Expression vector based on recombinant adenoviruses and construction method thereof

Abstract

The invention discloses a method for preparing an expression vector of adenoviruses. The method comprises the following steps: using a genome of wild type adenoviruses AdC6 as a base, deleting an E1 coding region and an E3 coding region according to a genome direct cloning method, and inserting I-Ceu I and PI-Sce I digestion sites in the E1 deleted region. Simultaneously, OFR6 of human-derived adenoviruses Ad5 is used for replacing ORF6 of AdC6, the success rate of packaging recombinant viruses and the titer of the packaged recombinant viruses in human-derived kidney blastocyte (293A) are improved, and the replication-deficient expression vector of the recombinant adenovirus is obtained. According to the direct molecular cloning method, long-fragment plasmids are constructed, the method issimple and easy to operate, and the method can be used for construction of any long-fragment vectors. The construction of the expression vector of the adenoviruses can be completed through several times of cloning connection, and the steps are simple; the E1 and E3 regions are deleted, the Ad5 ORF6 is replaced, and the deletion and replacing can be completed in the PCR amplification process of DNA fragments; the method is easier and quicker in horizontal bacteria screening and positive cloning, and the construction time of the adenovirus vector is shortened.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant adenovirus vector for gene therapy and vaccine vector and its construction method. Background technique [0002] The development of adenovirus (adenovirus, Ad) as a gene expression carrier began in the early 1960s, when virologists observed that the adenovirus genome can hybridize with the simian virus 40 (SV40) genome, indicating that the adenovirus genome can carry heterogeneity. source gene. Since then, adenovirus has gradually developed into an important vector system. [0003] At present, 57 different adenovirus serotypes have been identified. Recombinant adenovirus vectors have been widely used in gene therapy and vaccine vectors, especially human serotype 5 adenovirus, which is currently used in tumor gene therapy and human immunodeficiency The primary viral vector for viral (HIV) vaccine research. In addition, the development of vaccines for pathogens such as...

AI Technical Summary

Problems solved by technology

The traditional method is to carry out homologous recombination of exogenous gene and wild-type virus in the packaging virus cell line to obtain recombinant adenovirus, but this method is complicated to operate and it is not easy to obtain a single recombinant adenovirus; the second method is Based on homologous recombination in Escherichia coli, its disadvantage is that it is relatively time-consuming and prone to mutation; the third method is to directly clone the viral genome into a plasmid vector, and then linearize the transfection of the packaging cell line to package the recombinant adenovirus

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