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ACPP-anti-p21Ras single-chain antibody fusion protein capable of entering tumor cells and preparation method thereof

A p21ras and fusion protein technology, applied in the field of ACPP-anti-p21Ras single-chain antibody fusion protein and its preparation, can solve the problem of low immunogenicity, achieve the effects of increased expression, easier industrial production, and easier preparation

Active Publication Date: 2021-11-19
中国人民解放军联勤保障部队第九二〇医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And because there is no Fc fragment of the whole antibody, the immunogenicity is low, and the anti-mouse antibody reaction will hardly be generated when used in the human body

Method used

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  • ACPP-anti-p21Ras single-chain antibody fusion protein capable of entering tumor cells and preparation method thereof
  • ACPP-anti-p21Ras single-chain antibody fusion protein capable of entering tumor cells and preparation method thereof
  • ACPP-anti-p21Ras single-chain antibody fusion protein capable of entering tumor cells and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Preparation of single-chain antibody gene fragments

[0052]1.1 Immunization of Balb / c mice with p21Ras protein: Take 5 Balb / c mice aged 6-8 weeks (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and inject 100 μg of the laboratory-processed Prokaryotic expression of purified p21Ras-K protein (for the preparation method of p21ras-K protein, refer to the paper "Expression, Identification and Purification of Recombinant p21ras Protein and Preparation of Polyclonal Antibody"), add an equal amount of complete Freund's adjuvant for the initial injection , subcutaneous injection at 5 points. Two weeks later, the second injection was given, with the same dose as the first injection, plus an equal amount of incomplete Freund's adjuvant, and injected subcutaneously at 5 points. Two weeks later, the third injection was given, the dose was the same as the first one, without adjuvant, intraperitoneal injection. The fourth injection was g...

Embodiment 2

[0059] Example 2: Establishment and screening and identification of single-chain antibody library

[0060] 2.1 Construction of recombinant phagemid

[0061] 2.1.1 Double enzyme digestion of the recombinant pMD-ScFv vector and expression vector: extract the plasmid from the positive pMD-ScFv clone identified by PCR, and follow the instructions of Tiangen Plasmid Extraction Kit. Recombined pMD-ScFv plasmid and expression vector plasmid pCANTAB-5E (purchased from Pharmacia Company) were digested with Sfi I respectively, and 30 μl of expression vector plasmid / pMD-ScFv vector were added in 200 μl PCR reaction tube respectively; Sfi I enzyme ( 10U / μl) 4μl; 10×Buffer M 5μl, ddH 2 O 11 μl, after the above system is configured, react at 50°C for 4 hours. After gel recovery and purification of the digested product, add 30 μl of the purified product, 2 μl of Not I enzyme (10U / μl), 5 μl of 10× Buffer H, 2 μl of BSA, 2 μl of Trion X-100, ddH into a new 200 μl PCR reaction tube 2 O 9 μl,...

Embodiment 3

[0073] Embodiment 3: Preparation of ACPP-anti-p21Ras single-chain antibody fusion protein

[0074] 3.1 Design and construction of recombinant prokaryotic expression plasmid of ACPP-anti-p21Ras single chain antibody

[0075] Add the ACPP gene sequence (see SEQ ID NO: 6) directly to the 5' end of the anti-p21Ras single-chain antibody gene sequence obtained in the previous step, and finally obtain the gene sequence of the ACPP-anti-p21Ras single-chain antibody fusion protein. The specific sequence is shown in SEQID NO: 7. The ACPP-anti-p21Ras single-chain antibody fusion gene sequence was codon-optimized according to the codon preference of Escherichia coli through the online website (http: / / www.jcat.de / ), and the optimized sequence (see SEQ ID NO for the specific sequence: 1) Send to Kunming Qingke Biotechnology Co., Ltd. for chemical synthesis, and at the same time add Nde I at the 5' end of one sequence, add a Hind III restriction site at the 3' end, and add Kpn I at the 5' e...

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Abstract

The invention discloses a sequence, expression and purification preparation method of an ACPP-anti-p21Ras single-chain antibody fusion protein capable of entering tumor cells and an application thereof. According to the invention, an optimal fusion protein prokaryotic recombinant expression system is established by screening different combinations of prokaryotic expression vectors and strains, expression conditions are optimized, and a pilot-scale expression and purification process is established by using a fermentation tank and an AKTA chromatographic system. Compared with the prior art, the expression quantity and purity of the fusion protein are improved, a foundation is laid for large-scale production, and meanwhile, the ACPP-anti-p21Ras single-chain antibody fusion protein can enter tumor cells to be combined with p21Ras protein, so that an ras signal channel is blocked, and the purposes of inhibiting tumor cell growth and inducing tumor cell apoptosis are achieved. The ACPP-anti-p21Ras single-chain antibody fusion protein has a wide application prospect in the aspect of preparing a preparation for treating ras-related tumors.

Description

technical field [0001] The invention relates to the technical field of bioengineering, and more specifically relates to an ACPP-anti-p21Ras single-chain antibody fusion protein capable of entering tumor cells and a preparation method thereof. Background technique [0002] The ras gene is an important cell proto-oncogene, and its name comes from the English rat asrcoma of rat sarcoma. The ras gene family includes three main members: H-ras, K-ras, and N-ras, which are located on chromosomes 12, 11, and 1, respectively, and encode a protein with a molecular weight of about 21KD consisting of 188-189 amino acids. The amino acid sequence homology of the three Ras proteins is 85%. [0003] As an extremely important signal transduction protein, Ras protein regulates the normal differentiation and proliferation of cells. After the Ras protein is synthesized, its carboxy-terminus must undergo complex post-translational modifications, so that it can be accurately positioned on the i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12P21/02C07K1/22C07K1/14C12N1/21A61K39/395A61K47/64A61P35/00C12R1/19
CPCC07K16/32C12N15/70C12P21/02A61K47/64A61P35/00C07K2317/622C07K2319/10C12N2800/22A61K2039/505
Inventor 杨举伦冯强杜禹林芯芮潘鑫艳
Owner 中国人民解放军联勤保障部队第九二〇医院
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