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Method for constructing strict anaerobic salmonella, constructed strict anaerobic salmonella and application of constructed strict anaerobic salmonella

A technology of salmonella and anaerobic bacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, medical preparations with non-active ingredients, etc., can solve problems such as time-consuming, ineffective tumor treatment, and low safety

Pending Publication Date: 2022-05-24
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2002, the attenuated Salmonella VNP20009 (msbB-, purI-) was subjected to phase I clinical trials, and the results showed that the strain could colonize tumor tissues, but the effect on tumor treatment was not obvious
[0007] However, it takes up to 26 days for the YB1 strain of Bin Yu et al. to be completely eliminated in normal tissues and organs, which takes a long time and has low safety; compared with the PBS group, after injecting YB1 into the tail vein of tumor-bearing mice, the body weight of the mice was significantly reduced (greater than 5%)

Method used

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  • Method for constructing strict anaerobic salmonella, constructed strict anaerobic salmonella and application of constructed strict anaerobic salmonella
  • Method for constructing strict anaerobic salmonella, constructed strict anaerobic salmonella and application of constructed strict anaerobic salmonella
  • Method for constructing strict anaerobic salmonella, constructed strict anaerobic salmonella and application of constructed strict anaerobic salmonella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Construction of 9 strains of strict anaerobic strains (SL7207(ΔdapA)-Promoters-BBa_B0033-dapA) (primers used in the following experiments are shown in Table 2)

[0075] 1. Construction of pSC101-BBa_B0033-dapA plasmid

[0076] a. Using the pSC101-FbFp-KnaR-loxp+ promoter plasmid as a template, using the vector forward primer and the vector reverse primer as primers, obtain linearized vector fragment 1 by PCR;

[0077] b. Using the Salmonella SL7207 genome as a template, the dapA gene forward primer and the dapA gene reverse primer as primers, obtain the target fragment 1 by PCR;

[0078] c. One-step cloning method to obtain pSC101-BBa_B0033-dapA plasmid.

[0079] 2. Construction of pSC101-R1074-BBa_B0033-dapA plasmid

[0080] a. The pSC101-BBa_B0033-dapA plasmid was digested with BsaI to obtain the linearized vector fragment 2;

[0081] b. The primer annealing method to obtain the R1074 promoter fragment;

[0082] c. Ligase ligation to obtain pSC101-R1074...

Embodiment 2

[0096] Embodiment 2: In vitro characterization of 9 bacterial strains

[0097] Characterization under aerobic conditions: pick 1 single clone and add it to LB (DAP+) medium containing kanamycin; pick 3 clones and add it to LB (DAP-) medium containing kanamycin . Cultivate in an air shaker (37° C., 220 rpm) for a period of time.

[0098] Characterization under anaerobic conditions: 3 single clones were picked and added to LB (DAP+) medium containing kanamycin. Cultivate overnight in an air shaker (37°C, 220rpm). Put the overnight cultured bacterial solution into an anaerobic incubator, and transfer at a ratio of 1:100. Take 20 μl of bacterial liquid and add it to 2 ml of LB (DAP+) medium containing kanamycin; take 20 μl of bacterial liquid and add it to 2 ml of LB (DAP-) medium containing kanamycin, and repeat 3 times. Measure the initial OD600 value of the sample after transfer. In an anaerobic box, culture at 37°C for 24 hours. Measure the OD600 value of the samples aft...

Embodiment 3

[0103] Embodiment 3: In vivo characterization of 9 bacterial strains

[0104] C57BL / 6 mice were subcutaneously inoculated with 1×10 6 Mouse bladder cancer cells (MB49) / mouse were used to establish a mouse bladder cancer subcutaneous tumor model. The experiment was divided into PBS group, SL7207 strain group, Fnr-SP group, Hip1 group, I14018 group, Pept group, Ptet-arcA group, Ptet-Fnr group, R1074 group, Ssbp1 group, YsgAP group. Tail vein inoculation 1×10 7 Various bacteria of the present invention / mouse. The distribution of bacteria in normal tissues, organs and tumors of tumor-bearing mice, the change of tumor volume, the change of mouse weight, and the survival rate of mice were detected within 6 days. Experimental results (such as Figure 4A-Figure 4E ):

[0105] (1) The distribution of bacteria in tumor-bearing mice ( Figure 4A-Figure 4E , left column): The strain in the Fnr-SP group cleared slowly in vivo, and there were a large number of bacteria in normal tissu...

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Abstract

The invention relates to a method for constructing strict anaerobic salmonella, the strict anaerobic salmonella constructed by using the method and application of the strict anaerobic salmonella in tumor treatment.

Description

technical field [0001] The invention relates to the field of tumor targeting therapy, in particular to a method for constructing strictly anaerobic Salmonella, the strictly anaerobic Salmonella constructed by the method and its application in tumor treatment. Background technique [0002] Cancer is the leading cause of death worldwide. Compared with normal cells, cancer cells have the characteristics of unlimited proliferation, transformation and easy transfer. In addition to uncontrollable division of cancer cells (capable of multipolar division), cancer cells also partially invade surrounding normal tissues and even metastasize to other organs through the internal circulatory system or lymphatic system. The history of cancer treatment development shows that traditional cancer treatment methods, such as surgery, chemotherapy, radiation therapy, immunotherapy, hormone therapy, bone marrow / stem cell transplantation, etc., all have certain defects, such as surgical treatment ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21A61K35/74A61K45/06A61K45/00A61K47/46A61P35/00C12R1/42
CPCC12N15/74C12N9/80C12N9/88C12Y305/01047C12Y403/03007A61K35/74A61K45/06A61K45/00A61K47/46A61P35/00A61K2300/00Y02A50/30
Inventor 刘陈立王作伟盛方芊曾正阳卢伟琪郭旋
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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