Application of anti-HSPA4 (Heat Shock 70 KDa Protein 4) autoantibody as breast cancer diagnosis or prognosis evaluation marker
A technology of autoantibodies and prognosis assessment, applied in the field of biotechnology and medical diagnosis
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Embodiment 1
[0101] Example 1: Using protein profiling technology to screen the specific binding partners of serum autoantibodies of breast cancer tumor-bearing mice
[0102] The mouse breast cancer cell line 4T1 was purchased from ATCC (DMEM medium + 10% fetal bovine serum) in the United States. Balb / c mice were purchased from Shanghai Xipuer-Bikay Experimental Animal Co., Ltd. and raised in an SPF-grade environment.
[0103] Establishment of mouse breast cancer model: Inject 5×10 5 For each breast cancer cell line 4T1 cell, the enzyme-linked immunosorbent assay (ELISA) method was used to detect the antibody level in the mouse serum, and the lymph node metastasis and tumor growth of the mouse were observed.
[0104] Purification of mouse serum antibodies: Antibody purification kits (Melon Gel IgG Spin Purification Kit, Pierce, USA) were used to purify antibodies from mouse serum. The specific operation is carried out according to the manual.
[0105] Protein spectrometry detection of s...
Embodiment 2
[0109] Example 2: Expression and preparation of human specific binding partner (HSPA4)
[0110] The screened specific binding partner of the autoantibody (HSPA4) was synthesized by genetic engineering methods known to those skilled in the art. Because tumor antigens have different modifications, the expression system of HSPA4 is to select the commonly used human breast cancer cell line MBA-MD-231 (purchased from ATCC, USA) to obtain antigenic proteins with immunogenicity.
[0111] 1. Cloning plasmid construction: using the method based on PAS (PCR-based Accurate Synthesis), design a full-length splicing primer (the sequence of human HSPA4 is shown in Gene ID: 3308), and connect it into the expression vector pcDNA3.1; the obtained recombinant plasmid Transfer to cloned strains, and select positive clones on the resistance plate. Small plasmid extraction was performed on the positive clones, which were verified by double enzyme digestion and sequencing; the positive clone plasm...
Embodiment 3
[0116] Example 3: Binding coating of specific binding partners to solid phase substrates
[0117] Coating of microplates: Dilute specific binding partners with coating buffer (0.05mol / L carbonic acid buffer, pH 9.6), distribute to 96-well microtiter plates, and store overnight at 4°C. Remove the coating solution and wash twice with PBS. Add blocking solution (5% skimmed milk powder-PBS), room temperature for 2-4 hours. Shake off the blocking solution and wash with PBS.
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