Pre-T carrier used for preparing eukaryon expression constructing body and preparation method and application thereof

Abstract

The invention discloses a pre-T carrier used for preparing eukaryon expression constructing body and a preparation method and an application thereof, relating to the technical field of T carrier preparation. The current pre-T carrier introduces Kozak sequence and XcmI box or AhdI box into current plasmid eukaryon expression carrier, utilizes XcmI enzyme digestion or AhdI enzyme digestion to form a T carrier, and utilizes the T carrier to directly clone PCR product. The preparation method of the current pre-T carrier is as follows: (1) preparing a universal insertion sequence of eukaryon expression carrier containing Kozak sequence and XcmI box; and (2) preparing the eukaryon expression pre-T carrier. The pre-T carrier in the invention exists in the form of plasmid and can be obtained continuously by the amplification of bacteria; the pre-T carrier can be used for directly cloning PCR product, thus simplifying the double enzyme digestion process on the PCR product and the carrier; the design of primer is more simple and convenient; the expression efficiency of the exogenous protein can be improved; the invention is applicable to preparing various eukaryon expression constructing bodies rapidly by the method of TA cloning.

Description

technical field [0001] The invention relates to the technical field of T vector preparation, in particular to a pre-T vector for preparing eukaryotic expression constructs, a preparation method and application thereof. Background technique [0002] At present, the most commonly used expression vector for preparing eukaryotic expression constructs is generally a plasmid with a strong promoter and multiple cloning sites, and a linear vector is obtained by double enzyme digestion, so this vector has the advantage of being permanently used once. However, this vector cannot directly clone PCR products; when designing PCR primers for insert fragments, it is difficult to design high-quality primers if the double-digestion site and Kozak sequence are not considered. Then it may lead to low expression efficiency of the target fragment. This makes the use of existing eukaryotic expression vectors to prepare eukaryotic expression constructs more steps and inefficient. Contents of th...

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Problems solved by technology

However, this vector cannot directly clone PCR products; when designing PCR primers for insert fragments, it is difficult to design high-quality primers if the double-digestion site and Kozak sequence are not considered. Then it may lead to low expression efficiency of the target fragment

This makes the use of existing eukaryotic expression vectors to prepare eukaryotic expression constructs with many steps and low efficiency

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