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Preparation of specific antibodies aiming at PCDH10C terminal protein segment molecule

A protein fragment, specific technology, applied in the field of specific antibody preparation, can solve the problem of no specific antibody found

Inactive Publication Date: 2008-09-03
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when using primers targeting the C-terminus for PCR amplification, it was found that there was a significant transcriptional down-regulation in colorectal cancer stem cell-like cells, indicating that PCDH10 had splicing changes in colorectal cancer cells, and the PCDH10C-terminal protein molecular fragment may be linked to a Unique cell information transduction pathway, but no specific antibody against PCDH10C-terminal protein molecular fragment has been found

Method used

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Examples

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preparation example Construction

[0019] The preparation method for the specific antibody of PCDH10C terminal protein fragment molecule comprises the following steps:

[0020] 1) Using polymerase chain reaction technology to directly clone the base sequence of PCDH10C 3' terminal from in vitro cultured cell lines into prokaryotic cells, and construct the prokaryotic cell PCDH10C / pET15b expression vector of PCDH10C terminal fusion protein;

[0021] 2) Transform the PCDH10C / pET15b expression vector into BL21DE3pLysS Escherichia coli, establish the prokaryotic cell expression system of the PCDH10C-terminal fusion protein, purify the PCDH10C-terminal fusion protein with His metal chelating resin, and use Thrombin enzyme to excise the His-tag at the N-terminal of the PCDH10C-terminal fusion protein label to obtain the PCDH10C-terminal fusion protein;

[0022] 3) Use the tag-cleaved PCDH10C-terminal fusion protein as an antigen to immunize white rabbits to produce anti-PCDH10C-terminal antibodies, collect antiserum,...

Embodiment 1

[0032] Example 1: Construction of a prokaryotic cell expression vector for PCDH10C-terminal fusion protein

[0033] 1 x 10 of the expressed PCDH10 5 Centrifuge the human leukemia K562 cells, add 0.5ml Trizol Total RNA Extraction Reagent and mix thoroughly, extract the total RNA, and then use reverse transcriptase to reverse transcribe the RNA to cDNA; then design the upstream primers containing the Nde I endonuclease site CATATG and the downstream primers containing The BamH I endonuclease site GGATCC, the PCDH10C-terminal DNA sequence was directly cloned into the pET15b Nde I / BamH I site by polymerase chain reaction technology, and after Nde I and BamH I endonuclease digestion and identification, further use DNA sequencing verification, qualified is the PCDH10C / pET15b expression vector.

Embodiment 2

[0034] Example 2: Construction of a prokaryotic cell expression vector for PCDH10C-terminal fusion protein

[0035] 110 that will express PCDH10 7 Centrifuge the human leukemia K562 cells, add 1.5ml Trizol and mix thoroughly, extract the total RNA, and then use reverse transcriptase to reverse transcribe the RNA to cDNA; then design the upstream primer containing the Nde I endonuclease site CATATG and the downstream primer containing the BamH I endonuclease Enzyme site GGATCC, the PCDH10C-terminal DNA sequence was directly cloned into the pET15bNde I / BamH I site by PCR method, and after passing the Nde I and BamH I endonuclease digestion and identification, it was further verified by DNA sequencing, and the qualified one was ΔPCDH10C / pET15b expression vector.

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Abstract

The invention discloses a process for preparing specific antibody for PCDH10 C end albumen fragment molecule and. The method comprises, using polymerase chain reaction, directly cloning PCDH10C 3'end base sequence to the prokaryotic cell expression system from vitro cultured cell strain to purify the PCDH10 C end amalgamation albumen; using purified PCDH10 C end amalgamation albumen as antigen immunity white rabbit, producing anti PCDH10 C end antibody; collecting antiserum to obtain anti PCDH10 C end albumen specific antibody of rabbit with high-purity and high activity after affinity purification. Specific antibody for PCDH10 C end albumen fragment molecule prepared in the invention is one of the main instrumentalities for researching PCD10 mediated signal pathway transduction. The epitope of specific antibody for PCDH10 C end albumen fragment molecule is a potential target of molecular targeted therapy. The specific antibody for PCDH10 C end albumen fragment molecule can be used for clinically estimating the change of tumor gene expression and metastatic potential.

Description

technical field [0001] The invention relates to a method for preparing a specific antibody against PCDH10C-terminal protein fragment molecules. Background technique [0002] PCDH10 belongs to Ca 2+ A member of the Dependency-cadherin superfamily with six extracellular domains in tandem repeats and a unique intracellular domain. We found that pcdh10 was screened by viral library technology in gene expression profiling of primary cancer stem cells - / - Cells are easy to swim out and pass through the semi-permeable membrane of "Transwell", while pcdh10 + / + cell migration is weak. Through bioinformatics analysis, the full-length cDNA sequence of pcdh10 was further cloned and a eukaryotic cell expression vector expressing pcdh10 was constructed, and K562 cells and SW480 colorectal cancer cells were transiently transfected by liposomes, and it was found that ectopic expression of PCDH10 inhibited these cells In vitro proliferation and increased cell apoptosis. It was further f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/12C12N15/62C12N15/70C07K19/00
Inventor 朱永良
Owner ZHEJIANG UNIV
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