Plasmid pZF17-30 for constructing Brucella mutant strain, construction method of plasmid and application of plasmid

Abstract

The invention provides a plasmid pZF17-30 for constructing a Brucella mutant strain, a construction method of the plasmid and an application of the plasmid. The construction process mainly includes the steps: amplifying an rAPOBEC1-nCas9-UGI fragment from a commercial plasmid pCMV-BE3; integrating the fragment with a carrier containing a general host replicon pBBR1-MCS-5 and non-specific sgRNA byrecombination reaction; inserting a ccdB gene and a Chl resistance gene into the sgRNA; digesting the plasmid pZF17-30 by the aid of restricted enzyme BsaI; connecting specific sgRNA with linearized pZF17-30. The sequence of the constructed carrier is as shown in SEQ ID NO. 3, the carrier can be used for constructing the Brucella mutant strain, and a 478-site basic group in a CDS (coding sequence)area of a virB10 gene is mutated into T from C.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a plasmid pZF17-30 for constructing a Brucella mutant strain and a construction method thereof, which can be used for terminating mutation of the brucella gene coding region, and premature termination of protein translation. Background technique [0002] Brucellosis (brucellosis) is an important zoonotic disease that has spread to the world and has caused serious economic losses to the animal husbandry. a major threat. However, so far, no safe and effective vaccine has been developed. Brucella, also known as Brucella, is the pathogen that causes brucellosis. To develop safe and effective vaccines, it is very important to study the functions of various genes of the bacteria. [0003] At present, there are many difficulties in the functional research of Brucella genes. The first is that Brucella grows slowly. Under a stable culture environment in the labo...

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Problems solved by technology

[0003] At present, there are many difficulties in the functional study of Brucella genes

One is that Brucella grows slowly. In a stable culture environment in the laboratory, the bacteria need to grow for 36-48 hours to grow visible colonies on the plate. The wild strains isolated for the first time tend to grow more slowly.

The second is the zoonotic characteristics of Brucella patients, which makes the operation of Brucella quite cumbersome and time-consuming

These gene deletion mutants have improved the shortcomings of traditional attenuated brucellosis vaccines, such as poor safety and interference with serological immune diagnosis.

However, most of the current technologies for constructing the deletion of virulence genes in Brucella use traditional homologous recombination combined with suicide plasmids, but traditional homologous recombination technology has its own drawbacks, for example, it is necessary to rebuild complex targeting for different target genes And screening of vector plasmids makes the operation cumbersome, heavy workload, time-consuming, and increases the cost of experiments. At the same time, the construction of vector plasmids is also limited by the restriction enzyme sites on the genome where the cloning vector and target gene are located. Functional genomics During the experimental operation, the cloning vector may not be able to withstand large fragments of DNA. In addition, the traditional homologous recombination technology has a low probability of recombination (Copeland et al 2001), which limits the large-scale application of this technology in the laboratory, and also makes Bruce Progress in research on bacterial virulence genes is slow, so this traditional Brucella gene editing technology is expected to be improved

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