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Plasmid recombinant listeria monocytogenes as well as construction method and application thereof

A technology of mononuclear cells and Listeria, applied in the field of bioengineering, can solve the problems of reducing the extraction rate of plasmid DNA and the absence of bacterial plasmid extraction kits, etc., and achieve high purity

Pending Publication Date: 2022-07-29
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are many commercial bacterial plasmid DNA extraction kits, but they are all used for plasmid extraction of pure bacteria, and there are no kits for bacterial plasmid extraction in complex samples such as stool
Moreover, the existing plasmid DNA extraction kits for pure bacteria usually use the column method to remove impurities, which will reduce the extraction rate of plasmid DNA, which accurately reflects the diversity of bacterial plasmids in stool samples a key factor

Method used

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  • Plasmid recombinant listeria monocytogenes as well as construction method and application thereof
  • Plasmid recombinant listeria monocytogenes as well as construction method and application thereof
  • Plasmid recombinant listeria monocytogenes as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of recombinant Listeria monocytogenes

[0048] In Example 1, the GFP gene was amplified and connected to the vector pKSV7 to obtain the recombinant plasmid pKSV7-GFP, and then the recombinant plasmid pKSV7-GFP was transformed into Listeria monocytogenes strain LM1073 to obtain plasmid recombinant Listeria monocytogenes Special bacteria. The obtained plasmid-recombinant Listeria monocytogenes is preserved in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M20211306, Listeria monocytogenes CCTCC-Lm1055.

[0049] Specific steps are as follows:

[0050] 1. Amplification of GFP gene

[0051] According to the full-length GFP gene, PCR amplification primers P1 and P2 were designed, and the primer sequences are shown in Table 1. The PCR amplification products were obtained using the PrimeSTAR GXLPremix kit from Takara.

[0052] The reaction system of PCR is shown in Table 2, wherein the DNA template is the GFP gene sh...

Embodiment 2

[0089] Example 2 The method for extracting bacterial plasmid DNA from pig feces

[0090] In this example, using the plasmid recombinant Listeria monocytogenes LM-GFP prepared in Example 1 as the internal standard bacteria to extract bacterial plasmid DNA from pig feces samples, including the following steps:

[0091] 1. Bacterial culture and bacterial suspension preparation

[0092]The plasmid recombinant Listeria monocytogenes LM-GFP obtained in Example 1 was streaked and cultured on a BHI plate, single clones were picked, inoculated into BHI liquid medium, and placed in a constant temperature shaking incubator for shaking culture at 28°C 20h, LM-GFP bacterial liquid was obtained. The LM-GFP bacterial solution was serially diluted 10-fold, with a total of 9 gradients, and 100 μL of the bacterial solution of each gradient concentration was coated with chloramphenicol-resistant BHI solid medium plates, and three parallels were set for each concentration. , calculate the conce...

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Abstract

The invention discloses a plasmid recombinant listeria monocytogenes. A green fluorescent protein (GFP) gene is cloned on a plasmid carried by the plasmid recombinant listeria monocytogenes. The invention also discloses a construction method and application of the plasmid recombinant listeria monocytogenes. The plasmid recombinant listeria monocytogenes disclosed by the invention can be used as an internal standard bacterium for extracting bacterial plasmid group DNA (Deoxyribonucleic Acid) from animal waste. And the method for detecting the DNA extraction rate of the bacterial plasmid group in the excrement as an internal standard bacterium has specificity.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a plasmid recombinant Listeria monocytogenes and a construction method and application thereof. Background technique [0002] The antibiotic resistance of animal-derived bacteria is quite serious, which has caused animal production safety risks and public relations health safety risks. To clarify the source and transmission route of antibiotic resistance genes is the premise of preventing and controlling antibiotic resistance of animal-derived bacteria (Holmes AH, Moore L S P and Sundsfjord A, et. al. 2016. Understanding the mechanisms and drivers of antimicrobial resistance. The Lancet, 387, 176-87). The gut is the main storage site for bacteria. Therefore, the drug-resistant bacteria in animal feces are the main objects to study the drug resistance of animal-derived bacteria. The genetic basis of bacterial resistance phenotype is the antibiotic resistance g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/12C12N15/74C12Q1/6806C12Q1/689C12R1/01
CPCC07K14/43595C12N15/74C12Q1/6806C12Q1/689C12Q2600/166C12Q2521/531C12Q2527/125C12Q2537/16C12Q2545/101
Inventor 林颖峥魏人杰李树清桑益旸王恒安周琬陈志飞
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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