Single-chain antibody resisting HIV-1 outer membrane protein and recombinant immunotoxin

A single-chain antibody and outer membrane protein technology, applied in the direction of antiviral immunoglobulin, antibody, antiviral agent, etc., can solve the problems of destruction, poor curative effect of single drug, incomplete cure of AIDS, etc., and achieve strong antigen binding characteristics Effect

Inactive Publication Date: 2004-11-24
MILITARY VETERINARY INST MILITARY SUPPIES PLA
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AI Technical Summary

Problems solved by technology

[0005] Recombinant immunotoxins prepared by anti-HIV-1 outer membrane protein single-chain antibodies can be used as therapeutic drugs for HIV infection. At present, the drugs used for HIV-1 treatment at home and abroad are mainly chemical drugs. The nucleosides first started in 1987 Drugs, such as zidovudine (ADV), didanosine (ddI), zalcitacine (ddC), radevudine (3TC), etc., but in terms of clinical application, nucleoside drugs have no effect on HIV- 1, the curative effect was unsatisfactory; in 1994, after 4-5 years of research, non-nucleoside reverse transcriptase inhibitors and protease inhibitors began to be used clinically, mainly including nevirapine, saquinavir, Ritonavir and Indinavir, These drugs can reduce the amount of virus and increase the count of CD4 cells, but the effect of single drug is not good. In 1995, a combination of multiple drugs was used, that is, Highly active anti-retroviral therapy (Highly active anti-retroviral therapy HAART). Virus therapy can achieve better therapeutic effect, can effectively inhibit virus replication, and plays a very important role in the treatment of HIV-1 infection. It can basically completely remove free HIV-1 virions in the blood
But despite this, this method still cannot completely cure AIDS. The fundamental reason is that there is latent infection of HIV-1 in the resting CD4+ memory T lymphocytes, so when the therapeutic drugs of high-efficiency antiviral therapy are stopped, this A latently infected HIV-1 can replicate again with the activation of CD4+ memory T lymphocytes that were originally in a quiescent state, resulting in a rebound in the number of HIV-1 viruses in the body. For this situation, people have proposed two feasible adjuvant treatments The first way is to use IL2 to activate HIV-1 infection CD4 which was originally in a static state + Memory T lymphocytes, activated CD4 + Memory T lymphocytes can be recognized and killed by the body's CTL response, and infected HIV-1 itself can also target activated CD4 + Memory T lymphocytes cause certain damage, so that these cells disintegrate and release HIV-1 virions, which are killed by the body's neutralizing antibodies and HAART drugs; another treatment strategy is to use recombinant The toxin specifically kills and destroys the target cells infected by HIV-1. On the one hand, this recombinant toxin can directly destroy the resting CD4+ memory T lymphocytes infected by HIV-1, and on the other hand, it can activate them and accelerate the activation of HIV. -1 Disintegration of infected T lymphocytes

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  • Single-chain antibody resisting HIV-1 outer membrane protein and recombinant immunotoxin
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  • Single-chain antibody resisting HIV-1 outer membrane protein and recombinant immunotoxin

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Embodiment Construction

[0024] The implementation method of the present invention is described below through the isolation of anti-HIV-1 outer membrane protein single-chain antibody gene and the preparation and identification of recombinant toxin.

[0025] 1. Cultivation, storage and recovery of monoclonal antibody hybridoma cell lines

[0026] Conventional methods were used for culturing, preservation and recovery of hybridoma cell lines. For specific steps, refer to "Modern Immunology Experimental Techniques" Hubei Science and Technology Publishing House, pp. 24-25, edited by Shen Guanguan and Tong Rulin. The medium used for culturing hybridoma cell lines is M-1640 complete medium.

[0027] 2. Routine gene manipulation in molecular biology

[0028]Preparation and transformation of Escherichia coli competent cells, plasmid extraction and restriction endonuclease digestion, recovery of DNA fragments, ligation of linear DNA fragments, screening and identification of recombinant plasmids, PCR amplific...

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Abstract

A single-chain antibody resisting HIV-1 outer membrane protein and two recombinant immunotoxins resisting HIV-1 are disclosed. Said single-chain antibody has particular gene sequence and can be used for the diagnosis and immunotherapy of HIV infection. Said recombinant immunotoxins is prepared by using said single-chain antibody to respectively replace the receptor binding regions of pseudomonas aeruginosa exotoxin and diphtherin, and can be used as the medicine to treat HIV infection.

Description

Technical field: [0001] The invention provides a single-chain antibody gene against human type I immunodeficiency virus (HIV-1) outer membrane protein gp120 and two recombinant immunotoxins against HIV-1 infected cells, belonging to the field of biotechnology. Background technique: [0002] Monoclonal antibodies play an important role in the diagnosis and treatment of AIDS, but there are two problems in the practical application of mouse-derived monoclonal antibodies as drugs: one is that mouse-derived antibodies are heterologous proteins to humans, and repeated use of them will cause The anti-mouse antibody is HAMA (human anti-mouse antibody); the second is that the monoclonal antibody has a large molecular weight and poor tissue penetration. In addition, because the hybridoma cell lines of monoclonal antibodies will cause genetic variation during long-term storage, the ability to secrete monoclonal antibodies will be reduced, and human-human hybridomas cannot be immunized ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/395A61K39/42A61P31/18C07K16/08C07K16/10C12N15/09C12N15/13C12N15/62
Inventor 金宁一王宏伟金洪涛江文正张应玖金扩世郭志儒夏志平
Owner MILITARY VETERINARY INST MILITARY SUPPIES PLA
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