A method for screening non-integrating attenuated Listeria strains highly expressing foreign proteins

An exogenous protein, Listeria technology, applied in the fields of resistance to vector-borne diseases, instruments, and biological material analysis, can solve the differential expression between Listeria colonies, unfavorable rapid rough screening of large-scale samples, and complex preparation operations. and other problems, to achieve the effect of prominent effect, convenient operation and good therapeutic effect

Active Publication Date: 2022-07-08
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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  • A method for screening non-integrating attenuated Listeria strains highly expressing foreign proteins
  • A method for screening non-integrating attenuated Listeria strains highly expressing foreign proteins
  • A method for screening non-integrating attenuated Listeria strains highly expressing foreign proteins

Examples

Experimental program
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Effect test

Embodiment 1

[0112] Example 1: Solid and liquid media, antibiotic use and non-integrating attenuated Listeria monocytogenes Sterile culture system and strain preservation

[0113] Liquid culture medium BHI (brain heart infusion 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 sterilization at 121°C for 20min, when the temperature dropped to about 50°C, antibiotics (chloramphenicol resistance) were added, shaken well, and the plate was poured.

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

[0116] Culture system: about 10 7 CFU Listeria initial culture (such as LM-OVA 28 ) into 5ml of liquid BHI medium containing chloramphenicol resistance, shaker at 230rpm and 37°C for 14-16h, that is, the...

Embodiment 2

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

[0120] will contain about 10 7 The initial culture of Listeria monocytogenes in CFU was added to 10ml of liquid BHI medium containing chloramphenicol resistance, shaken at 230rpm and 37°C for 14-16h, centrifuged at 4500rpm for 15-20min to collect bacteria, and washed with equal volume of PBS. The cells were resuspended twice with 1 / 10 volume of PBS (containing 7% DMSO), aliquoted, and stored at -80°C.

[0121] Statistical method of CFU: Dilute the cells with PBS or medium according to a 10-fold gradient. When the gradient is diluted, it can only be diluted from the previous gradient to the next gradient, and mix well by shaking. Take 10 respectively -5 , 10 -6 , 10 -7 , 10 -8 Each concentration of 100ul was coated on BHI plates, and the number of colonies was counted. eg 10 -8 The number of colonies grown at the concentration is N, then CFU=N x 10x 10 ...

Embodiment 3

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

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

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Abstract

The present disclosure relates to a method for screening non-integrating attenuated Listeria strains that highly express foreign proteins. Specifically, the present disclosure relates to a method for detecting the amount of exogenous protein expressed by non-integrated attenuated Listeria, a method for screening non-integrated attenuated Listeria, and a method for screening the non-integrated attenuated Listeria. Non-integrating attenuated Listeria. The method adopted in the present disclosure has the advantages of short time consumption, simple operation, and large sample detection amount, and can meet the requirement of fast and large-scale primary screening of the produced samples while simultaneously producing large batches of samples. Even if there are obvious expression differences among non-integrating Listeria strains, the method of the present disclosure can still achieve rapid and mass screening of non-integrating Listeria, and then obtain the Listeria strain with the best expression effect, which is non-integrating Listeria Vaccine preparation is facilitated.

Description

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

Claims

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Application Information

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Patent Type & Authority Patents(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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