Adenovirus vector and construction method thereof

Abstract

The invention discloses an adenovirus vector and a construction method thereof. The construction method comprises the following steps: electrically converting pRed / ET(delta alpha) to DB3.1 competencecontaining a pAV.Desld vector for homologous recombination with a Kan resistant fragment containing a homologous arm, and screening positive clones with Amp+Kan resistance rather than Amp+Cm resistance under a continuous resistant pressure; and carrying out recombination with attR4-Cm-ccdB-attR2 or attR4-Cm-ccdB-attR3 with the homologous arm and screening correct positive clones with Amp+Cm resistance rather than Amp+Kan resistance by means of resistance so as to obtain the adenovirus vector. The adenovirus vector provided by the invention has very good accuracy, efficiency and high success rate. The manpower and time cost is lowered greatly and the user experience is improved.

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 widely exists in nature, and its genome is about 36kb in length. Adenovirus has almost 100% infection efficiency for most cells, including dividing and non-dividing cells and primary cells, and is not integrated into chromosomes in almost all known cells except egg cells, so it will not interfere with other host genes . Since the adenovirus genome is not integrated into the host 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 virus vector system, and the most widely used one is serotype 5 adenovirus vector. In order to improve the biological safety of th...

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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