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Preparation and application of apolipoprotein C-III monoclonal antibody

A technology of monoclonal antibody and apolipoprotein, applied in the field of bioengineering

Inactive Publication Date: 2017-06-09
西藏自治区人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently there are monoclonal antibodies against apolipoprotein C-III, but monoclonal antibodies with higher dilution, better affinity and higher expression are still needed

Method used

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  • Preparation and application of apolipoprotein C-III monoclonal antibody
  • Preparation and application of apolipoprotein C-III monoclonal antibody
  • Preparation and application of apolipoprotein C-III monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: Apolipoprotein C-III antigen polypeptide design

[0016] 1.1 According to the apolipoprotein C-III GenBank accession number GI: 4557323, obtain the human apolipoprotein C-III protein sequence (UniProt: P02656), which contains 79 amino acids.

[0017] 1.2 Use ProtParam (http: / / web.expasy.org / protparam / ) to analyze the characteristics of apolipoprotein C-III protein online. The analysis results are shown in Table 1. The protein has a molecular weight of 8.7646KD and an isoelectric point of 4.72, which is acidic protein.

[0018] Table 1 Analysis results of basic physicochemical properties of apolipoprotein C-III

[0019]

[0020] 1.3 DNAstar software was used to analyze the immunogenicity, hydrophilicity and surface accessibility of apolipoprotein C-III protein, and the results showed that there were 16 amino acids near the C-terminus of apolipoprotein C-III with antigenicity, hydrophilicity and surface accessibility stronger (see figure 1 ).

[0021]...

Embodiment 2

[0023] Example 2: Synthesis and coupling of apolipoprotein C-III antigen polypeptide

[0024] 2.1 Apolipoprotein C-III polypeptide synthesis

[0025] The peptide was synthesized by Shanghai Gil Biochemical Company with a purity of 93.85%.

[0026] 2.2 Coupling of polypeptide and carrier protein

[0027] Dissolve 5mg of polypeptide in 50μl DMSO, add coupling buffer to dilute to a final volume of 1ml, take out 200μl for ELISA detection, add 400μl of OVA solution with a concentration of 10mg / ml to the remaining 800μl of polypeptide solution and mix well, then add 200μl EDC with a concentration of 10 mg / ml was mixed immediately and reacted at room temperature for 2 hours.

[0028] 2.3 Ultrafiltration and packing of coupling products

[0029] Dilute the coupling product to 4ml with PBS, perform ultrafiltration on the coupling product with a 10KD Milipore ultrafiltration centrifuge tube, discard the filtrate in the lower tube after each ultrafiltration, and continue to use PBS wh...

Embodiment 3

[0030] Example 3: Preparation of hybridoma cells resistant to apolipoprotein C-III

[0031] 3.1 Immunization of animals

[0032] Select 6 clean grade BALB / c mice aged 6-8 weeks, take aliquoted coupling protein and equal volume of corresponding adjuvant to emulsify and mix the mice, and immunize the mice. The immunization procedure is shown in Table 2.

[0033] Table 2 Animal immunization program

[0034]

[0035] 3.2 Detection of serum antibody titer by ELISA method

[0036] 7 days after the third immunization, blood was taken from the tail of the immunized mice, and the serum antibody titer was detected by ELISA. The specific steps were as follows:

[0037] Coat the ELISA plate with synthetic apolipoprotein C-III polypeptide (2 μg / ml) as antigen, 100 μl per well, incubate overnight at 4 °C, discard the coating solution, add 200 μl 15% skimmed milk powder to each well, block at 37 °C for 1 h , washed 3 times, added serum samples diluted at a ratio of 1:1000, 1:2000, 1:40...

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Abstract

The invention discloses preparation and application of a mice anti human apolipoprotein C-III monoclonal antibody. Specifically, the invention provides an amino acid sequence of apolipoprotein C-III antigen polypeptide, a mice hybridoma cell strain WLC001 with a preservation number of CCTCC No. C2015191, and an apolipoprotein C-III monoclonal antibody generated by the hybridoma cell strain. The invention also relates to application of the apolipoprotein C-III monoclonal antibody in detecting immunoblotting of the apolipoprotein C-III.

Description

technical field [0001] The invention relates to the technical field of bioengineering. Specifically, the present invention relates to a monoclonal antibody, a hybridoma cell line secreting the monoclonal antibody, and the application of the monoclonal antibody in detecting apolipoprotein C-III immunoblotting. Background technique [0002] Apolipoprotein C-III is a small molecular glycoprotein composed of 79 amino acids. It is the most abundant type of apolipoprotein C family. It is mainly synthesized in the liver and only a small part is synthesized in the small intestine. Apolipoprotein C- III is mainly distributed in chylomicrons, very low-density lipoprotein and high-density lipoprotein. The apolipoprotein C-III gene is located in the q23 region of the long arm of chromosome 11, between apolipoprotein A-I and apolipoprotein A-IV, and the three together form a gene family. The transcription of apolipoprotein C-III is regulated by many factors such as insulin and peroxiso...

Claims

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Application Information

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IPC IPC(8): C12N5/20C12N15/06C07K16/18G01N33/92
Inventor 格桑罗布李康
Owner 西藏自治区人民医院
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