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Preparation method and application of polyclonal antibody against chicken caspase-1

A technology for polyclonal antibodies and uses, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibodies, etc., can solve problems such as restricting research, and achieve the effect of simple separation and purification method, low cost and short cycle.

Inactive Publication Date: 2017-08-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As for whether other RNA viral diseases or bacterial bacterial diseases can infect chicken cells to activate caspase-1 and induce the expression of inflammatory cytokines such as IL-1β, it is necessary to study the interaction between chicken viral diseases and bacterial diseases and chicken caspase-1 Mechanism, to explore the mechanism of action of caspase-1 in the process of performing biological functions, it is necessary to use various experimental methods such as Western-blot, ELISA, IFA, Co-IP, etc., and these research methods must have anti-chicken caspase-1 polyclonal Antibodies are used as support. However, no chicken caspase-1 antibody has been prepared in the field and in the biological market, which seriously restricts the research on chicken caspase-1. Therefore, an antibody against chicken caspase-1 is needed

Method used

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  • Preparation method and application of polyclonal antibody against chicken caspase-1
  • Preparation method and application of polyclonal antibody against chicken caspase-1
  • Preparation method and application of polyclonal antibody against chicken caspase-1

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1: Construction of pET-32a(+)-caspase-1 prokaryotic expression plasmid

[0023] 1. Design and synthesis of primers

[0024] According to the amino acid sequence of chicken caspase-1 mRNA (accession number: AF031351.1) in GenBank, and referring to the multiple cloning site sequence of the prokaryotic expression vector pET-32a(+), a pair of primers (underlined as enzyme cleavage sites) were designed using Oligo7 software point):

[0025] Forward-Primer: 5'- GAATTC ATGAGCAGGGCAAGATCTTC-3'

[0026] Reverse-Primer: 5'- CTCGAG GAGGCCTGGGAAGAGAT-3'

[0027] Forward-Primer and Reverse-Primer are upstream and downstream primers respectively, and EcoR I (GAATTC) and Xho I (CTCGAG) restriction sites are added to the 5' end respectively.

[0028] 2. Amplification of chicken caspase-1 gene

[0029] Extract total RNA from chicken lung tissue, and reverse it into cDNA; use the cDNA as a template, and use the above-mentioned primers to perform PCR amplification to ob...

Embodiment 2

[0034] Example 2: Induced expression and identification of expression products

[0035] Transform the recombinant prokaryotic expression plasmid into BL21 host bacteria, pick the positive monoclonal bacteria and inoculate them into the LB (Amp+) medium for culture, shake the culture at 200rpm / min, when the OD600 is about 0.6, add IPTG at the final concentration of 1mM, 37℃ Induce the expression for 12 hours, collect the bacteria after centrifugation, add 4°C pre-cooled PBS to the pellet and resuspend, after ultrasonic cracking, separate the supernatant and the pellet, fully dissolve the pellet with a phosphate solution containing 8M urea, and remove it after centrifugation at 4°C Precipitate and keep the supernatant. Add 5×SDS loading buffer to the supernatant, mix well, and then bathe in boiling water for 10 minutes to obtain unpurified protein samples. SDS-PAGE and western blot methods are used to identify protein expression, such as image 3 shown.

Embodiment 3

[0036] Embodiment 3: Purification of recombinant protein

[0037] Prepare several pieces of 10% 1.5mm SDS-PAGE protein glue, run the above unpurified protein samples on SDS-PAGE gel, stain with 0.25mol / L pre-cooled KCL after running the gel, shake on the shaker for 5min, you can see When an obvious and specific white band appears near the size of the target band, after it is determined to be the target band, carefully cut it off with a scalpel and move it to a clean plate, wash it with pre-cooled PBS for 3 times, and then Put the gel strip into a clean mortar, grind it with liquid nitrogen, add 500uL PBS, transfer to a clean EP tube, stay at 4°C overnight, centrifuge at 4°C to take the supernatant, which is the purified target protein . SDS-PAGE and western blot methods can be used to identify the purification of the target protein, such as Figure 4 shown.

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Abstract

The invention provides a preparation method for a polyclonal antibody against chicken caspase-1. According to the preparation method, caspase-1 recombinant protein is used as an antigen for immunization of an animal so as to obtain the polyclonal antibody against chicken caspase-1. According to the invention, chicken caspase-1 is used as a research object; the polyclonal antibody against chicken caspase-1 prepared through gene amplification and construction, expression, purification and identification of a recombinant vector can specifically recognize target protein, so a foundation is laid for deep research on the toxicological significance of the family of chicken caspase-1 genes and the role played by the caspase-1 genes in virus-mediated innate immune response, and a novel target is provided for research on novel vaccines and development of medicines. Meanwhile, the prokaryotic expression vector constructed in the invention has high efficiency in expression of recombinant protein, and a separating and purifying method thereof is simple, low in cost and short in period.

Description

technical field [0001] The invention belongs to the technical field of polyclonal antibody preparation, and in particular relates to a preparation method and application of chicken caspase-1 polyclonal antibody. Background technique [0002] The Caspase family is a class of conserved aspartic acid-specific proteolytic enzymes that can use the cysteine ​​residues in the autocatalytic site to specifically cut the aspartic acid residues of the target protein. Plays an important role in regulating the inflammatory response. Caspase-1, also known as IL-1β-converting enzyme, is also the first protease discovered in the caspase family. It was first discovered in mammalian cells. It is not only closely related to the occurrence of inflammation, but also involved in the programmed death of some cells. [0003] Like most proteases, caspase-1 exists in the cytoplasm as an inactive protease, which consists of a long N-terminal prodomain, a large subunit (p20) and a small subunit (p10) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C07K16/06C12N15/57C12N15/70C12N9/64A61K39/395A61P31/04A61P31/12G01N33/573
CPCC07K16/065C07K16/40C07K2317/33C07K2317/35C12N9/6478G01N33/573G01N2333/96472
Inventor 任涛朱雯娴戴旭廖明
Owner SOUTH CHINA AGRI UNIV
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