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Single-domain antibody aiming at apolipoprotein A1 and application thereof

A technology of apolipoprotein and single-domain antibody, which is applied in the field of biomedicine or biopharmaceuticals, and can solve problems such as low sensitivity, poor antibody stability, and limited detection of apolipoprotein A1

Inactive Publication Date: 2014-06-04
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this traditional antibody has poor stability, low sensitivity, and high production cost, all of which limit the detection of apolipoprotein A1

Method used

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  • Single-domain antibody aiming at apolipoprotein A1 and application thereof
  • Single-domain antibody aiming at apolipoprotein A1 and application thereof
  • Single-domain antibody aiming at apolipoprotein A1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction of a single domain antibody library against apolipoprotein A1:

[0027] (1) Apolipoprotein A1 extracted from human blood (purchased from Nanjing Huibiao Biotechnology Co., Ltd.), the concentration of apolipoprotein A1 used for immunization was 500 μg / mL, and 0.5 mg of apolipoprotein A1 was added to each immunization Mix with Freund's adjuvant in equal volume, immunize a Xinjiang dromedary (Jurong Shenglong Livestock Breeding Factory), once a week, and immunize 5 times in total, except for the first use of complete Freund's adjuvant (purchased from sigma ), Freund's incomplete adjuvant (purchased from sigma) was used for the remaining times to stimulate B cells to express antigen-specific single domain antibodies during the immunization process. (2) After the 5 times of immunization, extract 100 mL of camel peripheral blood lymphocytes and extract total RNA referring to the RNA extraction kit provided by QIAGEN. (3) According to the Super-Script ...

Embodiment 2

[0037] Example 2: Screening process for single domain antibodies against apolipoprotein A1:

[0038] (1) Coat 100 μg / mL apolipoprotein A1 dissolved in PBS on a NUNC plate and place it at 4°C overnight, and set up a negative control at the same time. (2) On the second day, add 200 μL of 1% milk to the two wells respectively, and block at room temperature for 2 hours. (3) After 2 hours, add 100 μL of phage (8×10 11 tfu immunized camel single-domain antibody phage display gene library) for 1 hour at room temperature. (4) Wash 5 times with PBST (0.05% Tween 20 in PBS) to wash away unbound phages. (5) Use triethylamine (100mM) to dissociate the phage that specifically binds to apolipoprotein A1, and infect Escherichia coli TG1 in logarithmic phase growth, produce and purify the phage for the next round of screening, The same screening process was repeated for 2 rounds. The result is as Figure 4 Display: In the process of continuous screening, positive clones will be continuou...

Embodiment 3

[0039] Example 3: Using phage enzyme-linked immunosorbent method (ELISA) to screen specific single positive clones:

[0040] The schematic diagram of the experiment is shown in Figure 5 As shown, the specific detection is as follows:

[0041] (1) From the cell culture dish containing phage after 3-4 rounds of selection, pick 96 single colonies and inoculate them in TB medium containing 100 μg / mL ampicillin (1 LTB medium contains 2.3 g potassium dihydrogen phosphate, 12.52g dipotassium hydrogen phosphate, 12g peptone, 24g yeast extract, 4mL glycerol), grow to the log phase, add IPTG with a final concentration of 1mmol, and culture overnight at 28°C. (2) Use the infiltration method to obtain the crudely extracted antibody, and transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1 hour. (3) Wash off unbound antibodies with PBST, add primary mouse anti-HA tag antibody (anti-mouse anti-HA antibody, purchased from Beijing Kangwei Century B...

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Abstract

The invention discloses a VHH chain of a single-domain antibody for apolipoprotein A1. The VHH chain comprises a frame region FR and a complementary determining region CDR. The invention discloses amino acid sequences of the frame region FR selected from the following group of FR and the amino acid sequences of the complementary determining region CDR. The invention also discloses two types of single-domain antibodies for the apolipoprotein A1, also discloses two types of DNA molecules for coding the VHH chain of the single-domain antibody for the apolipoprotein A1 mentioned in the invention or the single-domain antibody for the apolipoprotein A1 mentioned in the invention, also discloses a host cell for expressing the single-domain antibody for the apolipoprotein A1, and also discloses the application of the single-domain antibody for the apolipoprotein A in detection of the apolipoprotein A1. Through the gene sequences and the host cell of the single-domain antibody disclosed by the invention, the single-domain antibody can be efficiently expressed in colon bacillus and applied in research and development of an apolipoprotein A1 detection reagent.

Description

technical field [0001] The invention belongs to the technical field of biomedicine or biopharmaceuticals, and relates to a single domain antibody against apolipoprotein A1. Background technique [0002] Apolipoproteins have important physiological functions in lipoprotein metabolism. Apolipoproteins are mainly divided into five types: A, B, C, D, and E. They are mainly synthesized in the liver (partially in the small intestine), and are an important component of plasma lipoproteins. Apolipoprotein A1 levels are affected by factors such as gender, race, age, body mass index, alcohol intake, hormones, and smoking. The research results show that apolipoprotein A1 can bind free cholesterol in surrounding tissues, promote the clearance of cholesterol in arterial wall cells, and accelerate cholesterol metabolism in the liver. Therefore, the clinical significance of apolipoprotein A is the same as that of high-density lipoprotein. It is negatively correlated with coronary heart d...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/63G01N33/68
Inventor 万亚坤孙燕燕李光辉母亚雯
Owner SOUTHEAST UNIV
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