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Anti-cd3 nanobody cd3/nb29 and its preparation method and application

A technology of nano-antibodies and recombinant vectors, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibodies, etc., and can solve problems such as easy contamination, limited application range, and difficulty in large-scale production

Active Publication Date: 2020-12-08
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there are many problems in the application of antibody drugs, such as long research and development cycle, high production cost, difficulty in large-scale production, poor stability and easy degradation, high storage cost, easy contamination, high maintenance cost, and immunogenicity, etc. its scope of clinical application

Method used

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  • Anti-cd3 nanobody cd3/nb29 and its preparation method and application
  • Anti-cd3 nanobody cd3/nb29 and its preparation method and application
  • Anti-cd3 nanobody cd3/nb29 and its preparation method and application

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Embodiment 1

[0074] Embodiment 1, the preparation of nanobody

[0075] The present invention provides a nanobody derived from camels, its name is CD3 / Nb29, the amino acid sequence of the nanobody CD3 / Nb29 is shown in SEQ ID No.8 in the sequence table, composed of the nucleotides of SEQ ID No.9 acid sequence code.

[0076] Nucleotide electrophoresis of nanobody CD3 / Nb29 as shown in figure 1 As shown, the first channel is a 2000bp molecular marker, the second channel is a CD3 Nanobody DNA electrophoresis band, and the PCR product band is about 450bp.

[0077] Replace the DNA fragment between the PstI and NotI recognition sequences of the vector pComb3 (Biovector product) with the DNA molecule shown in SEQID No.9, and keep the other sequences unchanged to obtain the recombinant vector pComb3-CD3 / Nb29, pComb3-CD3 / Nb29 and The difference of pComb3 is only that the DNA segment between the PstI and NotI recognition sequences of pComb3 is replaced by the DNA molecule shown in SEQ ID No.9. The ...

Embodiment 2

[0086] Example 2, Determination of Nanobody and CD3 Binding Rate

[0087] Determination of Binding Rate of Nanobody CD3 / Nb29 to CD3 (Direct Method)

[0088] Isolate T cells and B cells from the peripheral blood cells of healthy volunteers, and add the Nanobody CD3 / Nb29 (1 μg) of Example 1 to 1×10 6 Incubate the above T cells and B cells at 4°C in the dark for 30min, wash twice with PBS, add 5μl PE anti-HA tag antibody (abcam, Clone: ​​20B12) and incubate at 4°C for 30min, wash twice with PBS, put the samples on BACKMAN flow cytometry, the results were as Figure 3A , shown in 3B. Figure 3A is the binding percentage of blank control and CD3 Nanobody CD3 / Nb29 to B cells respectively; Figure 3B is the binding percentage of blank control and CD3 Nanobody CD3 / Nb29 to T cells respectively; Figure 3A and 3B The horizontal axis is the fluorescence intensity (PE), the vertical axis is the percentage of the number (% of Max), S2 represents the blank control, and S1 represents the ...

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Abstract

The invention discloses an anti-CD3 nanobody CD3 / Nb29 and a preparation method and an application thereof. The nanobody provided by the invention comprises a determinant complementary area and a framework area; the determinant complementary area of the nanobody is composed of CDR1, CDR2 and CDR3; and through a nucleotide sequence and a host cell of the nanobody disclosed by the invention, the nanobody can be highly expressed in Escherichia coli, is applied to research and development of a CD3 molecular detection reagent, and is used for preparation of tumor inhibitors or tumor cell inhibitorsand preparation of drugs for inhibiting CD3 activity and promoting T cell proliferation.

Description

technical field [0001] The invention relates to anti-CD3 nanobody CD3 / Nb29 in the field of biomedicine and its preparation method and application. Background technique [0002] CD3 molecule is an important differentiation antigen on the T cell membrane and a characteristic mark of mature T cells. It forms a complex with the T cell surface receptor TCR and plays an important role in intracellular signal transduction. CD3 antibody can specifically recognize CD3 molecules on the surface of T cells, causing cross-linking of T cell TCR-CD3 complexes, directly generating activation signals, leading to activation and proliferation of T cells. Current research shows that CD3 antibody plays an important role in tumor therapy. [0003] However, there are many problems in the application of antibody drugs, such as long research and development cycle, high production cost, difficulty in large-scale production, poor stability and easy degradation, high storage cost, easy contamination,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCA61P35/00C07K16/2809C07K2317/22C07K2317/55C07K2317/565C07K2317/567C07K2317/569C07K2317/622C07K2317/74C07K2317/76C07K2317/92
Inventor 卢小玲杨晓梅段斯亮
Owner GUANGXI MEDICAL UNIVERSITY