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Construction of PP2 strict anaerobic salmonella strain and application of PP2 strict anaerobic salmonella strain in tumor treatment

A Salmonella, strict technology, applied in the construction of PP2 strict anaerobic Salmonella strain and its application in tumor therapy, can solve the problems of weight loss, time-consuming, and low safety in mice

Active Publication Date: 2022-05-06
SHENZHEN INST OF ADVANCED TECH
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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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  • Construction of PP2 strict anaerobic salmonella strain and application of PP2 strict anaerobic salmonella strain in tumor treatment
  • Construction of PP2 strict anaerobic salmonella strain and application of PP2 strict anaerobic salmonella strain in tumor treatment
  • Construction of PP2 strict anaerobic salmonella strain and application of PP2 strict anaerobic salmonella strain in tumor treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Construction and electrophoresis verification of SL7207(ΔdapA)-PP2-BBa_B0033-dapA strain and SL7207(ΔdapE)-PP2-BBa_B0033-dapE strain

[0080] 1. Construction of pSC101-BBa_B0033-dapA plasmid

[0081] a. Using the pSC101-FbFp-KnaR-loxp+promoter plasmid as a template, the vector forward primer 1 and vector reverse primer 2 in the above table are primers, and PCR obtains a linear vector 1 ( figure 2 (A));

[0082] b. With the Salmonella SL7207 genome as a template, the dapA gene forward primer and the dapA gene reverse primer in the above table are primers, and PCR obtains the dapA linear fragment ( figure 2 (B));

[0083] c. One-step cloning method to obtain pSC101-BBa_B0033-dapA plasmid. Colony PCR identification results ( figure 2 of (C))

[0084] 2. Construction of pSC101-PP2-BBa_B0033-dapA plasmid

[0085] a. The pSC101-BBa_B0033-dapA plasmid was digested with BsaI to obtain a linearized vector fragment 2 ( figure 2 (D));

[0086] b. The primer ...

Embodiment 2

[0097] Example 2: In vitro characterization of SL7207(ΔdapA)-PP2-BBa_B0033-dapA

[0098] Characterization under aerobic conditions: Pick 5 single clones and resuspend them in 10 μl LB medium respectively. Add 5 μl of the bacterial suspension to the LB (DAP+) medium containing kanamycin, and add the remaining 5 μl of the bacterial suspension to the LB (DAP-) medium containing kanamycin. Cultivate in an air shaker (37° C., 220 rpm) for a period of time.

[0099] 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...

Embodiment 3

[0104] Example 3: In vivo characterization of SL7207(ΔdapA)-PP2-BBa_B0033-dapA (abbreviated as PP2)

[0105] C57BL / 6 mice were subcutaneously inoculated with 1×10 6 Mouse bladder cancer cells (MB49), to establish a mouse bladder cancer subcutaneous tumor model. The experiment was divided into three groups, PBS group, SL7207 strain group, SL7207(ΔdapA)-PP2-BBa_B0033-dapA group. Tail vein inoculation 1×10 7 bacteria. Detect 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. Experimental results (such as Figure 4A , Figure 4B , Figure 4C and Figure 4D ):

[0106] (1) The distribution of bacteria in tumor-bearing mice ( Figure 4A ): within 14 days, normal tissues and organs of mice can clear the strain. In the SL7207 group, bacteria rapidly multiplied in normal tissues and tumors within 7 days, and finally all mice died within 7 days.

[0107...

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PUM

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Abstract

The invention relates to an anaerobic activation promoter PP2 gene sequence, an anaerobic gene loop regulated and controlled by the PP2 promoter, strict anaerobic salmonella typhimurium containing the anaerobic gene loop regulated and controlled by the PP2 promoter, a carrier, application of the strict anaerobic salmonella typhimurium and the carrier, a method for changing facultative anaerobic bacteria into strict anaerobic bacteria, and a method for treating cancers by using the bacteria regulated and controlled by the anaerobic loop. The invention also relates to an application in tumor treatment.

Description

technical field [0001] The present invention relates to the field of tumor targeting therapy, specifically, the present invention relates to an anaerobic activation promoter PP2 gene sequence, an anaerobic gene circuit regulated by the PP2 promoter, and a strictly anaerobic mouse comprising an anaerobic gene circuit regulated by the PP2 promoter Salmonella typhi and vectors and their applications, methods of making facultative anaerobic bacteria strictly anaerobic, methods of treating cancer using bacteria regulated by anaerobic circuits. 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 lympha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74A61K45/06A61K48/00A61K31/7088A61P35/00A61P35/02C12R1/42
CPCC12N15/74A61K45/06A61K48/0008A61K31/7088A61P35/00A61P35/02C12N2830/002A61K2300/00Y02A50/30
Inventor 刘陈立盛方芊王作伟曾正阳卢伟琪郭旋黄雄亮
Owner SHENZHEN INST OF ADVANCED TECH
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