A kind of anti-phosphatase 2cm monoclonal antibody and its application

A technology of monoclonal antibody and phosphatase, which is applied in the direction of anti-enzyme immunoglobulin, instruments, biochemical equipment and methods, etc., can solve the problems of incomplete research on molecular mechanism of action, no specific detection of monoclonal antibody, etc., and achieve the goal of preparing The method is simple and efficient, highly specific, and has strong specific effects

Inactive Publication Date: 2020-04-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although long-term studies have found that PP2Cm plays a key regulatory role in the catabolism of BCAA, in the past 20 years of research, the research on the molecular mechanism of BCKDH and PP2Cm in the metabolism of BCAA is not perfect enough, and there is no specificity. Monoclonal Antibodies for Sex Detection

Method used

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  • A kind of anti-phosphatase 2cm monoclonal antibody and its application
  • A kind of anti-phosphatase 2cm monoclonal antibody and its application
  • A kind of anti-phosphatase 2cm monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation and Purification of Recombinant Rat PP2Cm Protein

[0025] 1. Gene cloning

[0026] Using the existing template plasmid PP2Cm, the source gene is on the T vector, and the plasmid information comes from the literature: A novelmitochondrial matrix serine / threonine protein phosphatase regulates themitochondria permeability transition pore and is essential for cellular survival and development. Genes Dev 2007;21:784– 96. [PMC free article] [PubMed] (provided by Dong Bing, China Agricultural University), selected a partial gene fragment 377-1405bp for cloning and recombination. Ncol and UTR restriction enzyme sites were added at the 5' and 3' ends of the gene, respectively, and 3'Xhol cloned the gene into the vector pET28a (Kan) by cutting the 5'Ncol and 3'Xhol, and handed it over to Suzhou Jin Synthesized by Weizhi Biotechnology Co., Ltd., the primer sequence is as follows:

[0027] Upstream primer (5'-3'): TAATACGACTCACTATAGG;

[0028] Downstream p...

Embodiment 2

[0032] The establishment of embodiment 2 hybridoma cell lines

[0033] 1. Immunity

[0034]The protein purified in Example 1 was emulsified with complete Freund's adjuvant (Sigma Company), and immunized with 4-6 week-old female Balb / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) by intraperitoneal injection of each Mice were immunized with 50 μg of protein every 14 days, and the second immunization antigen was emulsified with Freund’s incomplete adjuvant (Sigma Company), and then directly immunized with the antigen at a dose of 50 μg / mouse. Three days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the multi-antibody titer of the anti-immunogen in the mouse serum. 50μg / piece. Serum titer detection of 5 mice after the third immunization see image 3 . The antigen used for immunizing mice is recombinant PP2Cm antigenic protein, which is encoded by the nucleotide sequence in Sequence Table 1 an...

Embodiment 3

[0041] Example 3 Cell line establishment and ascites induction method to prepare monoclonal antibody

[0042] 1. Establishment of cell lines

[0043] After routine ELISA detection, 5 positive reaction cell lines were obtained. After 5 times of subcloning, one of them was found to have a higher reaction value. It was named 7P3 and expanded for culture. It was used for liquid nitrogen cryopreservation and preparation of monoclonal antibody ascites. . The 7P3 hybridoma cell line was collected on March 30, 2017 in the General Microbiology Center of the China Committee for Microbial Culture Collection (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, CGMCC for short, zip code 100101), classified as mouse hybridoma cell line, and its deposit number is CGMCCNo.13818.

[0044] 2. Ascites preparation

[0045] Cells in the logarithmic growth phase were washed with serum-free medium and suspended, counting 5...

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Abstract

The invention provides an anti-phosphatase 2Cm monoclonal antibody and also provides the preparation and application of the monoclonal antibody. A high-purity phosphatase 2Cm recombinant protein is obtained through a genetic engineering method. The most stable and most active hybridioma cell line of a phosphatase-secreting 2Cm protein antibody is screened out by using the recombinant protein, the preservation number of the hybridioma cell line is CGMCC NO.13818. The monoclonal antibody produced by the hybridioma cell line has high specificity and strong affinity, the content of rate-limiting enzyme phosphatase 2Cm of the BCAA metabolic process can be detected, and the anti-phosphatase 2Cm monoclonal antibody is used for auxiliary diagnosis of relevant diseases caused by BCKDH lack and can be also used for early screening of animal selection and breeding.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to an anti-phosphatase 2Cm monoclonal antibody monoclonal antibody, which can be used to detect phosphatase 2Cm. Background technique [0002] Phosphatase 2Cm (PP2Cm) plays a key regulatory role in the catabolism of branched-chain amino acids (BCAA). Studies have found that PP2Cm is essential for cell survival and normal development, and its activity can be down-regulated under stress conditions. Using proteomics to study mitochondrial phosphatases, it was found that PP2Cm can specifically bind to α-branched-chain ketoacid dehydrogenase complex (BCKDH) and catalyze the dephosphorylation of Ser293 in the presence of substrates. The absence of PP2Cm abolishes the ability of substrates to induce dephosphorylation of α-branched-chain ketoacid decarboxylase (E1α) in vivo and in vitro, and the BCAA metabolism deficiency in PP2Cm-deficient mice is similar to that of maple diabetes melli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/40G01N33/577G01N33/573C12R1/91
CPCC07K16/40G01N33/573G01N33/577G01N2333/916G01N2800/042
Inventor 董冰陈思潭刘亚京朴香淑尹靖东
Owner CHINA AGRI UNIV
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