Rabies viruses CTN strain complete genome infectious cDNA cloning, and preparation and use thereof

Abstract

The present invention provides a rabies virus CTN strain whole genome cDNA infectious clone, as well as a method of using reverse genetics technology to save the active rabies virus. The present inventive method includes: using the single restriction enzyme sites in the CTN strain of rabies virus full-length genome to divide the full-length virus into four segments to amplify, simultaneously using the green fluorescent protein gene to substitute 423bp base at the pseudo-gene point, cloning in the pVAX-R expression vector, to construct a recombinant full-length plasmid of the virus genome; performing PCR amplification of nucleoprotein, phosphoprotein, glycoprotein and transcription large protein gene sequence of the CTN strain of the rabies virus, and cloning the amplified fragment into a vector pVAX1, to constitute four auxiliary plasmids in the reverse heredity system; and then using the five plasmids to jointly transfect cells, to rescue the rabies virus. The present invention successfully saves the rabies virus, and has a great significance in the study of the pathogenic mechanism of rabies virus, development of new vaccines, viral vectors and the like areas.

Description

technical field [0001] The invention relates to a rabies virus, more specifically uses reverse genetic technology to rescue recombinant rabies virus, and belongs to the field of bioengineering. Background technique [0002] Rabies (RV) is a zoonotic disease caused by rabies virus, and the case fatality rate is almost as high as 100%. The disease is prevalent in more than 100 countries and regions. The number of rabies deaths in the world reaches 55,000 people every year. India is the country with the most severe rabies epidemic, accounting for half of the deaths. The number of rabies cases in China is second only to India, accounting for the world's second. Rabies virus has neurotropism, and the infection in animals can be divided into two processes: centripetal diffusion and centrifugal diffusion. The former is that the virus invades the peripheral nerve from the invasion site through the nerve-muscle junction or nerve sensor, and then reaches the central nervous system a...

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Problems solved by technology

First, the minimum structures of the active centers of HamRz and HdvRz include 45nt and 85nt, respectively, such long primers will inevitably cause length mismatch between upstream and downstream primers; GC% imbalance between primers; dimer and hairpin structures between primers are inevitable; Secondly, due to the difference in the length of the upstream and downstream primers, it is difficult to find a suitable annealing temperature, and the PCR reaction conditions are also difficult to optimize

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