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Monoclonal antibody against human alpha1 acid glycoprotein and preparation method thereof

A technology of monoclonal antibody and glycosylation, which is applied in the direction of anti-animal/human immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc. It can solve the problem of poor specificity of antigenic glycoprotein antibodies and complicated experimental operations To achieve the effect of increasing the production of antibodies, enhancing the immune response, and improving the production of antibodies

Active Publication Date: 2013-02-27
BEIJING PROTEIN INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above research does not provide a widely applicable method, and the experimental operation is more complicated
[0003] Taking human AGP as an example, the present inventors tried to establish a plan for producing antibodies that specifically recognize glycosylated proteins. The monoclonal antibodies thus produced can basically overcome the defects of low antigenicity of glycoproteins and poor specificity of glycoprotein antibodies

Method used

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  • Monoclonal antibody against human alpha1 acid glycoprotein and preparation method thereof
  • Monoclonal antibody against human alpha1 acid glycoprotein and preparation method thereof
  • Monoclonal antibody against human alpha1 acid glycoprotein and preparation method thereof

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Embodiment 1

[0019] The gene cloning of embodiment 1 glycoprotein

[0020] The inventors designed PCR primers according to the published gene sequence (SEQ ID NO.1) of human AGP:

[0021] AGP 5' Forward Primer

[0022] 5'-GCCAAGCTTATGGCGCTGTCCTGGGTTCT-3' (SEQ ID ON.2)

[0023] AGP 3' reverse primer

[0024] 5'-GGCGGATCCTAGGATTCCCCCTCCTC-3' (SEQ ID ON.3)

[0025] Using the human liver cDNA library as a template, the full-length AGP gene was amplified by PCR (PCR parameters: 95°C for 5 minutes; 94°C for 30s, 54°C for 30s, 72°C for 1 minute, a total of 40 cycles; 72°C and then extended for 10 minutes). After Hind III and BamH I double digestion, it was connected to the pLNCX2-RFP plasmid (the RFP gene came from the pDsRed2 plasmid, and after the RFP gene was cut out from the plasmid with Hind III and Not I endonucleases, it was connected to the pLNCX2-RFP plasmid after the same double digestion pLNCX2 plasmid), chemically transformed competent cells E.coli DH5α, picked a single clone to e...

Embodiment 2

[0026] Example 2 Construction, screening and verification of cell lines stably expressing and secreting exogenous glycoproteins

[0027] Extract the constructed pLNCX2-RFP-AGP plasmid and the plasmid pVSV-G encoding retroviral envelope protein from E.coli DH5α, according to the amount of DNA and transfection reagent Lipofectamine2000 (11668-019, Invitrogen) is 1:5 The ratio of the two plasmids was co-transfected. With the help of chloroquine, the two plasmids were transformed into retroviral packaging cells GP2-293. After 8 hours, the medium was changed to eliminate the toxic effect of chloroquine on the cells. 48 hours after the withdrawal of chloroquine, the culture supernatant of GP2-293 was collected, and the retroviral particles containing foreign genes were released in the culture medium at this time. The precipitated retroviral particles were enriched by prolonged high-speed centrifugation (4°C, 50,000g, 90 minutes). After renatured, these viruses were added to the c...

Embodiment 3A

[0028] The preparation of embodiment 3AGP recombinant antigen

[0029] Design PCR primers according to the published gene sequence of human AGP:

[0030] AGP 5' Forward Primer

[0031] 5'-GCCGGATCCATGGCGCTGTCCTGGGTTCT-3' (SEQ ID ON.4)

[0032] AGP 3' reverse primer

[0033] 5'-GGCAAGCTTCTAGGATTCCCCCTCCTC-3' (SEQ ID ON.5)

[0034] Using the human liver cDNA library as a template, the full-length AGP gene was amplified by PCR, and then double-digested with BamH I and Hind III after recovery from the gel. After recovery, it was ligated with the same double-digested pET28a vector, and chemically transformed into competent cells E.coli DH5α, positive clones were selected after double enzyme digestion verification and plasmid sequencing. The plasmids of the selected positive clones were chemically transformed into competent cells BL21(DE3). Inoculate the BL21 strain that can express AGP, and when the OD value of the bacterial solution reaches about 0.6, add 1 mmol / L IPTG for in...

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Abstract

The invention aims to prepare a specific monoclonal antibody aiming at alpha1 acid glycoprotein (AGP, Serum alpha1-acid glycoprotein) and establish a corresponding experimental method. Particularly, the monoclonal antibody can selectively identify glycosylation modified natural AGP, but has low affinity with prokaryotically expressed non-glycosylation modified AGP. The subtype of the monoclonal antibody is IgG1, and the affinity constant is 9*108. The invention provides a hybridoma cell line BPI-AGP for secreting the monoclonal antibody, wherein the collection number of the hybridoma cell line is CGMCC 2868, and the hybridoma cell line can stably secrete high-valence monoclonal antibody against AGP natural protein. The BPI-AGP can be used for inspecting the AGP in the blood. Meanwhile, the invention also provides a method for preparing monoclonal antibody hybridoma aiming at the glycosylation modified protein.

Description

technical field [0001] The present invention belongs to the fields of immunochemistry, protein chemistry and cell biology. Specifically, the present invention relates to a hybridoma cell line capable of producing a monoclonal antibody specific to human α1 acid glycoprotein (AGP, Serum α1-acidglycoprotein) and a preparation method thereof. The monoclonal antibody prepared by the hybridoma can be used in disease diagnosis or related fields. technical background [0002] The post-translational modification status of proteins is closely related to protein functions, in which glycoproteins play a variety of important biological functions, such as glycoproteins on cell membranes involved in embryonic development, cell movement, immune response, and cell division. In addition, most plasma proteins have varying degrees of glycosylation modifications. It is worth noting that the proteins known as markers for detection of various diseases are all glycoproteins, such as PSA, AFP, CA1...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18G01N33/577C12N15/06C12P21/08C12N5/20
Inventor 任艳韦汉福潘秦刘玲徐宁志刘国振吴琳刘斯奇
Owner BEIJING PROTEIN INNOVATION
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