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Porcine CD127 polypeptide and coding gene and application thereof

A coding and gene technology, applied in the field of molecular biology, can solve the problems of flow monoclonal antibody without porcine CD127 gene, lymphocyte subgroup typing and detection, and cannot be used universally

Active Publication Date: 2019-08-13
CHONGQING ACAD OF ANIMAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CD127 antibodies against human and mouse on the market are not universal among species, and there is no flow cytometric monoclonal antibody against porcine CD127 gene
Due to the lack of antibodies, the typing and detection of Treg and other lymphocyte subsets in pigs cannot be performed

Method used

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  • Porcine CD127 polypeptide and coding gene and application thereof
  • Porcine CD127 polypeptide and coding gene and application thereof
  • Porcine CD127 polypeptide and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Acquisition of porcine CD127 extracellular fragment recombinant protein

[0038] According to the cDNA sequence (NM_001146128.2) and protein sequence (NP_001139600.1) of the porcine CD127 gene on GenBank. SignalP 4.1 server was used to analyze the sequence and predict the protein signal peptide. The transmembrane domain was predicted with TMHMM Server v.2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / ). According to the comprehensive analysis results, the porcine CD127 gene protein No. 22 to No. 240 is the extracellular fragment of the protein. Analysis showed that the full length of porcine CD127cDNA was 1999bp, and the coding region (ORF) was from 49th bp to 1428th bp, encoding a protein with a length of 459aa. Signal peptide prediction shows that the first 21aa of CD127 protein is signal peptide ( figure 1 A). The mature CD127 protein is 438aa, the molecular weight is 49kD, and the isoelectric point (pI) is 6.52. Analysis of the transmembrane region showed t...

example 2

[0041] The mouse immunization of example 2 recombinant CD127 protein

[0042] After the recombinant protein of the purified extracellular fragment of CD127 was completely emulsified with complete Freund's adjuvant, 100 μg of CD127 protein per mouse was used for intraperitoneal immunization. After the initial immunization, the CD127 protein emulsified with Freund's incomplete adjuvant was used to immunize once again on the 7th, 14th and 28th day respectively; the mouse serum was collected 3 days after the last immunization, and after the prokaryotic expressed CD127 protein was coated, Antibody titer of mouse serum was detected by Elisa. The results showed that the antibody titers to the recombinant protein in the sera of the six mice were all greater than 1:8000 (Table 1). At the same time, the co-labeling of mouse antiserum and porcine CD3 antibody on lymphocytes in porcine blood was detected by flow cytometry.

[0043] Table 1: Antibody titers of sera from 6 mice

[0044] ...

example 3

[0046] Example 3. Monoclonal Antibody Screening

[0047] Take the spleen cells of mice M2 and M4 and mix them with SP2 / 0 myeloma cells at a ratio of 1:10, centrifuge at 1000g for 3 minutes, discard the supernatant, shake the cells to make them completely evenly mixed, put them in a water bath at 37°C, and Add 1 mL of preheated PEG within 45 s, then add 10 mL of RPMI1640 medium to stop the reaction after standing for 45 s, centrifuge at 1000 g for 10 min, discard the supernatant, add HAT medium containing 20% ​​serum, and culture in a cell culture bottle at 37°C for 48 h , the culture supernatant of the mixed cultured cells was taken for Elisa detection, and the results showed that the two mixed pooled cells could both secrete antibodies against porcine CD127. The OD values ​​of M2 and M4 mixed pool culture supernatants were 2.813 and 2.713 respectively (0.088 and 0.079 for the negative control). At the same time, the medium supernatant was diluted 1:100 as anti-CD127 antibody...

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Abstract

The invention provides a porcine CD127 polypeptide. The porcine CD127 polypeptide is an extracellular partial fragment of a porcine CD127 protein. In a process of screening a monoclonal antibody, thepolypeptide is used as an immunogen, and in combination with Elisa and a flow cytometer for detection, the monoclonal antibody capable of being used for detecting the porcine CD127 gene through the flow cytometer is prepared. By utilizing the antibody, detection and typing of porcine lymphocyte subpopulations, particularly Treg cells, can be carried out by the flow cytometer while the cell activity is maintained. In order to further research the functions of porcine lymphocytes, research and application of cellular immunity are carried out on pigs, and further research on the relationship between porcine disease resistance characters and lymphocytes has great practical significance.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a porcine CD127 gene monoclonal antibody and a preparation method thereof. Background technique [0002] CD127 (leukocyte differentiation antigen molecule 127) is an important regulatory factor for the homeostasis of mature T cells in the periphery. IL-7 responds to the α chain of receptors on T lymphocytes, and is a subset of lymphocytes, especially Treg (regulatory T lymphocytes). Detection and typing of an important marker gene. Anti-CD127 flow monoclonal antibody is an important tool antibody for the study of lymphocyte subsets by flow cytometry. Currently, there are only CD127 commercial antibodies against mouse and human on the market, and there is no CD127 monoclonal antibody against porcine. [0003] Regulatory T cells (Treg) are T cell subsets with immunoregulatory functions in T lymphocytes, and play an important role in maintaining autoimmune tolerance and regulating...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/715C07K16/28C12N15/12C12N15/11G01N15/14
CPCC07K14/7155C07K16/2866G01N15/14Y02A50/30
Inventor 赵久刚蓝静龙熙张凤鸣潘红梅张亮杨溢欢陈磊王金勇潘晓
Owner CHONGQING ACAD OF ANIMAL SCI
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