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CD123 single domain antibody, nucleic acid sequence, expression vector and kit

A nucleotide sequence, single-domain antibody technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, introduction of foreign genetic material using vectors, biological testing, etc. Transformation, low binding force and other problems, to achieve the effect of high expression, easy transformation and low production cost

Active Publication Date: 2020-02-18
SHENZHEN PREGENE BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In related technologies, CD123 ordinary antibodies can be realized with relatively large molecular weight, low binding force to antigens, difficult modification and poor stability

Method used

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  • CD123 single domain antibody, nucleic acid sequence, expression vector and kit
  • CD123 single domain antibody, nucleic acid sequence, expression vector and kit
  • CD123 single domain antibody, nucleic acid sequence, expression vector and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Anti-CD123 Antigen Specific Antibody Library Construction

[0033] It should be noted that the CD123 antigen used in this example was purchased from Beijing Sino Biotech. The QIAGEN kit is the QIAamp RNA Blood Mini Kit (50) from Beijing Yaanda Biotechnology Co., Ltd., Cat. No. 52304. The cDNA synthesis kit was MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0 from TAKARA Company.

[0034] 1. Immunization experiment.

[0035] 1) Select a healthy alpaca as the immune target;

[0036] 2) Using 2 mg of CD123 antigen mixed with an equal volume of Freund's adjuvant to immunize a healthy alpaca, a total of 6 immunization experiments were carried out;

[0037] 3) After the 4th immunization experiment, the alpaca serum was sampled, and its antigen immune titer was determined, until the measured immune titer of the alpaca serum reached more than 10,000 times, 100ml of alpaca whole blood was taken, and isolate lymphocytes;

[0038] 4) lyse the lymphocytes in step 3), e...

Embodiment 2

[0067] The screening of embodiment 2 specific phage

[0068] 1) Preliminary preparation. Prepare 5*10 CD123-negative control cell lines and modified CD123-positive cell lines respectively 6 , use CPBSH or 2% BSA-PBS to incubate on a drum at room temperature for 2 hours, and centrifuge for later use.

[0069] 2) Screening. The phage (5*10 11 ~1*10 12 ) into negative cells, CPBS or 2% BSA-PBS to 0.5ml, incubate on a drum at room temperature for 2 hours; take supernatant, add to positive cells, CPBS or 2% BSA-PBS to 0.5ml , incubated on a drum at room temperature for 2 hours; so that the shell of the phage can specifically secrete CD123 antibody and bind to CD123 antigen; wash 5 times with PBST, wash 3 times with PBS, centrifuge, and discard the supernatant to remove the non-binding The phages were washed away.

[0070] 3) Titer determination. The above cell-bound phages were resuspended, and 2ml of TG1 in the logarithmic growth phase was taken, infected, and allowed to st...

Embodiment 3

[0076] Example 3 Screening of specific positive monoclonal antibodies

[0077] 1) Screen the highly expressed recombinant plasmids.

[0078] The specific CD123 antibody gene fragment obtained in Example 2 was amplified by PCR to obtain PCR products with restriction endonucleases BbsI and BamHI sites; the above PCR products and pSJF2 were treated with restriction endonucleases BbsI and BamHI respectively Vector; T4 ligase is used to connect the above-mentioned digested gene fragments and recombine them to obtain a recombinant plasmid sdAb-pSJF2 capable of high-efficiency expression in Escherichia coli.

[0079] 2) Screen CD123 positive cloned antibodies.

[0080] Pick a single colony from the agar plate where the colony grows, inoculate it in a 96-well deep-well culture plate containing Amp-containing 2YT liquid medium, and cultivate it for 4 hours; inoculate the single clones one by one in the numbered cells separated by small grids on the LB solid plate containing Amp; add ...

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Abstract

The invention discloses a CD123 single domain antibody, a nucleic acid sequence, an expression vector and a kit. An amino acid sequence of the CD123 single domain antibody includes an FR1 region, a CDR1 region, an FR2 region, a CDR2 region, an FR3 region, a CDR3 region and an FR4 region which are formed in sequence, wherein the FR1 region, the FR2 region, the FR3 region and the FR4 region are composed of frame-region amino acid sequences, the CDR1 region, the CDR2 region and the CDR3 region are composed of variable-region amino acid sequences, and the amino acid sequences of the FR1 region, the CDR1 region, the FR2 region, the CDR2 region, the FR3 region, the CDR3 region and the FR4 region are shown in a table 1. According to the technical scheme, in combination with a genetic engineeringmethod, the single domain antibody capable of specifically binding to a CD123 protein is obtained, and the single domain antibody has high specific binding capability of specifically binding to the CD123 protein and high affinity.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and antibody medicine, in particular to a CD123 single domain antibody, nucleotide sequence, expression vector and kit. Background technique [0002] CD123 is the alpha chain of interleukin-3 receptor (IL-3R), which can specifically recognize interleukin-3 (Interleukin-3, IL-3) and bind to IL-3. [0003] IL-3 is mainly produced by helper T cells activated by antigen stimulation, which can promote cell growth and proliferation. It is related to the occurrence of tumors, allergic inflammation, and autoimmune diseases. CD123 is expressed on the primary leukemic cells of most AML patients. Moreover, CD123 is highly expressed in AML LSCS, but not or weakly expressed in normal hematopoietic stem cells. In addition to being expressed in AML, CD123 is also expressed in chronic myeloid leukemia, myelodysplastic syndrome and mastocytosis, and B-cell acute lymphoblastic leukemia. Regarding t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/70G01N33/68
CPCC07K16/2866C07K2317/565C07K2317/569C12N15/70G01N33/6854G01N33/6869G01N2333/7155
Inventor 栗红建李莹莹张继帅包朝乐萌宋宗培蔡清华丁怡瑾曾卉
Owner SHENZHEN PREGENE BIOPHARMA CO LTD
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