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Surface antigen 1 of Toxoplasma gondii human antibody Fab fragment and encoded gene thereof

A surface antigen and human antibody technology, applied in the field of anti-Toxoplasma gondii surface antigen 1 human antibody Fab fragment and its encoding gene, anti-T. applied clinical problems

Inactive Publication Date: 2009-10-28
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because of the immunogenicity of mouse-derived antibodies and rabbit-derived antibodies, when applied to the human body, they can cause the body's immune response against foreign proteins, and produce human anti-mouse antibodies or human anti-rabbit antibodies, which not only affect the curative effect, but also induce allergic reactions, so it is difficult to clinical application
Although antibody engineering technology can be used to humanize it, the technical process is complicated, time-consuming and labor-intensive, and it is difficult to achieve short-term results; transgenic animals also have problems such as high price and limited output.

Method used

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  • Surface antigen 1 of Toxoplasma gondii human antibody Fab fragment and encoded gene thereof
  • Surface antigen 1 of Toxoplasma gondii human antibody Fab fragment and encoded gene thereof
  • Surface antigen 1 of Toxoplasma gondii human antibody Fab fragment and encoded gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1. Construction of antibody library

[0084] Using 5 ml of peripheral anticoagulated blood from a patient with toxoplasma encephalitis, lymphocytes were separated with Ficoll-Paque (Pharmacia, Uppsala, Sweden), and total RNA was extracted with a kit (QIAGEN GmbH, Hilden, Germany). The total RNA was reverse-transcribed into cDNA using the Gene-Amp RNA PCR kit (Perkin-Elmer Cetus, Norwalk, Conn) with Oligo(dT)16, and the upstream and downstream primers (Invitrogen ) (Table 1) for PCR amplification of immunoglobulin γ, κ, λ chain genes. After the PCR product was purified by a kit (QIAGEN GmbH, Hilden, Germany), the κ chain and λ chain products were double-digested with AscI and NheI (NEWENGLAND BioLabs), respectively. The digested κ chain and λ chain products were combined with human immunoglobulin Fab expression vector pFab-His2( figure 1 ) connection, followed by electrotransformation into Escherichia coli JM109 (Takara, Dalian, China) to form a light chain li...

Embodiment 3

[0092] Example 3. Screening of anti-T. gondii human immunoglobulin G

[0093] Take 10ng of antibody library DNA with independent clone titer of 3×106, transform 100μl JM109 Escherichia coli, spread the bacterial solution on Luria broth (10g sodii chloride, 10g tryptone, 5g yeast extract / L, PH 7) plate ( containing ampicillin 50 μg / ml), cultured at 37°C for 7 hours, and to be cloned (about 5×10 3 Clones / 90mm diameter plate) when the diameter is about 0.3mm, cover the plate with a diameter of 82mm nitrocellulose membrane (Armacia / Pharmacia). After the clones are completely transferred to the membrane, place the membrane on an LB plate containing 1.0mMIPTG On, induced expression at 30°C for 6 hours, and then treated with lysozyme, DNase and bovine serum albumin (100mM Tris-HCl [pH 7], 150mM NaCl, 5mM MgCl 2 , 1.5% BSA, 1 mg of DNase, 40 mg lysozyme / ml) to lyse the membrane. After washing to remove residual bacterial debris on the membrane, block with bovine serum albumin (BSA),...

Embodiment 4

[0096] Example 4. Characteristic analysis of the Fab fragment Tox1403L-11H of the anti-T. gondii tachyzoite SAG1 antibody

[0097] Take the plasmid of the positive clone Tox1403L-11H, digest with AscI-NdeI and SfiI-NotI restriction endonucleases, respectively, to obtain the light chain and heavy chain genes, and then combine with the modified sequencing vectors CV-2 and CV-1 Ligation and transformation of Escherichia coli JM109, extraction of plasmid DNA containing light chain or heavy chain genes, respectively, and sequencing with M13Reverse primer (5'-GGATAACAATTTCACACAGG-3'), the sequencing work was done by Invitrogen. And calculate amino acid sequence (sequence 2,4) with VectorNTI 10 software, carry out homology analysis (table 2) with IgBlast, and according to Kabat system to positive clone Tox1403L-11H light chain variable region and heavy chain variable region CDR, FR are divided ( image 3 , 4).

[0098] Table 2 is the homology comparison of the positive clone Tox140...

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Abstract

The present invention belongs to the field of biotechnology, and relates to a surface antigen 1 (SAG1) of Toxoplasma gondii human antibody Fab fragment, encoded gene and use thereof. According to the invention, the surface antigen 1 (SAG1) of Toxoplasma gondii human antibody Fab fragment is filtered from a base through establishing a Toxoplasma gondii human immunoglobulin, ELISA, diluting the prothrombin time, sequencing analysis, etc. Through expression purifying and authenticating, the human antigen Fab fragment is authenticated to specifically identify the tachyzoite-bradyzoite recombination SAG1 of Toxoplasma gondii and have higher affinity with the tachyzoite-bradyzoite recombination SAG1 of Toxoplasma gondii, for being identified with the specificity of Toxoplasma gondii tachyzoite-bradyzoite. The human antigen Fab fragment of the invention does not contain Fc segment and does not activate the alexin or cause the histopathological damages of human immune response, etc. when the function of restricting the invasion of Toxoplasma gondii to the host cell is exerted. The surface antigen 1 (SAG1) of Toxoplasma gondii human antibody Fab fragment is safe and reliable when applied for the human body. The antigen medicine for treating toxoplasmosis or the antigen targeted medicine can be prepared.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to an anti-toxoplasma gondii antigen antibody, in particular to a Fab fragment of an anti-toxoplasma gondii surface antigen 1 (SAG1) human antibody, its coding gene and its application. Background technique [0002] Toxoplasma gondii (Toxoplasma gondii) is called Toxoplasma gondii for short, belongs to Apicomplexa, Eucoccidia, and Toxoplasmidae. Suffering from toxoplasmosis. Immunodeficiency patients (such as patients with malignant tumors, organ transplantation and immunosuppressants, and immunodeficiency patients) are infected with Toxoplasma gondii, leading to serious systemic toxoplasmosis such as meningitis, hepatitis, pneumonia, and even death. Since toxoplasmosis is one of the most important complications of AIDS patients, the incidence of toxoplasmosis has also been increasing with the increase of AIDS infection rate in recent years. According to investigations, 30%-40% ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/20C12N15/63C12N1/21C12N1/19C12N5/10A61K39/395A61K47/48A61P33/02C12R1/19A61K47/68
Inventor 付永锋程训佳橘裕司
Owner FUDAN UNIV
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