Controllable Genome Modification Plasmodium, Recombinant Expression Vector and Construction Method, Application

Abstract

The invention relates to a controllable genome-modified plasmodium, a recombinant expression vector and the construction method and application of the controllable genome-modified plasmodium and the recombinant expression vector. The recombinant expression vector comprises a gene targeting long homologous arm, a gene targeting short homologous arm, a tetracycline repression protein gene expression cassette, a pyrimethamine resistance gene expression cassette and a target gene expression cassette, wherein the tetracycline repression protein gene expression cassette, the pyrimethamine resistance gene expression cassette and the target gene expression cassette are located between the gene targeting long homologous arm and the gene targeting short homologous arm, and tetracycline operator gene sequences are inserted in multiple transcriptional start sites of a target gene promoter, so that the recombinant expression vector can be used for conditional research of the functions of a certain functional gene in a plasmodium genome. Furthermore, a functional gene expression sequence, corresponding to a target gene, in the plasmodium genome is knocked out by means of the gene knockout technique; meanwhile, the recombinant expression vector is transfected into a plasmodium with genes knocked out, so that the controllable genome-modified plasmodium is obtained; a new technical scheme is provided for further research of the functions of all functional genes in the plasmodium genome, and application prospects are broad.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a controllable genome-modified malaria parasite, a recombinant expression vector, a construction method, and an application. Background technique [0002] Malaria, caused by Plasmodium infection, is one of the three major infectious diseases worldwide. Malaria infects hundreds of millions of people every year and kills more than a million people, most of them children under the age of five. One of the main reasons why malaria is so difficult to effectively control and eradicate is the limited understanding of the biological complexity of its causative agent, Plasmodium. Plasmodium has a complex life cycle, and its growth and development need to be completed in hosts such as female Anopheles mosquitoes and humans (or other vertebrates): Plasmodium reproduces sexually in Anopheles mosquitoes, and in humans or other vertebrate hosts. reproduce asexually. The development process...

AI Technical Summary

Problems solved by technology

Other studies have combined the Cre / loxP or Flp / FRT system with gene knockout to achieve gene conditional knockout, but this gene conditional knockout technology is limited to the use of specific or tissue-specific promoters, and this conditional knockout It is irreversible except in a specific period or in a specific tissue. Therefore, for most of the malaria parasites that are haploid in the growth period, there are still important gene deletions that cause the parasites to die or be severely damaged, so that transgenic parasites cannot be screened and cloning problems, and genes that are important for each growth stage of Plasmodium are more difficult to study in this way

The gene-induced expression method used in animal cells often uses some special promoters, which can be induced under certain physical and chemical conditions, such as the heat shock protein promoter can be induced at high temperature, glucocorticoid and heavy metal response Promoters, etc. These gene expression induction methods have the following defects: poor specificity of induced expression, expression leakage when the system is closed, and the toxicity of the inducer itself can cause damage to cells, etc.

In 2005, the tet-off system constructed by Meissner et al. regulated the expression of the green fluorescent protein (GFP) gene in human Plasmodium falciparum (Proc.Natl.Acad.Sci.USA.2005,102(8):2980-2985), However, the transfection plasmid is not integrated into the Plasmodium genome and exists in the form of episomes, which are easily lost, resulting in unstable transfection results, and the induced expression rate of regulated genes is very low (about 20%) and persists. The time is short, and the endogenous gene of Plasmodium remains intact without knockout

However, the Plasmodium whose genes crucial to growth and development are knocked out is difficult to produce in large quantities because the defective strains cannot grow and develop normally. , purification and storage, etc., and once a small number of sporozoites escape the immunity against the liver stage and invade blood red blood cells, they will enter the important stage of malaria parasite pathogenicity, resulting in the failure of vaccine protection

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