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A method for preparing nanobodies against carcinoembryonic antigen

A technology of nano-antibody and antigen, applied in the field of preparation of antibody protein

Active Publication Date: 2021-05-04
深圳市国创纳米抗体技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the unique characteristics of nanobodies in terms of molecular weight and spatial structure, it is difficult to express them compared with traditional Kluyveromyces lactis. So far, there has been no report on the expression of nanobodies against carcinoembryonic antigen (Anti-CEA) in yeast cells.

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  • A method for preparing nanobodies against carcinoembryonic antigen
  • A method for preparing nanobodies against carcinoembryonic antigen
  • A method for preparing nanobodies against carcinoembryonic antigen

Examples

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Embodiment 1

[0030] Example 1. Preparation of Nanobodies Against Carcinoembryonic Antigen

[0031] 1. Anti-carcinoembryonic antigen nanobody gene cloning

[0032] 1.1 Primer Design

[0033] According to the sequence that can detect the CEA gene obtained from the previous screening, the nucleotide sequence was optimized (SEQ ID NO. 1) without changing the amino acid sequence (SEQ ID NO. 2), and then based on the expression of Kluyveromyces lactis Based on the characteristics of the multi-cloning site of the vector pKLAC2, the following pair of primers were designed and synthesized:

[0034] The pKL1 forward primer is:

[0035] 5'- ccatgg GCGGCCGCGAATCTGGTGGTGGTTTGGTTCAA-3';

[0036] The pKL2 reverse primer is:

[0037] 5'-AGG gaattc TCAATGATGATGATGATGATGTTGAGTACCTTGACCGAAGTAACC-3’

[0038] Both ends of pKL1 and pKL2 are designed with NotI and EcoRI restriction sites respectively (see the lowercase and underlined parts in the above sequence)

[0039] 1.2 PCR amplification of Anti-C...

Embodiment 2

[0086] Example 2. Affinity determination of Kluyveromyces lactis-expressing nanobody 11C12 protein against carcinoembryonic antigen

[0087]The affinity of Nanobodies against carcinoembryonic antigen was tested by direct coupling method (Biacore). The carcinoembryonic antigen used Recombinant Carcino Embryonic Antigen CEA protein (Abcam, product number is ab99235).

[0088] Prepare 20ug / ml of anti-carcinoembryonic antigen nanobodies with running buffer as the sample on the machine, select pH=1.5 glycine hydrochloride system, use the template method that comes with the instrument, and set the parameters as follows: injection time 60 seconds, flow rate 30ul / min, dissociation time 600s, regeneration time 30s, flow rate 30ul / min, the affinity of anti-carcinoembryonic antigen nanobodies to carcinoembryonic antigen was tested. like Image 6 As shown, the affinity of the nanobody against carcinoembryonic antigen measured by the direct coupling method was 5.042×10 -9 .

[0089] se...

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Abstract

The invention provides a method for preparing a nanobody against carcinoembryonic antigen, said method comprising the following steps: (1) amplifying the coding gene of the carcinoembryonic antigen nanobody; (2) converting the coding gene obtained in step (1) to Linked to the expression vector pKLAC2; (3) transducing the vector obtained in step (2) into the host cell Kluyveromyces lactis; (4) inducing the host cell obtained in step (3), and harvesting the expressed anti-carcinoembryonic antigen nano Antibody. The invention also provides an expression vector containing the anti-carcinoembryonic antigen nanobody and a host cell Kluyveromyces lactis containing the vector. Using the method of the present invention, the expressed nanobody is secreted to the outside of the cell, the foreign protein is less, and the target band detected by Western Blot is single, thereby reducing the cost of separation and purification, and greatly improving the efficiency of expression; Under the conditions of bottle culture, the amount of extracellular secretion was 0.8 mg / l, and the affinity of the nanobody against carcinoembryonic antigen measured by direct coupling method was 5.042×10 ‑9 .

Description

technical field [0001] The present invention relates to a method for preparing an antibody protein, and more particularly to a method for preparing a nanobody protein using Kluyveromyces lactis. Background technique [0002] Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a glycoprotein with a molecular weight of about 180 kDa. CEA, a member of the immunoglobulin superfamily, was originally classified as a protein expressed only in fetal tissues and has now been identified in normal adult tissues. Overexpression of CEA has been detected in colorectal, pancreatic, lung, gastric, hepatocellular, breast, and thyroid cancers. Therefore, CEA has been identified as a tumor-associated antigen, while CEA has also been used as a tumor marker, and immunological assays measuring elevated CEA in the blood of cancer patients have been used clinically for cancer prognosis and control. [0003] At present, CEA has become a potential tumor-associated antigen for targete...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/13C12N15/81C12N1/19C07K16/30C12R1/645
CPCC07K16/3007C07K2317/569C12N15/815
Inventor 宋海鹏
Owner 深圳市国创纳米抗体技术有限公司
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