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Antigen and antibody prepared by taking PADI4 as tumor marker, and applications
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A tumor marker and antibody technology, used in applications, biological testing, genetic engineering, etc., can solve the problems of obstacles, low yield, and only 80% purity, and achieve the effect of good test repeatability and high specificity
Active Publication Date: 2020-10-02
SHANDONG XINCHUANG BIOLOGICAL TECH CO LTD
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However, at present, there is no kit for detecting PADI4 in the patient's serum. The main technical obstacle is that the antigen is human PADI4 protein, which is expressed recombinantly using the insectbaculovirus expressionsystem, and the N-terminal is added with a Twin Strep tag for purification. Cells, need to obtain soluble protein as antigen
After expression and purification tests, PADI4 is expressed in sf9 cells. The optimal expression condition is that 30ul virusplasmid (M.O.I.~1) infects Sf9 cells for 3 days. The technical obstacle lies in the low yield after expression and purification, and the yield is only 0.34mg / L. The purity is only 80%, and the amount of antigen required for the preparation of monoclonal antibodies is at least 3.5 mg, so a larger volume is required to obtain sufficient antigen; at the same time, antibody preparation needs to go through immunization, fusion, subcloning, screening and strain determination, and antibody production stages. Due to the high homology between PADI4 protein and PADI2 protein, the need to remove cross-reaction during screening is also a major problem hindering the development of this technology
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[0069] The protein PADI4 has a total of 663AAs, a molecular weight of 74.47KDa, no signalpeptide, no transmembrane helix, and a region with strong hydrophobicity at 265-271AAs. Comprehensive homology comparison results require soluble proteins as antigens, and immunize mice with 1-260AAs (remove the hydrophobic region and have low homology) of the soluble expression protein PADI4 in the E. coli system to prepare monoclonal antibodies. The carrier uses pET28b, C-terminal The 6His tag on the linker, the enzymecutting site is NcoI / XhoI.
[0070] b) Plasmid extraction (kit name: Endo-free plasmid Mini Kit I (50), OMEGA bio-tek):
[0071] 1) Take 4 mL of the overnight cultured bacterial solution, centrifuge at 12000 rpm for 1 min, and collect the bacterial cells;
[0072] 2) Add 250 μL Solution I to resuspend the cells;
[0073] 3) Add ...
Embodiment 2
[0141] The steps for using the kit described in Example 1 are as follows:
[0142] 1) The antibody immobilized plate was restored to room temperature equilibrium.
[0143] 2) Add the standard sample diluent (PADI4), and use the diluent to dilute a total of 7 gradients from 10ng / ml to 0.015625ng / ml, 100μl per well, the last well is the blank control, add 100μl of the diluent, and measure 100μl of the sample, 37 ℃, 1.5h.
[0144] 3) Wash the plate, wash the plate 5 times with 300 μl of PBST, and pat the liquid dry.
[0145] 4) Add 197-C11-A5-Antibody-Bio, dilute to 2ug / ml with diluent for use, 100 μl per well, 37°C, 1h.
[0146] 5) Wash the plate, wash the plate 5 times with 300 μl of PBST, and pat the liquid dry.
[0147] 6) Add Streptavidin-HRP, dilute 1:2000 with diluent for use, 100 μl per well, 37°C, 30min.
[0148] 7) Wash the plate, wash the plate 5 times with 300ul of PBST, and pat the liquid dry.
[0149] 8) TMB color development, 100 μl per well, 37° C. for 10 min...
Embodiment 3
[0153] Embodiment 3 uses the clinical sample detection of PADI4 detection kit of the present invention:
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technical field [0001] The invention relates to an antigen, antibody and application prepared based on PADI4 as a tumor marker, and belongs to the technical field of molecular biological detection. Background technique [0002] Peptidylarginine deiminase (Peptidylarginine deiminase, PAD or PADI) is an enzyme in human tissues. Currently, five PAD enzymes (ie, PAD1, 2, 3, 4 and 6) have been discovered. These enzymes are encoded by a cluster of genes located on the lp36 region of the human chromosome and have variable tissue distribution. It can perform post-translational modification on some other tissue proteins in the presence of calcium ions. This enzyme can catalyze the amino group of arginine (arginine) in the polypeptide chain into a carbonyl group, thereby converting arginine into citrulline (citrulline). Citrulline is an unnatural amino acid. This process of converting arginine to citrulline in polypeptides catalyzed by PAD is called citrullination. After citrullin...
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