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Single-chain antibody of broad-spectrum anti-p21ras protein and preparation method thereof

A p21ras, single-chain antibody technology, applied in the field of medical biology, can solve the problems of weak vascular permeability, long half-life and high toxicity

Inactive Publication Date: 2010-11-24
成都军区昆明总医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Due to the large molecular weight, weak vascular permeability, poor ability to diffuse into tumors, long half-life and high toxicity of intact antibodies used in tumor biotherapy, their targeted delivery and tumor antagonism application effects are limited.

Method used

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  • Single-chain antibody of broad-spectrum anti-p21ras protein and preparation method thereof
  • Single-chain antibody of broad-spectrum anti-p21ras protein and preparation method thereof
  • Single-chain antibody of broad-spectrum anti-p21ras protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: Trizol method extracts hybridoma cell line total RNA and reverse transcription synthesizes complementary DNA

[0073] 1.1 Extraction of total RNA from hybridoma cell lines

[0074] Trizol reagent can dissolve broken cells, and release substances such as RNA, DNA, and genomic proteins in the cells. The lysate was treated with isopropanol and absolute ethanol, and centrifuged at low temperature at high speed to obtain the total RNA of the hybridoma cell line. The specific steps include: put the cell culture bottle on ice, pour out the culture medium, wash the cells twice with D-Hanks solution, add 1ml trizol, and pump it several times with a pipette, and observe that the cells fall off like sand Transfer the cells to a 1.5ml centrifuge tube. After 10-15 minutes, add 200ul of chloroform, invert up and down to mix thoroughly, and place on ice for 5 minutes. Centrifuge at 4°C, 12000g, for 15 minutes. Remove from centrifuge and place on ice, aspirate superna...

Embodiment 2

[0077] Example 2: Synthesis of single-chain antibody gene fragments by overlap extension method

[0078] 2.1 Using cDNA as a template, PCR amplifies the variable regions of the light and heavy chains of the monoclonal antibody hybridoma cell line respectively

[0079]The process of gene amplification by PCR method is similar to the replication process of DNA in vivo. It mainly utilizes the characteristic of DNA polymerase that depends on the DNA template, imitates the replication process in vivo, and conducts the polymerization reaction under the induction of additional primers. When the reaction temperature is the optimal temperature of DNA polymerase, DNA polymerase will start to incorporate single nucleotide from the 3' end of the primer and move from the 5' end to the 3' end of the template in the presence of four dNTP substrates. End-to-end extension to synthesize a new strand of DNA. After 25-30 cycles (the three steps of denaturation, renaturation, and primer extensio...

Embodiment 3

[0084] Example 3: The single-chain antibody gene fragment was connected to the pMD19-T vector and transformed into competent Escherichia coli DH5α to amplify the target single-chain antibody gene fragment and prepare for DNA sequencing

[0085] 3.1 Ligation reaction of single chain antibody gene fragment and pMD19-T vector

[0086] The special vector pMD19-T for PCR product cloning was rebuilt from pUC19. The EcoR V recognition site was inserted between the XbaI recognition sites at the multi-cloning site of the pUC19 vector. Add a "T" to the 3' end of the side. Most heat-resistant DNA polymerases have the characteristic of adding an "A" at the 3' end of the product when performing PCR reactions. A and T are paired to complete the connection of the PCR product and the carrier, improving the efficiency of ligated cloning. The specific steps are: prepare the following DNA ligation solution in a microcentrifuge tube, the total volume is 5ul. Experimental group: (T-A vector 1ul;...

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Abstract

The invention discloses a single-chain antibody of broad-spectrum anti-p21ras protein and a preparation method thereof. The gene segment of the single-chain antibody is constructed by connecting light and heavy chain variable region gene segments of monoclonal antibody hybridoma cell strains of the broad-spectrum anti-p21ras protein through flexible oligonucleotide linkers; the gene segment of the single-chain antibody is connected with a phagemid expression vector to construct recombinant phagemid; the recombinant phagemid is converted into amber inhibiting colon bacillus TG1 to perform fusion expression; the positive recombinant phagemid is identified by using indirect ELISA screening through phage library exhibition; the positive recombinant phagemid is converted into non-amber inhibiting colon bacillus BL21 (DE3) to perform soluble expression of the single-chain antibody; indirect enzyme-linked immunosorbent assay and immune cell and tissue chemical experiments prove that the single-chain antibody for the soluble expression has strong binding specificity with three p21ras proteins of H-ras, K-ras and N-ras and high affinity; and the single-chain antibody is used for constructing small molecular antibodies such as intracellular antigens and the like and used for diagnosis and research of ras gene related tumors.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a broad-spectrum anti-p21ras gene expression protein single-chain antibody and a preparation method of the antibody. Background technique [0002] Ras gene is an important cell proto-oncogene, and its name comes from the English rat sarcoma of rat sarcoma. The Ras gene family includes three main members, namely 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. Namely p21Ras protein, the amino acid sequences of the three p21Ras proteins have about 85% homology. As an extremely important signal transduction protein, p21Ras protein regulates the normal differentiation and proliferation of cells. [0003] After the p21Ras protein is synthesized, its carboxy-terminus must undergo complex post-translational modifications, so that it can be accurat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/32C12N15/13C12N15/70G01N33/574
Inventor 杨举伦王丽陈玥甄时建周云刚刘杜先赵稳兴
Owner 成都军区昆明总医院
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