CIAPIN1 monoclone antibody for multidrug resistant differential diagnosis of stomach cancer and method for preparing the same
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A monoclonal antibody, mouse anti-human technology, applied in the field of genetic engineering technology and medical detection reagents, can solve the problems of high expression and complex MDR of gastric cancer
Inactive Publication Date: 2009-07-29
FOURTH MILITARY MEDICAL UNIVERSITY
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This result shows that although the occurrence of MDR in gastric cancer is a common phenomenon, the expression of classical MDR-mediated molecules is not as high as expected, suggesting that the generation of MDR in gastric cancer has a complex mechanism, which may involve some non-classical MDR-related molecules , we need to further search and verify
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Embodiment 1
[0070] Example 1: Preparation of mouse anti-human CIAPIN1 monoclonal antibody
[0071] 1. Cloning of CIAPIN1 gene from human gastric cancer multidrug resistant cell line SGC-7901 / VCR by RT-PCR
[0075] The forward primer introduces an EcoR I restriction site, and the reverse primer introduces a Sal I restriction site (underlined part) to facilitate subcloning operations. Primers were synthesized by Shanghai Sangong.
[0076] 1.2 Reverse transcription to synthesize single-stranded DNA (Superscript reverse transcription kit from Invitrogen):
[0077] The reaction was carried out according to the following conditions: human gastric cancer multidrug-resistant cell SGC-7901 / VCR total RNA (operated and prepared according to Invitrogen TrizolRNA purification kit) (1 μg / μl) 2 μl, followed by hexanucleotide primer (10 μmol / L) 7...
Embodiment 2
[0132] Example 2, CIAPIN1 Monoclonal Antibody Differential Diagnosis of Gastric Cancer Multidrug Resistance
[0133] 1. Immunohistochemical section staining:
[0134] Formalin-fixed, paraffin-embedded human gastric resection specimens→Xylene dewaxing and gradient alcohol dehydration→PBS wash twice, 5min / time→0.3%H 2 o 2 In methanol at room temperature for 20 minutes, inactivate endogenous peroxidase → wash 3 times with PBS, 5 minutes each time → pre-balance the wet box at 37°C for 30 minutes, add 10% normal goat serum blocking solution in the wet box at 37°C for 20 minutes → Discard the blocking solution and do not wash, add the purified CIAPIN1 monoclonal antibody at a ratio of 1:200, overnight at 4°C in a wet box → wash 3 times with PBS, 5 min each time → add biotin-labeled goat anti-mouse IgG, and place in a wet box at 37°C Medium 30min→PBS wash 3 times, 5min / time→drop SP working solution, incubate in a wet box at 37°C for 30min→PBS wash 3 times, 5min / time→DAB color devel...
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Abstract
The invention relates to a mouse anti-human CIAPIN1 monoclonal antibody for confirming multi-drug resistant gastric cancer, and a preparation method thereof. The invention is characterized in that the monoclonal antibody is obtained by adopting the hybrid tumor technology; the heavy chain is 1gG1; the light chain is k; the molecular weight is 39KD; and the affinity is 3.6x10. The preparation method comprises the following steps of: (1) synthesizing primers; (2) synthesizing single-chain DNA by carrying out reverse transcription on SGC-7901 / VCR general RNA of a human gastric-cancer multi-drug resistant cell; (3) expanding human CIAPIN1 gene cDNA by using the primers F and R and by adopting a PCR method; (4) constructing CIAPIN1 gene induction expression plasmids pET28-CIAPIN1 and pGEX-CIAPIN1; (5) constructing engineering bacteria B21-CIAPIN1 and DE3-CIAPIN1; (6) using the engineering bacteria B21-CIAPIN1 and DE3-CIAPIN1 to carry out prokaryotic expression and purification on CIAPIN1 protein; (7) using CIAPIN1 protein to immunize a 4-week-old Balb / C mouse so that mouse anti-human CIAPIN1 antibody is generated; and (8) using the serum of the immunized mouse to prepare the mouse anti-human CIAPIN1 monoclonal antibody. The invention can better identify multi-drug resistant gastric cancer and non-multi-drug resistant gastric cancer of clinical gastric cancer patients, and predict clinical prognosis of the gastric cancer patients.
Description
technical field [0001] The invention relates to the technical field of genetic engineering and medical detection reagents, and is a new marker for confirming multidrug-resistant gastric cancer in clinical pathological differential diagnosis of gastric cancer—rat anti-human CIAPIN1 monoclonal antibody and its preparation method. Background technique [0002] Gastric cancer is one of the most common malignant tumors in the world. Although its incidence has been on a downward trend, the incidence of gastric cancer remains high in Asian countries such as China, North Korea, and Japan, as well as in some European and South American countries. Chemotherapy is one of the main treatment methods for gastric cancer. Although people use a variety of chemotherapeutic drugs, the treatment of gastric cancer has not been fundamentally improved. The main reason is that gastric cancer can develop resistance to chemotherapeutic drugs. Drug resistance, especially multidrug resistance (MDR) i...
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