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Recombinant Escherichia coli for expressing Caspase-3 recombinant scFv78 and functional verification method of recombinant Escherichia coli

A technology for recombining Escherichia coli and scfv78, applied in the field of molecular biology, to achieve great application value, clear genome and genetic background, and easy transformation

Pending Publication Date: 2020-05-01
NANCHANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The present invention aims at the technical defects of the prior art, and provides a recombinant Escherichia coli expressing Caspase-3 recombinant scFv78 and its function verification method to solve the problem of human toxin Caspase-3 recombinant single-chain antibody (scFv78) in the prior art. , the technical problem of not being able to specifically accumulate in tumors

Method used

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  • Recombinant Escherichia coli for expressing Caspase-3 recombinant scFv78 and functional verification method of recombinant Escherichia coli
  • Recombinant Escherichia coli for expressing Caspase-3 recombinant scFv78 and functional verification method of recombinant Escherichia coli
  • Recombinant Escherichia coli for expressing Caspase-3 recombinant scFv78 and functional verification method of recombinant Escherichia coli

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Embodiment 1

[0035] 1. Using pET302-78-Caspase-3 as a template and using the sequences of SEQ ID No.3 and SEQ ID No.4 as primers, obtain the 78-Caspase-3 fragment shown in SEQ ID No.1 by PCR amplification.

[0036] Among them, 78 is a single-chain antibody that specifically recognizes TEM1 that has been screened by our laboratory in the early stage, and is named 78; Caspase3 is an apoptosis-inducing factor. The gene sequence of the Caspase3 fragment was obtained through GeneBank, and then handed over to the company (Nanjing GenScript ) to carry out chemical synthesis of the whole sequence to obtain pUC57-78-Csapase3 recombinant plasmid or pET302-78-Caspase-3 recombinant plasmid. Further, pET302-78-Caspase-3 or pUC57-78-Csapase3 was used as a template, and the sequences of SEQ ID No.3 and SEQ ID No.4 were used as primers to obtain the 78-Caspase- 3 fragments.

[0037] 2. The obtained 78-Caspase-3 fragment and plasmid pET302 were double digested with Xho I and AvrII. After purification, the...

Embodiment 2

[0042] Recombinant Escherichia coli Nissle 1917 expressing Caspase-3 recombinant scFv78, which was constructed by the following method:

[0043] 1) Ligate the gene fragment 78-Caspase-3 encoding the human toxin Caspase-3 recombinant single-chain antibody scFv78 into the pET302 plasmid by enzyme digestion and ligation to obtain the pET302-78-Caspase-3 recombinant plasmid;

[0044] 2) Transform the pET302-78-Caspase-3 recombinant plasmid into Escherichia coli Nissle 1917 to obtain recombinant E. coli Nissle1917-pET302-78-Caspase-3, which is the recombinant scFv78 expressing Caspase-3 Recombinant Escherichia coli Nissle 1917.

[0045] Wherein, the gene fragment 78-Caspase-3 encoding the recombinant single-chain antibody scFv78 of human toxin Caspase-3 in step 1) is a DNA fragment whose nucleotide sequence is shown in SEQ ID No.1.

[0046] The coding gene fragment 78-Caspase-3 of human toxin Caspase-3 recombinant single-chain antibody scFv78 in step 1) uses the DNA fragments with...

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Abstract

The invention provides recombinant Escherichia coli for expressing Caspase-3 recombinant scFv78 and a functional verification method of the recombinant Escherichia coli. The method comprises the following steps: firstly, connecting a recombinant single-chain antibody scFv78 encoding gene integrated with a Caspase-3 encoding gene to a pET302 vector through an enzyme digestion enzyme-linked approach, and converting obtained recombinant plasmid into Escherichia coli Nissle1917 so as to obtain a bacterial strain capable of expressing the Caspase-3 recombinant scFv78. The invention adopts the intracellular invasion and aggregation characteristics of the Escherichia coli Nissle1917 and the fixed-point targeting characteristic of the scFv78 on TEM1 positive cells; the human-derived toxin Caspase-3 protein site plays a role specifically, so that the TEM1 positive tumor cells are killed more accurately, and damage to normal cells of an organism is reduced. On the basis, the method utilizes recombinant bacteria to express the human-derived toxin Caspase-3 recombinant single-chain antibody scFv78 protein, and then the protein and MS1-TEM1 cells are co-incubated in an in-vitro environment, sothat the affinity and lethality of the to bacterial strain tumor cells are verified.

Description

technical field [0001] The invention relates to the technical field of molecular biology, and further relates to biopharmaceutical and tumor medical technology, in particular to a recombinant Escherichia coli expressing Caspase-3 recombinant scFv78 and a function verification method thereof. Background technique [0002] At present, there are chemical, physical and biological methods for tumor treatment, but the above-mentioned treatments have not achieved the ideal effect of treating tumors. Therefore, it is imminent to explore safer and more effective new tumor treatment methods. Since William Coley first used bacteria to treat tumors, bacterial targeted tumor therapy has become a hot spot in international research in recent years. A variety of bacteria have been found to specifically invade tumor tissues and multiply in large numbers, and the idea of ​​using live bacteria to treat tumors has emerged. . [0003] Targeted therapy means that anti-tumor drugs can be targeted...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/66C12N15/70G01N33/573C12R1/19
CPCC12N9/6472C12N15/66C12N15/70G01N33/573
Inventor 陈廷涛田溥塬
Owner NANCHANG UNIV
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