A kind of antigen protein specifically combined with glutamate dehydrogenase 65 antibody
A glutamate dehydrogenase, specific binding technology, applied in the field of truncated glutamate decarboxylase 65, antigen-antibody protein, can solve long, at least 6 hours, or even overnight reaction, lack of specificity, efficiency low level problem
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Embodiment 1
[0030] Eukaryotic expression of truncated glutamic acid decarboxylase (GAD65) gene
[0031] (1) Main reagents and equipment
[0032] PCR kit (including Taq enzyme, buffer, dNTP, T4 ligase) and DNA markers were purchased from full gold; protein markers were purchased from Beyond Biotech; restriction endonucleases such as BamH I, XhoI, EcoR I, Nco I, HindⅢ Enzymes were purchased from Takara (Dalian Bao Biology); calf intestinal alkaline phosphatase (CIAP) kit (including components: calf intestinal alkaline phosphatase, alkaline phosphatase buffer) was purchased from Takara; gel recovery kit and plasmid extraction kits were purchased from OMEGA; kanamycin and ampicillin were purchased from BBI; IPTG was purchased from BBI; PCR primers and recombinant plasmid sequencing were completed by BGI or Invitrogen; other reagents were purchased from Shenzhen New Industry Biomedical Engineering Co., Ltd. Limited offer.
[0033] The ABI Veriti PCR instrument was purchased from ABI, the ho...
Embodiment 2
[0046] Determination of the sensitivity and specificity of the 17 antigens expressed by the above insect cells to the hGAD65 antibody
[0047] The principle of the chemiluminescent immune sandwich method: the serum sample to be tested, the buffer solution and the antigen-coated magnetic microspheres are added to the reaction cup for reaction, and at 37°C, the antibody in the serum sample to be tested and the antigen coated on the magnetic microspheres are immune In the case of a magnetic field, the nano-magnetic beads will be magnetized rapidly, and the complex will be washed to obtain the complex of the antigen and the antibody to be tested. Other components will be washed away, and then the labeled protein A-NHS-ABEI will be added. Protein A can Quickly and specifically bind to the C-terminus of the antibody to be tested to form a "sandwich" immune complex. When the detected antibody content in the serum sample is more, the relative light intensity RLU detected by the instru...
Embodiment 3
[0068] Comparison of prokaryotic and eukaryotic expressed proteins for detection of type 1 diabetes
[0069] hGAD65 and its truncated forms were expressed in Escherichia coli, and the optimized segments screened by the above hGAD65 were respectively connected to the pGEX4T-1 expression vector, and the expression strain of Escherichia coli was BL21(DE3). After the expression product is purified by GST tag, the GST tag is cut off with Thrombin, and the untagged target protein is obtained by affinity chromatography.
[0070] The sensitivity and specificity of prokaryotic expressed hGAD65 to hGAD65 antibody were determined.
[0071] The prokaryotic expression segments of hGAD65 and its truncated forms are numbered PA1, PB1, PG1, and PH1 in sequence, which correspond to A1, B1, G1, and H1 of eukaryotic expression, respectively.
[0072] The detection platform and scheme details are the same as in Example 1, and the results are shown in Table 4.
[0073] Table 4
[0074]
[00...
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