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Toxoplasma transgenosis carrier based on piggyback transposon and construction method

A gene transfer and Toxoplasma gondii technology, applied in the biological field, can solve problems such as low efficiency, complicated operation, and heavy workload, and achieve the effects of simple operation, high transformation efficiency, and efficient integration

Inactive Publication Date: 2012-10-17
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Construction of Toxoplasma gondii Tbd by chemical mutagenesis - Mutants show that the ability of bradyzoite formation is weakened, but the molecular mechanism cannot be determined; some virulence and metabolism-related genes of Toxoplasma gondii have been identified by gene targeting technology, but this method can only construct one mutant at a time, and the efficiency Not high, heavy workload
RNAi technology is also very complicated in operation. First, a large number of siRNA molecules need to be designed according to the target gene sequence, and then their gene silencing effects are verified one by one, and it is difficult to achieve complete gene silencing in terms of effect.

Method used

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  • Toxoplasma transgenosis carrier based on piggyback transposon and construction method
  • Toxoplasma transgenosis carrier based on piggyback transposon and construction method
  • Toxoplasma transgenosis carrier based on piggyback transposon and construction method

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Experimental program
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Effect test

Embodiment 1

[0028] Construction and Application of Toxoplasma Gondii Tachyzoite Gene Transfer Vector

[0029] According to attached figure 1 , 2 The shown technical route prepares the transfer carrier of Toxoplasma gondii tachyzoites, and the specific steps are as follows:

[0030] 1. Cloning and sequence analysis of Toxoplasma gondii SAG1 promoter sequence and polyadenylation signal sequence

[0031]Toxoplasma gondii RH strain was inoculated intraperitoneally into Kunming mice, ascites was extracted 3-4 days later, the parasites were collected by centrifugation, and the genome of Toxoplasma gondii was extracted according to conventional methods. Using SAG1-F and SAG1-R as primers, PCR method was used to amplify Toxoplasma gondii SAG1-5, the 5′ non-coding region of the SAG1 gene; using GRA2-F and GRA2-R as primers, the PCR method was used to amplify the 3′ non-coding region GRA2-3 of the Toxoplasma gondii GRA2 gene, and the amplified product was passed through agarose After the gel ele...

Embodiment 2

[0051] Construction and Application of Toxoplasma Gondii Bradyzoite Gene Transfer Vector

[0052] According to attached image 3 The shown route prepares the toxoplasma gondii bradyzoite transfer vector, and the specific steps are as follows:

[0053] 1. Cloning of Toxoplasma gondii BAG1 promoter sequence

[0054] Toxoplasma gondii RH strain was inoculated intraperitoneally into Kunming mice, ascites was extracted 3-4 days later, the parasites were collected by centrifugation, and the genome of Toxoplasma gondii was extracted according to conventional methods. Using SAG1-F and SAG1-R as primers, PCR method was used to amplify Toxoplasma gondii SAG1-5, the 5-terminal non-transcoding region of the SAG1 gene; using GRA2-F and GRA2-R as primers, the PCR method was used to amplify the 3′-terminal non-transcoding region GRA2-3 of the Toxoplasma gondii GRA2 gene, and the amplified product was passed through agarose After the gel electrophoresis was confirmed, the obtained genes wer...

Embodiment 3

[0074] Construction and Application of Toxoplasma Gondii Tachyzoite-Bladyzoite Gene Transfer Vector

[0075] According to attached Figure 4 The shown route prepares the expression vector of the toxoplasma gondii tachyzoite-bradyzoite transfer vector, and concrete steps are as follows:

[0076] 1. Cloning of Toxoplasma gondii GRA2 gene promoter sequence

[0077] Toxoplasma gondii RH strain was inoculated intraperitoneally into Kunming mice, ascites was extracted 3-4 days later, the parasites were collected by centrifugation, and the genome of Toxoplasma gondii was extracted according to conventional methods. Using GRA2P-F and GRA2P-R as primers, PCR method was used to amplify Toxoplasma gondii GRA2-5, the 5′ non-transcoding region of the GRA2 gene; using GRA2-F and GRA2-R as primers, the PCR method was used to amplify the 3-terminal non-transcoding region GRA2-3 of the GRA2 gene of Toxoplasma gondii, and the amplified product was gelatinized by agarose After the gel electrop...

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Abstract

The invention provides a toxoplasma gene transfer vector which is based on piggyBac transposon and a preparation method thereof. The toxoplasma gene transfer vector is composed of a piggyBac transposon expression vector and an auxiliary plasmid for expressing piggyback transposase. The piggyBac transposon expression vector is constructed of toxoplasma promoter sequences such as the regulating elements of tachyzoite and bradyzoite specific expressing protein SAG1, BAG1 and GRA, purpose genes as well as the piggyBac inverted repetitive sequence; the auxiliary plasmid is constructed of the regulating elements of the SAG1 and the GRA and piggyBac transposase coded genes. Compared with the prior art, the invention has the advantages that the operation is simple, and the conversion efficiency is high, thereby providing a new path for the research and certification on the insect drug target and vaccine candidate antihelion against toxoplasma.

Description

technical field [0001] The invention aims to provide a toxoplasma gondii gene transfer vector based on piggyBac transposon, and also discloses a construction method of the vector, which belongs to the field of biotechnology. Background technique [0002] Toxoplasmosis is a worldwide zoonotic parasitic disease caused by Toxoplasma gondii. The infection rate of human Toxoplasma gondii is extremely high, and in some cases it can cause serious diseases, such as pregnant women infected with Toxoplasma gondii can affect the development of the fetus, severe cases cause teratogenicity or even death, and can cause miscarriage or premature delivery in pregnant women; for immunosuppression or immunodeficiency In patients, Toxoplasma gondii is a major opportunistic pathogen. After pregnant women are infected with toxoplasma, there is a 30% to 46% chance of transmission from mother to fetus, causing congenital infection of the fetus, resulting in miscarriage, stillbirth or congenital to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66
Inventor 刘全商立民朱兴全
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI