A kind of adenovirus vector and its construction method

Abstract

The invention discloses and provides a recombinant adenovirus vector and a construction method thereof. The method is to electroporate pRed / ET(Δα) into DB3.1 competent cells containing pAV.Des1d vector, perform homologous recombination with a Kan-resistant fragment containing a homology arm, and screen out only the Positive clones with Amp+Kan resistance but no Amp+Cm resistance. The second step of recombination uses attR4‑Cm‑ccdB‑attR2 or attR4‑Cm‑ccdB‑attR3 with homology arms to recombine, and then use resistance to screen out those with Amp+Cm resistance but no Amp+Kan resistance Correct positive clones, thereby obtaining the adenovirus vector of the present invention. The adenovirus expression vector of the invention has good accuracy, high efficiency and high success rate. Greatly reduces labor time costs and improves user experience.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an adenovirus vector and a preparation method thereof. Background technique [0002] Adenovirus is a non-enveloped linear double-stranded DNA virus that exists widely in nature, and its genome is about 36kb in length. Adenoviruses are almost 100% efficient in infecting most cells, including dividing and nondividing cells and primary cells, and are not integrated into chromosomes in almost all known cells except egg cells, so they do not interfere with other host genes . Since the adenovirus genome does not integrate into the host's genome, the adenovirus is transiently expressed in the host. Adenoviruses can directly infect individual animals and are commonly used in vaccines and gene therapy. Due to the above advantages, adenovirus vector is a widely used viral vector system, among which the most widely used is serotype 5 adenovirus vector. In order to improve the biosafety of ade...

AI Technical Summary

Problems solved by technology

Multi-fragment fusion PCR is often prone to unsuccessful fusion, which not only increases the cost of consumables and time, but also cannot guarantee the result, which is not conducive to efficient production

[0014] In short, the existing adenoviral vectors and their construction methods have the following disadvantages: First, the existing adenoviral vectors contain attR1-attR2 recombination sites, so one LR reaction can only clone one exogenous For the target fragment, if multiple foreign fragments need to be cloned at the same time, these fragments need to be fused into one fragment by fusion PCR method, and then subsequent construction is carried out, which not only increases the difficulty of construction, but also increases time and labor costs. Not conducive to improving production efficiency

Second, after enzymatic digestion of the backbone, it is difficult to use the existing gel recovery kits on the market to recover the gel. Even if it is recovered, there is no guarantee that mechanical damage will be caused to the backbone fragments, resulting in the loss of the fragments.

Third, the traditional Red / ET homologous recombination technology has a high probability of unexpected intramolecular rearrangements and translocations, a high probability of false positives, and a low true positive rate

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