A kind of heavy/light chain variable region of pinp recombinant antibody and coding gene and recombinant antibody

A technology of recombinant antibodies and encoding genes, applied in the field of genetic engineering, to achieve the effect of small batch difference of antibodies, high stability and consistency, and high yield

Active Publication Date: 2021-04-30
WUHAN CLOUD CLONE CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The present invention aims to solve this series of problems

Method used

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  • A kind of heavy/light chain variable region of pinp recombinant antibody and coding gene and recombinant antibody
  • A kind of heavy/light chain variable region of pinp recombinant antibody and coding gene and recombinant antibody
  • A kind of heavy/light chain variable region of pinp recombinant antibody and coding gene and recombinant antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072]Example 1 Preparation of mice PINP recombinant antibody

[0073](1) PINP monoclonal antibody variable region gene fishing

[0074]1, hybridoma cell RNA extraction and reverse transcription results

[0075]Multiple immunization and screening, a high-specific anti-PINP mouse spleen cell and SP20 fused hybridoma cells, Trizol method extracts Hybridoma Cell RNA, which can be seen in agarose gel electrophoresis, clear 28s and 18s Band, indicating that RNA integrity is better. Seefigure 1 . The RNA concentration and purity measurement were d (260 nm) / d (280 nm) = 1.85, which can meet the requirements.

[0076]The synthesis of CDNA is synthesized by RNA as a template, which is cDNA as a template, and the inner paragraph gene β-actin in mice is used for PCR amplification, and the destination band of length 380 bp is amplified.figure 2 . Note Reverse transcription cDNA can be used for subsequent experiments.

[0077]2, PINP monoclonal antibody variable region PCR amplification

[0078]Primer sequence:...

Embodiment 2

[0133]Example 2, PINP recombinant antibody and PINP natural mice antibody for ELISA method test results comparison

[0134]Since there is no commercial anti-P1NP antibody can be compared, we use the PinP recombinant antibody compared to hybridoma cells in mice in the mice, which purifies the natural anti-PINP monoclonal antibody (hereinafter referred to as PinP natural small) Rats and commercial ELISA kits products (NBP2-76465, R & D) to verify the performance of PINP recombinant antibodies.

[0135]Antibody detects the recognition ability of natural P1NP and recombinant P1NP, ELISA method: PinP recombinant antibody and P1NP natural mouse mono-binds are 96-well plates, and then use the same PINP multi-anti-marking As the detection antibody, the PINP recombinant antibody was used as a standard, TMB as a substrate, and ELISA was detected, as follows:

[0136]1, linear range

[0137]PINP recombinant antibody range is: 7.8pg / mL-1,000 pg / ml;

[0138]PINP Natural mouse single-resistant linear range ...

Embodiment 3

[0173]Example 3, PINP recombinant antibody and PinP natural mice for CLIA method test results

[0174]Targets were determined by clinically CLIA quantitative amounts. PINP recombinant antibodies and PinP natural mice are suitable for CLIA methods. We used PINP recombinant antibodies and PINP natural mice to be used as a 96-well plate, and then used the same PINP multi-anti-marking, Mamino as a substrate, recombinant Pinp as a standard , Detect antibodies, CLIA detection, as follows:

[0175]1, detection range

[0176]PINP recombinant antibody is used for CLIA method detection range: 1.5pg / ml-10,000 pg / ml;

[0177]PINP Natural Mice Amount is used in CLIA method detection range: 4.6pg / mL-30,000 pg / ml.

[0178]2, CLIA method standard curve regression coefficient R2

[0179]PINP recombinant antibody R20.9999;

[0180]Pinp Natural mouse single anti-R2It is 0.9984.

[0181]3, CLIA method: This value is a concentration corresponding to the concentration of two standard deviations measured by 20 blank samp...

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Abstract

The invention discloses a heavy / light chain variable region of a PINP recombinant antibody and a coding gene and a recombinant antibody. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 1, and the heavy chain variable region nucleoside The acid sequence is shown in SEQ ID NO:2, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3, and the gene sequence encoding the light chain variable region is shown in SEQ ID NO:4. The variable region of the heavy chain and the variable region of the light chain are connected to their respective expression vectors and then co-transfected into animal cells to obtain recombinantly expressed PINP recombinant antibodies. The recombinant antibody expression plasmid is easy to preserve, and the recombinant expression method leads to a high yield rate of the antibody. The antibody has strong specificity, good stability, high sensitivity in various detection methods, and is suitable for wide-scale clinical use.

Description

Technical field[0001]The present invention relates to the field of genetic engineering, and more particularly to a light chain variable region, a heavy chain variable region, a heavy chain variable region, and a recombinant antibody thereof in the preparation of vital diagnostic reagents.Background technique[0002]With the increase of global aging population, bone metabolism-related diseases have jumped before the world's common diseases, so bone metabolism has also become one of the hotspots of medical research. More and more researchers and clinicians have begun to find clinical diagnostic markers for bone metabolism. At present, a large number of studies have shown that bone formation markers - PinP, closely related to multiple bone metabolism and metabolic diseases. The expression level of PINP reflects the formation of fresh bones, so it can be judged to osteoporosis and post-treatment; at the same time, PINP can also be used to determine malignant disease related to bone metabo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/11
CPCC07K16/18C07K2317/56
Inventor 何峰容乐鑫孙颖操媛刘勇姚新欣乐音谱严小丽姚雯李华渊
Owner WUHAN CLOUD CLONE CORP
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