus can be used as vaccines or gene therapy vectors, as well as for drug and neutralizing antibody development and reporter-tracer system.
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