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AIF expression strain integrated with recombinant single-chain antibody of AIF and application thereof

A single-chain antibody and recombinant strain technology, applied in the field of molecular biology, can solve the problems of large relative molecular weight, limit the therapeutic effect of solid tumors, and cannot distinguish normal cells, etc., and achieve the effect of treating tumor diseases

Active Publication Date: 2017-04-26
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibody immunotherapy is highly targeted, but due to its relatively large molecular weight, it limits the therapeutic effect of solid tumors
Small-molecule chemical drugs have a high-intensity killing effect on cancer cells, but because they cannot distinguish normal cells, they also have a certain killing effect on normal cells, and the side effects are more obvious

Method used

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  • AIF expression strain integrated with recombinant single-chain antibody of AIF and application thereof
  • AIF expression strain integrated with recombinant single-chain antibody of AIF and application thereof
  • AIF expression strain integrated with recombinant single-chain antibody of AIF and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] 1.1 Construction of recombinant expression system and expression of Aif and Aif-T18 proteins

[0054] (1) Chemically synthesize the pET302 (SEQ ID No.4) plasmid containing Aif-T18 (SEQ ID No.2) fragment and Aif (SEQ ID No.1) figure 1 As shown), transfer to E.coli BL21, pick pET302-Aif-T18 and pET302-Aif monoclonal into 10mL LB medium (containing Amp), 37°C, 200rpm, shake the bacteria overnight;

[0055] (2) Add the bacterium solution obtained in step (1) to a new expansion medium (200mL LB) in a ratio of 5%, and add the inducer IPTG (1 / 1000) (IPTG concentration is 1M, added at 1 / 1000, the final concentration of IPTG in the bacterial solution is 1mM), 37°C, 200rpm, 4h;

[0056] (3) Add 20-25mL of Binding Buffer, ultrasonically break, beat for 5s, stop for 8s, 95 times, power 200W, 2 cycles. When the bacterial solution becomes less viscous and clearer, stop breaking, centrifuge at 10,000 rpm for 10 minutes, transfer the supernatant to a new 50mL centrifuge tube, and st...

Embodiment 2

[0067] A bacterial strain for expressing AIF and a recombinant single-chain antibody integrated with AIF, the bacterial strain is prepared by the following method:

[0068] 1) Take the Aif expression gene whose sequence is shown in SEQ ID No.1 and the Aif-T18 expression gene whose sequence is shown in SEQ ID No.2, respectively, and connect them to the pET302 plasmid by restriction enzyme digestion, respectively, to obtain the recombinant plasmid pET302- Aif, pET302-Aif-T18;

[0069]2) Take the recombinant plasmids pET302-Aif and pET302-Aif-T18 obtained in step 1), respectively, and transfer them into E.coliBL21 to obtain recombinant bacteria pET302-Aif-B21 and pET302-Aif-T18-B21 respectively, which are the obtained The above-mentioned bacterial strains for expressing AIF and recombinant single-chain antibody integrated with AIF.

[0070] A method for preparing AIF and AIF-integrated recombinant single-chain antibody using the above bacterial strain, comprising the following s...

Embodiment 3

[0092] A bacterial strain for expressing AIF and a recombinant single-chain antibody integrated with AIF, the bacterial strain is prepared by the following method:

[0093] 1) Take the Aif expression gene whose sequence is shown in SEQ ID No.1 and the Aif-T18 expression gene whose sequence is shown in SEQ ID No.2, respectively, and connect them to the pET302 plasmid by restriction enzyme digestion, respectively, to obtain the recombinant plasmid pET302- Aif, pET302-Aif-T18;

[0094] 2) Take the recombinant plasmids pET302-Aif and pET302-Aif-T18 obtained in step 1), respectively, and transfer them into E.coliBL21 to obtain recombinant bacteria pET302-Aif-B21 and pET302-Aif-T18-B21 respectively, which are the obtained The above-mentioned bacterial strains for expressing AIF and recombinant single-chain antibody integrated with AIF.

[0095] A method for preparing AIF and AIF-integrated recombinant single-chain antibody using the above bacterial strain, comprising the following ...

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Abstract

The invention provides an AIF expression strain integrated with a recombinant single-chain antibody of AIF and application thereof. The technical scheme utilizes the characteristic of specific binding of a single-chain antibody T18 with a tumor endothelial cell marker molecule TEM1 to construct an AIF-T18 protein compound, thereby realizing targeted drug delivery of apoptosis inducing factor AIF to tumor cells, and enhancing the inducing apoptosis effect on tumor cells. According to the invention, firstly pET302 plasmid is adopted as the vector to construct a recombinant plasmid integrated with AIF-T18 expression gene, then a prokaryotic expression system E.coli BL21 is taken as the host and the recombinant plasmid is guided therein, thus obtaining a recombinant strain with stable expression ability. On the basis, the invention designs an efficient protein expression method surrounding the characteristics of the recombinant strain, and appropriate denaturation, renaturation and purification conditions are matched for a protein expression product existing in the form of inclusion body, thus realizing effective preparation of AIF-T18.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, and further relates to gene recombination technology and protein expression of recombinant plasmids in host cells, in particular to a bacterial strain for expressing AIF and a recombinant single-chain antibody integrated with AIF and the application of the bacterial strain . Background technique [0002] Endosialin (TEM1 / CD248) is a type I transmembrane protein, which consists of a 80.9KDa protein core region modified by glycosylation, and the mature glycoprotein after modification is about 175KDa. TEM1 is one of the recently discovered human tumor markers, which plays an important role in the growth, angiogenesis, invasion and metastasis of tumor cells. [0003] The outer portion of TEM1 consists of five globular domains (N-terminal C-type lectin domain, sushi-like domain, and three epidermal growth factor (EGF)-like repeats) and a mucin-like domain in which the core protein i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P21/00C12R1/19
CPCC07K14/4747C07K16/30C07K2319/33C12N15/70C12N2800/101
Inventor 陈廷涛辛洪波王鑫
Owner NANCHANG UNIV
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