Preparation of anti-human seminoprotein specificity single chain antibody and application method thereof

A single-chain antibody and seminal plasma protein technology, applied in the field of bioengineering, can solve the problems of large molecular weight, long preparation cycle, and difficulty in passing through cancer cell membranes, etc., and achieve the effect of increased sensitivity and small size

Inactive Publication Date: 2008-08-06
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a preparation and application method of an anti-human seminal plasma protein-specific single chain antibody with a transmembrane peptide in view of the shortcomings of the traditional prostate cancer antibody, which has a long preparation period, a large molecular weight of the antibody, and is not easy to pass through the cancer cell membrane.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] According to the above technical scheme, the anti-γ seminal plasma protein single-chain antibody was prepared by combining the polymerase chain reaction 1 and the polymerase chain reaction 2 with the preparation reaction 3, and the specific steps were as follows:

[0031] The first step is to design primers and use polymerase chain reaction technology to obtain anti-human γ-seminal plasma protein single-chain antibody gene fragments

[0032]In order to make the prepared antibodies easier to purify and penetrate the cell membrane, the primers were designed with a histidine tail and a transmembrane peptide KTAT (acid-resistant-threonine-alanine-threonine) at the amino terminus. The upstream primer is: 5'-CGCTTAATTAAA CAG ACC AAG GCG ACC CAT ATG ACC CAG GTC CAA CTG CAG-3', the downstream primer is: 5'-TTAGTTAGTTACCGGATCCC ACG TTT GAT CTC CAG-3', the total length is 783bp (base pairs).

[0033] Use polymerase chain reaction 1 to obtain specific gene fragments: 3 μl of 10×...

Embodiment 2

[0046] Using the same technical means as in Example 1, a biologically active anti-semiplasmin single-chain antibody with the transmembrane peptide KTAT was prepared, and then mixed with the dendrimer-modified cadmium telluride quantum dot solution, the reaction The conditions are: 1ml 0.85mg / ml single-chain antibody is mixed with 1ml 2.5mg / ml dendrimer-modified quantum dots (0.05M phosphate buffer, pH 7.4, 0.04% Tween-20), reacted at 4°C for 10 hours, and then , Add aminoethanol and stir the reaction at 25°C for 2h. The single-chain antibody-coated quantum dots are separated by a chromatographic column filtration method. Then, 5 μl of single-chain antibody-coated quantum dots were added to the cultured prostate cancer cell line LNCaP cell flask and the control gastric cancer cell line 7901, and they were co-cultured for 1 hour, and then washed repeatedly with phosphate buffer (pH 7.4) for 4 Again, under a fluorescent microscope, it can be seen that the prostate cancer cells h...

Embodiment 3

[0048] Using the same technical means as in Example 1, an anti-γ seminal plasma protein single-chain antibody with a transmembrane peptide KTAT was prepared, and then connected with dendrimer-modified magnetic nanoparticles to prepare an anti-γ seminal plasma protein single-chain antibody Antibody-modified magnetic nanoparticles. By injecting 200 μl of prostate cancer cell line LNCaP cells into the back of nude mice subcutaneously and feeding them for 2 weeks, tumor formation can be seen. Then, the prepared magnetic nanoparticles modified by anti-γ seminal plasma protein single-chain antibody were directly injected into the tail vein of nude mice. After 4 hours, under nuclear magnetic resonance, the magnetic nanoparticles could be seen to gather at the prostate cancer site, indicating that anti-γ Seminal protein scFv-modified magnetic nanoparticles can be used as contrast agents for prostate cancer.

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PUM

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Abstract

The invention relates to a preparation method of anti-human seminoprotein specific single-chain antibody and an application method, wherein the preparation method comprises that the inducer has histidine tail and transmembrane peptide threonine-alanine-threonine at the terminal of amido, the ascending inducer is 5'-CGCTTAATTAAA AAG CAG ACC GCG ACC CAT ATG ACC CAG GTC CAA CTG CAG-3', the descending inducer is 5'-TTAGTTAGTTACCGGATCCC ACG TTT GAT CTC CAG-3', the total length is 783bp, using polymerase chain reaction to obtain anti-human gamma-seminoprotein single-chain antibody gene segment, using the gene segment as template to prepare anti-human gamma-seminoprotein single-chain antibody. The antibody can be used to build variable prostate cancer antigen serology detection kit and can be connected with nanometer particles as magnetic nanometer particle and quantum dot to prepare the magnetic nanometer particle and quantum dot covered by gamma-seminoprotein single-chain antibody.

Description

technical field [0001] The invention relates to a method in the technical field of bioengineering, in particular to a preparation and application method of an anti-human seminal plasma protein-specific single-chain antibody with a transmembrane peptide KTAT. Background technique [0002] Human γ-semiplasmin is a specific antigen secreted by prostate cancer, present in prostate cancer cells and their metastatic cancer cells, and is a specific marker of prostate cancer. Applied iodine 131 Radioimmunoimaging of prostate cancer with labeled anti-human seminal protein monoclonal antibody has high sensitivity and specificity, which is superior to B-mode ultrasound and computer-controlled tomography. However, the application of murine antibodies in the human body will lead to the production of human anti-mouse antibodies, which limits its clinical application. Aiming at the current technical deficiencies, the prepared single-chain antibody has many advantages that the parental an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K17/06C12N15/13C12N15/70G01N33/53A61K49/16A61K47/42A61P35/00
Inventor 崔大祥韩月东武国军高峰贺蓉
Owner SHANGHAI JIAO TONG UNIV
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