Expression type pre-T vector, preparation thereof and applications

Abstract

The invention provides an expression type pre-T-vector for quickly cloning and expressing the product PCR of the target gene, and a preparation and an application thereof. The advantages of the expression type pre-T-vector mainly lie as follows: the target gene can be cloned and expressed by one step method, thereby omitting complicated recombination processes; the product PCR is not needed to bepurified and enzyme cut in construction process, and linearized T-vector can be obtained by the method that the pre-T-vector is single enzyme cut by XcmI with high enzyme cutting efficiency without using other restrictively restriction enzymes; the target gene is not needed to be induced by expensive IPTG, which can ferment engineering bacteria in large scale with lower cost; and the sequencing can be identified after the target gene is expressed, and then transformants whose expression results are up to expectation are sent to the DNA sequencing company to be sequenced, so the sequencing success rate can be improved, and the expenses can be saved.

Description

(1) Technical field [0001] The invention relates to an expression type pre-T vector which can be used for fast cloning and expressing PCR products of target genes and its preparation and application. (2) Background technology [0002] Genetic engineering is to recombine the target gene into an expression vector, and transform the recombinant expression vector into an appropriate host to express the required protein, thereby exerting benefits. [0003] PCR technology is the most commonly used method to obtain target gene fragments, and is a basic technique in molecular biology and genetic engineering research. It is widely used in vitro to amplify known or unknown specific DNA fragments. There are already T vectors used for rapid cloning of PCR products on the market, such as pUCm-T vectors. Applicants such as Lin Chenshui of the present invention have applied for a pre-T vector for cloning target PCR products and its preparation method. Because the rapid and effective cloni...

AI Technical Summary

Problems solved by technology

This method has obvious disadvantages: first, it cannot guarantee that 100% of the ends of the carrier molecules can be added with dT, and second, it is difficult to control exactly 1 dT to be added.

[0013] The following problems may exist in the preparation of T vectors by enzymatic methods: First, if the quality of the digested plasmid DNA is not high, the foreign gene cannot be inserted into the vector, and the vector can grow on the resistant plate after transforming competent cells

This method requires the use of expensive X-gal, and it is troublesome to prepare blue-white screening plates

Second, if the spatial position of the two XcmI restriction sites is relatively close, it will affect the restriction efficiency of the vector. Even if the purity of the carrier DNA is high and the quality of the enzyme is also good, only one restriction site will be digested In some cases, this linear vector cannot be ligated with the PCR product, but the vector is self-ligated, resulting in the generation of negative clones

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