Recombinant adenovirus expression vector SAd23-L

Abstract

The invention discloses a recombinant adenovirus expression vector, which is characterized in that an E1 coding region and an E3 coding region are deleted through a molecular cloning method based on agenome of a wild-type adenovirus SAd23, I-Ceu I and PI-Sce I cleavage sites are inserted in the E1 deletion region, and meanwhile, an E4 coding region of SAd23 is replaced with the E4 coding region of an adenovirus human serotype Ad5, so that the success rate of packaging the recombinant virus and the titer of the recombinant virus in human kidney embryo cells (HEK293) are improved, and the replication-defective recombinant adenovirus expression vector is obtained. The vector has the advantages that: 1. a large fragment of plasmids are constructed through a direct molecular cloning method, the method is simple and easy to operate, and can be used for constructing any large fragment of the vector; 2. the adenovirus expression vector can be constructed through multiple cloning connections,the steps are simple; and 3. screening positive clones at the bacterial level is easier and faster, and the construction time of the adenovirus vector is shortened.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a recombinant adenovirus vector used for gene therapy and vaccine vectors; specifically, the invention relates to a recombinant adenovirus expression vector SAd23-L. Background technique [0002] The development of adenovirus (adenovirus, Ad) as a gene expression vector began in the early 1960s. Adenoviruses can stimulate and induce effective T cell and B cell immune responses in different animals, but different types of adenoviruses have different inducing abilities. Adenoviruses in subgroup C induced the strongest specific T cell and B cell immune responses against foreign genes. Expression vectors based on adenovirus human serotype 5 (Ad5) and 2 (Ad2) are widely used in vaccine research and treatment, but due to the ubiquitous immune response against Ad5 adenovirus itself in the human body, this pre-existing immune response Including anti-adenovirus neutralizing antibody and kil...

AI Technical Summary

Problems solved by technology

However, the inventors have found in practice that the carrier Sad23 constructed by this method encountered difficulties in amplifying the virus and preparing for purification after the virus was successfully packaged. After the virus infected many HEK293 cells, the cells were recovered and frozen. Melt the cells, and the virus purified from the supernatant cesium chloride always fails to reach the ideal virus titer. After Sad23-eGFP virus infects 90 75T HEK293 cells, the recombinant virus obtained is purified, and the final titer is determined by plaque formation experiments. degrees only 10 9 PFU / ml

That is, in the process of amplifying the virus with the Sad23 virus vector, the amount of virus produced is too low, which then affects its actual clinical application, so further improvement is needed

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