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Method for screening non-integrated attenuated Listeria monocytogene strains of high-expression foreign proteins

A Listeria and foreign protein technology, which is applied in the fields of resistance to vector-borne diseases, instruments, and biological material analysis. It can solve the problems of large sample preparation, small number of samples, and long experiment time, and achieve large sample detection volume. , The effect of high insertion efficiency and convenient operation

Active Publication Date: 2019-12-17
SUZHOU ROYALTECH MED CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of protein expression detection, the experimental process of Western blot technology is complicated and time-consuming. In terms of Listeria expression detection, Western blot experiments require a large amount of sample preparation, complicated preparation operations, and a small number of samples prepared at a time, which is not conducive to large batches. Rapid coarse screening of samples
[0005] Since the above problems still exist in the existing solutions, it is necessary to provide a method for rapidly screening non-integrated attenuated Listeria strains with high expression of foreign proteins, and then solve the problem of Listeria produced during the preparation of Listeria strains. There may be a problem of differential expression among bacterial colonies

Method used

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  • Method for screening non-integrated attenuated Listeria monocytogene strains of high-expression foreign proteins
  • Method for screening non-integrated attenuated Listeria monocytogene strains of high-expression foreign proteins
  • Method for screening non-integrated attenuated Listeria monocytogene strains of high-expression foreign proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1: Solid and liquid culture media of non-integrated attenuated Listeria, use of antibiotics and non-integrated attenuated Prunus Culture System and Strain Preservation of Sterella

[0113] Liquid medium BHI (brain heart extract broth): 74g / L, natural pH value, sterilized at 121°C for 20min.

[0114] Solid medium BHI: On the basis of liquid BHI, add 1.5g / L agar powder. After sterilizing at 121°C for 20 minutes, when the temperature drops to about 50°C, add antibiotics (chloramphenicol resistance), shake well, and pour the plates.

[0115] Chloramphenicol (cm): stock solution (1000x) was prepared with absolute ethanol at 25-34mg / ml, filtered through a 0.22μm filter, and stored at -20°C. Use a final concentration of 25-34 μg / ml. Pay attention to avoid light.

[0116] Culture system: about 10 7 CFU Listeria starter culture (e.g. LM-OVA 28 ) into 5ml of liquid BHI medium containing chloramphenicol resistance, and cultured on a shaker at 230rpm and 37°C for 1...

Embodiment 2

[0119] Example 2: Preparation of Non-Integrated Attenuated Listeria Vaccine and CFU Concentration Determination

[0120] will contain about 10 7 Add the initial culture of CFU Listeria to 10ml of liquid BHI medium containing chloramphenicol resistance, culture on a shaker at 230rpm at 37°C for 14-16h, centrifuge at 4500rpm for 15-20min to collect the bacteria, wash the bacteria with an equal volume of PBS Cells were resuspended twice with 1 / 10 volume of PBS (containing 7% DMSO), aliquoted, and stored at -80°C.

[0121] CFU statistical method: use PBS or culture medium to dilute the bacteria in a 10-fold gradient, and only dilute from the previous gradient to the next gradient in the gradient dilution, and mix thoroughly by shaking. Take 10 respectively -5 、10 -6 、10 -7 、10 -8 100ul of each concentration was applied to the BHI plate, and the number of colonies was counted. such as 10 -8 The number of colonies grown at the concentration is N, then CFU=N x 10x 10 8 piec...

Embodiment 3

[0122] Example 3: Detection of exogenous protein expression secreted in the supernatant of non-integrated attenuated Listeria using the disclosed method amount

[0123] Pick a single colony from the plate for cultivating Listeria, add it to 10ml of liquid BHI medium containing chloramphenicol resistance, shake it at 230rpm at 37°C for 14-16h, and centrifuge at 4500rpm for 15-20min to precipitate the bacteria. Take 1 ml of the supernatant and mix with 3 times the volume of 10% TCA / acetone solution, and precipitate overnight at -20°C. Centrifuge at 15,000 rpm for 30 minutes to collect the precipitated protein, and wash twice with ice-cold acetone to remove residual TCA. The excess acetone was evaporated in a fume hood, and the precipitate was dissolved with 30 μl protein loading buffer containing 0.01N NaOH. After boiling and denaturation, 30 μl samples were spotted on the NC membrane, air-dried and washed 3 times with TBST, 5 min each time. Block with 5% skimmed milk TBST...

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Abstract

The invention relates to a method for screening non-integrated attenuated Listeria monocytogene strains of high-expression foreign proteins. Specifically, the invention relates to a method for detecting the amount of foreign proteins expressed by non-integrated attenuated Listeria monocytogenes, a method for screening non-integrated attenuated Listeria monocytogenes, and the non-integrated attenuated Listeria monocytogenes obtained by adopting the screening method. The method disclosed by the invention has the advantages of short consumed time, simple operation and large sample detection amount, can meet the requirement for preliminarily screening produced samples in quantity while producing mass samples simultaneously. Even if obvious expression difference exists among the non-integratedListeria monocytogene strains, the method disclosed by the invention can still realize rapid and large-scale screening of the non-integrated Listeria monocytogenes, further obtains the Listeria monocytogene strains with the optimal expression effect, and brings convenience for the preparation of Listeria vaccine.

Description

technical field [0001] The present disclosure relates primarily to the field of biotechnology. Specifically, the present disclosure provides a screening method for bacterial strains. More specifically, the present disclosure provides a method for screening non-integrating attenuated Listeria strains that highly express foreign proteins. Background technique [0002] Listeria monocytogenes (Listeria monocytogenes, Lm) is an important foodborne pathogen, which may cause severe listeriosis in the elderly, children, pregnant women and immunosuppressed people[1]. Listeria is a Gram-positive intracellular parasite that can survive and multiply in epithelial cells and phagocytes. Due to its unique infection process, Listeria can simultaneously induce an inflammatory response and activate the combination of MHC class I and class II antigen presentation pathways, making Listeria a promising vaccine vector [2-4]. [0003] At present, there are mainly two ways to construct tumor vac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68G01N2333/195Y02A50/30
Inventor 代楠关剑赵勇刚
Owner SUZHOU ROYALTECH MED CO LTD
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