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Single domain antibody resisting to human nerve dynein 1 and preparation method of single domain antibody

A ciliary protein and single-domain antibody technology, which is applied in the fields of genetic engineering and antibody engineering, can solve the problems of difficult penetration, affecting the targeting effect, unstable quality of market demand, etc., and achieve the effect of broad application prospects

Active Publication Date: 2016-01-13
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, a variety of monoclonal antibodies have been used in the clinical treatment of some malignant tumors, but there are still many limitations in the application of antibody drugs
First of all, the molecular weight of the intact antibody itself is relatively large, and it is difficult to penetrate into the interior of solid tumors
Secondly, the immunogenicity of monoclonal antibodies is strong, and there are toxic and side effects during the treatment process. For example, repeated use of mouse-derived monoclonal antibodies in the human body can induce human anti-mouse antibody (Humananti-murine antibody, HAMA) reaction
Furthermore, the presence of the Fc fragment of an intact antibody can make it bind to non-specific tissue cells with Fc receptors in the body, affecting its targeting effect
In addition, the large-scale production of monoclonal antibodies takes a long time, and the large-scale production of antiserum cannot guarantee market demand and the quality is unstable

Method used

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  • Single domain antibody resisting to human nerve dynein 1 and preparation method of single domain antibody
  • Single domain antibody resisting to human nerve dynein 1 and preparation method of single domain antibody
  • Single domain antibody resisting to human nerve dynein 1 and preparation method of single domain antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: anti-human neuropilin 1 single domain antibody gene (i.e. heavy chain variable region gene, V H ) clone

[0031] (1) Cells used and mouse-derived anti-human neuropilin 1 monoclonal antibody

[0032] The anti-human neuropilin 1 monoclonal antibody hybridoma cell line was developed by the Anticancer Research Center of Xiamen University School of Medicine. The above cells were routinely cultured in RPMI-1640 medium with 10% fetal bovine serum (Gibco) at 37 °C, 5% CO 2 to cultivate.

[0033] (2) Primer design

[0034] Since the CDR sequence of the variable region gene sequence of the antibody to be amplified is unknown, the primers used in the present invention adopt primer sequences popularized internationally.

[0035] at V H A restriction site NcoI:CATG is added to the 5' end of the primer CCATGG (containing the protective base CATG), at V H A restriction site XhoI: CCG is added to the 3' end of the primer CTCGAG (Contains protective base CCG).

...

Embodiment 2

[0046] Example 2: Construction of expression vector for anti-human neuropilin 1 single domain antibody gene

[0047] Purify V with Gel Purification Recovery Kit (OMEGA) H gene product, then V H The gene product and the pET-22b(+) plasmid (Novagen) were digested with restriction endonucleases NcoI and XhoI (NEB), respectively, and the gel was recovered. Under the catalysis of T4DNA ligase (NEB), the anti-human neuropilin 1 single domain antibody gene (V H Gene) cloned into plasmid pET-22b(+) to construct recombinant plasmid pET-22b(+)-V H , transformed into E.coliBL21(DE3), positive clones were screened by PCR and sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing.

Embodiment 3

[0048] Example 3: Prokaryotic expression and purification of anti-human neuropilin 1 single domain antibody gene

[0049] Select the correct sequencing containing pET-22b(+)-V H Single colony of E.coliBL21(DE3) with recombinant plasmid, cultured with shaking at 37°C overnight, diluted 1:100 into LB culture medium, and cultured with shaking until OD 600nmWhen the value is about 0.6-0.8, add IPTG (Sigma) with a final concentration of 0.5mM to induce expression for 4h. The bacteria were collected for SDS-PAGE electrophoresis. After staining and decolorization, it was found that the expression product of E.coliBL21 (DE3) had an obvious band at 16kDa, which was consistent with the expected target protein band size, indicating that the anti-human neuropilin 1 monoclonal Domain antibody genes are expressed (see figure 2 ).

[0050] The results of SDS-PAGE electrophoresis also showed that most of the prokaryotic expression products of the anti-human neuropilin 1 single domain anti...

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Abstract

The invention discloses a single domain antibody resisting to human nerve dynein 1 and a preparation method of the single domain antibody and belongs to the technical field of genetic engineering and antibody engineering. The preparation method comprises the steps of cloning genes of the single domain antibody resisting to the human nerve dynein 1, introducing the genes into a vector, and constructing a recombinant vector; introducing the recombinant vector into a host bacterium, and constructing a recombinant bacterium which expresses the single domain antibody resisting to the human nerve dynein 1; fermenting and culturing the recombinant bacterium, separating and purifying fermentation liquor, and obtaining the single domain antibody resisting to the human nerve dynein 1. According to the single domain antibody resisting to the human nerve dynein 1 and the preparation method of the single domain antibody, a hybridoma cell strain A6 of the monoclonal antibody antibody resisting to the human nerve dynein 1 serves as the template, and RT-PCR cloning is conducted, so that the heavy chain variable region gene (V<H>) of the antibody is obtained; the gene is introduced into the plasmid vector pET-22b (+), and the recombinant vector pET-22b(+)-V<H> is constructed; the recombinant vector is introduced into the expression strain E. coli BL21(DE3), IPTG induces expression of the single domain antibody resisting to the human nerve dynein 1, and then purification and identification are conducted.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and antibody engineering, and relates to an anti-human neuropilin-1 antibody, in particular to an anti-human neuropilin-1 single-domain antibody and a preparation method thereof. Background technique [0002] At present, a variety of monoclonal antibodies have been used in the clinical treatment of some malignant tumors, but there are still many limitations in the application of antibody drugs in treatment. First of all, the molecular weight of the intact antibody itself is relatively large, and it is difficult to penetrate into the interior of solid tumors. Secondly, monoclonal antibodies are highly immunogenic and have toxic side effects during treatment. For example, repeated use of mouse-derived monoclonal antibodies in humans can induce human anti-mouse antibody (Humananti-murine antibody, HAMA) reactions. Furthermore, the presence of the Fc fragment of an intact antibody can ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K1/22C12N15/70C12R1/19
Inventor 颜江华王生育罗芳洪吴婷倪二茹王勇军
Owner XIAMEN UNIV
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