Gene drive carrier and construction method thereof

A construction method and carrier technology, applied in the field of bioengineering, can solve problems such as destroying microbial flora, and achieve the effect of eliminating methicillin resistance genes

Inactive Publication Date: 2018-10-26
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, broad-spectrum antibiotics will kill most of the bacteria indi

Method used

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  • Gene drive carrier and construction method thereof
  • Gene drive carrier and construction method thereof
  • Gene drive carrier and construction method thereof

Examples

Experimental program
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Embodiment 1

[0023] In view of the frequent occurrence of drug-resistant strains caused by the abuse of antibiotics at present, the present invention attempts to combine the new chromosome cassette recombinase CcrC2 and CRISPR-Cas9 technology to construct the SCC mec killer carrier system, that is, the Gene drive carrier, which acts on methoxy The methicillin-resistant strain MRSA achieved the targeted deletion of the staphylococcal chromosome cassette SCC mec in the methicillin-resistant strain MRSA, thereby eliminating the methicillin-resistant gene. And the strain carrying the SCC mec killer carrier system can avoid re-acquiring the mobile element containing the mecA gene, and then can treat patients with existing antibiotics.

[0024] Such as figure 1 Shown, a kind of construction method of Gene drive carrier comprises the following steps:

[0025] Step 1), artificial synthesis of cap5A promoter and sgRNA fragment

[0026] Take 1 μL of 10 μM primers SK1 to SK8 respectively, mix them ...

Embodiment 2

[0046] Such as figure 1 , figure 2 and image 3 As shown, the methicillin resistance gene was eliminated in livestock-derived MRSA strain BA01611 using the SCCmec killer vector.

[0047] (1) Preparation of competent cells of MRSA strain BA01611

[0048] Inoculate a single clone of BA01611 strain in 10 mL of BHI. Shake the bacteria overnight at 37°C, inoculate about 6mL of the resulting liquid into 100mL fresh BHI medium the next day, and adjust the OD 600 Value is equal to 0.5. Continue shaking and culturing for 30 minutes, then centrifuge at 4°C at 5000g for 5 minutes, collect the bacteria and discard the culture medium, wash the bacteria once with an equal volume of sterilized water, and centrifuge again according to the first centrifugation conditions, collect the bacteria and discard the culture The cells were washed twice with an equal volume of 10% glycerol. Finally, resuspend the bacteria in 500uL of 10% glycerol, divide 50uL into 10 centrifuge tubes, and store t...

Embodiment 3

[0055] Such as figure 1 , figure 2 and Figure 4 As shown, the SCCmec killer vector was used to eliminate the methicillin resistance gene in the hospital-derived MRSA strain Mu50.

[0056] (1) Prepare competent cells of MRSA bacterial strain Mu50

[0057] Inoculate a single clone of Mu50 strain in 10 mL of BHI. Shake the bacteria overnight at 37°C, inoculate about 6mL of the resulting liquid into 100mL fresh BHI medium the next day, and adjust the OD 600 Value is equal to 0.5. Continue shaking and culturing for 30 minutes, then centrifuge at 4°C at 5000g for 5 minutes, collect the bacteria and discard the culture medium, wash the bacteria once with an equal volume of sterilized water, and centrifuge again according to the first centrifugation conditions, collect the bacteria and discard the culture The cells were washed twice with an equal volume of 10% glycerol. Finally, resuspend the bacteria in 500uL of 10% ethanol, divide 50uL into 10 centrifuge tubes, and store the...

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Abstract

The invention belongs to the field of bioengineering and particularly relates to a Gene drive carrier and a construction method thereof. The construction method includes the steps of 1), artificiallysynthesizing a cap5A promoter and an sgRNA fragment; 2), amplifying a cas9 gene; 3), amplifying a plasmid skeleton; 4), amplifying an rpsL promoter; 5), assembling an empty carrier; 6), inserting a spacer targeted at the formula described; 7), constructing the carrier. In the method, a novel chromosome box recombinase CcrC2 and CRISPR-Cas9 technology are combined in an attempting manner, an SCCmeckiller carrier system, namely the Gene driver carrier is constructed, the Gene drive acts on methicillin-resistant staphylococcus aureus MRSA, staphylococcal chromosome box SCCmec is targeted to remove from the methicillin-resistant staphylococcus aureus MRSA, and methicillin-resistant genes are thereby eliminated.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a Gene drive carrier capable of eliminating the methicillin resistance gene in MRSA and a construction method. Background technique [0002] Staphylococcus aureus can cause pneumonia, bacteremia, endocarditis and other infectious diseases, and the abuse of antibiotics in the medical process has led to a high incidence of drug-resistant strains, among which methicillin-resistant Staphylococcus aureus (methicillin-resistant Staphylococcus aureus) -resistant Staphylococcus aureus, MRSA) are particularly harmful. MRSA is produced by the exogenous acquisition of the mecA gene from susceptible strains, which encodes a new penicillin-binding protein-PBP2a, endowing Staphylococcus aureus with broad-spectrum resistance to β-lactam antibiotics, resulting in β-lactam antibiotics lose their effectiveness in clinical treatment. Among them, the mecA gene is located in the mobile gen...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66
CPCC12N15/63C12N15/66
Inventor 赵辛吴兆韡
Owner NORTHWEST A & F UNIV
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