Humanized monoclonal antibody IgG Fab fragment of dermatophagoides farinae 2 allergoid specificity as well as preparation method and application thereof

An allergen-like and dust mite technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, anti-animal/human immunoglobulin, etc., can solve the problems of unclear specific mechanism and achieve the reduction of allergic asthma Symptoms, effects of prolonging half-life

Active Publication Date: 2013-07-10
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, more and more evidences have shown that maternal allergen-specific IgG can effectively protect offspring from allergic symptoms, but the specific mechanism is still unclear. Phagocytosis, or inhibition of B cell function, or regulation of fetal immune response to exert its protective effect

Method used

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  • Humanized monoclonal antibody IgG Fab fragment of dermatophagoides farinae 2 allergoid specificity as well as preparation method and application thereof
  • Humanized monoclonal antibody IgG Fab fragment of dermatophagoides farinae 2 allergoid specificity as well as preparation method and application thereof
  • Humanized monoclonal antibody IgG Fab fragment of dermatophagoides farinae 2 allergoid specificity as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Construction of Antibody Library

[0041] Select 300 informed healthy people (no history of allergic diseases and no allergen immunization) who have not suffered from infectious diseases such as colds in the past 2 months. , Sweden) to separate lymphocytes, 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 (NEW ENGLAND 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 JM1...

Embodiment 3

[0048] Example 3. Screening of positive clones specific to dust mite type 2 allergen (Der f2)

[0049] Take 9×10 8 Transform 100 μl JM109 Escherichia coli with DNA 10ng of antibody library independent of clonal titer, spread the bacterial solution on Luria broth (10g sodii chloridum, 10g tryptone, 5g yeast extract / L, PH 7) plate (containing 50 μg ampicillin / ml), cultured at 37°C for 7 hours, to be cloned (about 5×10 3 Clones / 90mm diameter plate) when the diameter is about 0.1-0.3mm, cover the plate with a nitrocellulose membrane (Armacia / Pharmacia) with a diameter of 82mm. On the LB plate, induce expression at 30°C for 6 hours, and then use lysozyme, DNase and bovine serum albumin (100mM Tris-HCl [pH 7], 150mM NaCl, 5mM MgCl 2 , 1.5% BSA, 1mg of DNase, 40mg lysozyme / ml) to lyse the membrane. After washing to remove residual bacterial debris on the membrane, block with bovine serum albumin (BSA), react with 250 μg of purified recombinant protein, and then react with positiv...

Embodiment 4

[0052] Example 4. Characteristic Analysis of Fab Fragments of Positive Clones

[0053] Take the plasmid of the positive clone AM1L-Hsh, digest it with AscI-NdeI and SfiI-NotI restriction endonucleases, respectively, to obtain the light chain and heavy chain genes, and then combine them with the modified sequencing vectors CV-2 and CV-1 Ligation, transformation of Escherichia coli JM109, extraction of plasmid DNA containing light chain or heavy chain genes, respectively, with M13Reverse primer (5'-GGATAACAATTTCACACAGG-3'), sequenced by Invitrogen. And calculate the amino acid sequence with VectorNTI 10 software ( figure 2 , 4), homology analysis (table 2) was carried out with IgBlast, and according to Kabat system, the CDR and FR of the light chain variable region and the heavy chain variable region of AM1L-Hsh were divided ( image 3 , 5)

[0054] Since the Fab antibody contains 6 His-Tags and the heavy chain is linked to His, His-binding resin (Novagen, Madison, Wis.) was ...

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Abstract

The invention belongs to the field of biotechnology, relating to a humanized monoclonal antibody IgG Fab fragment of dermatophagoides farinae 2 allergoid specificity. In the invention, by constructing a humanized immunoglobulin G gene library through a clone blotting method, an ELISA (Enzyme-Linked Immuno Sorbent Assay) sifts the humanized monoclonal antibody IgG Fab fragment of the dermatophagoides farinae 2 allergoid specificity from the library, through expression, purification and identification, the humanized monoclonal antibody IgG Fab fragment is proved to identify the dermatophagoidesfarinae 2 allergoid specificity and have higher affinity therewith and identify the specificity of dermatophagoides farinae crude antigen at the same time. A mast cell degranulation inhibition test shows that the fragment obviously inhibits the combination of dermatophagoides farinae allergen and the allergic disease patient serum IgE attached to the mast cell surface and plays the role of competitively inhibiting antigen combination. Early prevention can be carried out on allergic asthma patients through the Fab antibody fragment, and a medicament composition for preventing and treating the allergic asthma is further prepared.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to an IgG Fab fragment of human antibody of dust mite type 2 allergen (Der f2) and its coding gene, as well as its preparation method and application. Background technique [0002] Asthma is a common chronic airway disease that is difficult to cure. The treatment of acute asthma attacks is mainly to control symptoms and reduce inflammation. Most patients with moderate to severe asthma need long-term treatment to control their condition. Therefore, asthma patients not only have to bear expensive treatment costs, but also have to bear a series of social problems including mental health and loss of learning and work ability caused by recurrent asthma attacks, insomnia, fatigue, and limited daily activities. This creates a huge burden on individuals, families and society. [0003] Allergic asthma has become a serious public health problem, and its morbidity and mortalit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/63C12N1/21C12N1/19C12N5/10C12P21/08A61K39/395A61P11/06C12R1/19
Inventor 程训佳邵红霞付永锋橘裕司
Owner FUDAN UNIV
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