Phospholamban gene anti-sense RNA recombinant AAV vector, virus, construction method and application

Abstract

The invention belongs to the biotechnological field, and specifically relates to a phospholamban gene anti-sense RNA recombinant AAV vector, a virus, a construction method and application. The phospholamban gene anti-sense RNA recombinant AAV vector is prepared by the following steps: performing EcoRI and BamHI double digestion on the PLB gene fragment shown by SEQ ID NO.1 and adeno-associated virus skeleton plasmid pAAV-MCS respectively, and connecting the digestion products of the PLB gene fragment and the adeno-associated virus skeleton plasmid pAAV-MCS. The recombinant adeno-associated virus is prepared by co-transfecting engineering cells by the phospholamban gene anti-sense RNA recombinant AAV vector and helper plasmid and then packaging. The recombinant adeno-associated virus prepared in the invention overcomes the low transfection efficiency of a non-viral vector; the prepared rAAV-asPLB also overcomes the shortcomings of the recombinant adenovirus in immunogenicity, expressiontime and the like, and has the advantages of high transfection efficiency, safety, lasting expression, etc.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a phospholamban gene antisense RNA recombinant AAV vector, virus, construction method and application. Background technique [0002] With the development of the Human Genome Project and the gradual deepening of molecular biology research, the molecular mechanisms of many diseases have been elucidated and the causative genes have been continuously discovered. Gene therapy has developed from the treatment of single-gene genetic diseases to the treatment of polygenic common and frequently-occurring diseases. , especially in the treatment of cardiovascular and cerebrovascular diseases and various tumors, showing good application prospects. Three key issues in gene therapy are: 1. To further clarify the etiology and pathogenic mechanism of human diseases, especially to study and propose an effective gene therapy program from the perspective of molecular mechanism. 2. Optimization and rea...

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Problems solved by technology

[0009] The purpose of the present invention is to provide a phospholamban gene antisense RNA recombinant AAV vector, virus, construction method and application, which solves the problem of hepatotoxicity and inflammation caused by adenovirus, retrovirus and lentivirus in the prior art. The traditional preparation of recombinant AAV uses adenovirus-assisted transfection method, but it is easy to cause technical problems of adenovirus contamination

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