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a fusion antigen protein

A technology for fusing antigens and proteins, applied in the direction of hybrid peptides, oxidoreductases, enzymes, etc., can solve the problems of insufficient specificity, low efficiency, long time, at least 6 hours, or even overnight reactions

Active Publication Date: 2018-06-05
SHENZHEN NEW INDS BIOMEDICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional radioimmunoassay or ELISA method takes a long time to detect, at least 6 hours, or even overnight reaction to complete the test. It mainly relies on a series of operations such as pure manual sample addition, which has low efficiency and insufficient specificity.

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Gene fusion and expression

[0031] (1) Main reagents and equipment

[0032] PCR kits (including Taq enzyme, buffer, dNTP, T4 ligase) and DNA markers were purchased from full gold; protein markers were purchased from Beyond Biotech; BamH I, Xho I, EcoR I, Nco I, HindⅢ and other restriction internal Dicer was 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 reagent Kits 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 provided by Shenzhen New Industry Biomedical Engineering Co., Ltd. Inc. provided.

[0033]The ABI Veriti PCR instrument was purchased from ABI, the horizontal electrophoresis instrument RDY-SP1Z w...

Embodiment 2

[0054] Determination of the sensitivity and specificity of the antigenic protein expressed by the above insect cells to its corresponding antibody

[0055] 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 ...

Embodiment 3

[0088] Comparison of prokaryotic and eukaryotic expressed proteins for detection of type 1 diabetes

[0089] hIA-2 and its truncated forms were expressed in Escherichia coli, and the optimized segment of hIA-2 screened above was 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.

[0090] The sensitivity and specificity of prokaryotic expressed hIA-2 to hIA-2 antibody were determined.

[0091] The prokaryotic expression segments of hGAD65 and its truncated forms are numbered PA1, PB1, PG1, and PH1 in sequence, corresponding to A1, B1, G1, and H1 expressed in eukaryotes.

[0092] The truncated prokaryotic expression segment of hIA-2- has a sign bit PN2, which corresponds to N2 in eukaryotic expression.

[0093] The detection platform and scheme detai...

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Abstract

The invention relates to fusion antigen protein. The fusion antigen protein is protein obtained by fusion expression of a glutamate dehydrogenase 65 gene and a tyrosine phosphatase gene or a truncated tyrosine phosphatase gene. Besides, the invention also provides a kit which contains the fusion antigen protein and is used for detecting diabetes.

Description

technical field [0001] The present invention relates to a fusion antigen protein, in particular to a fusion protein expressed by the fusion of glutamate dehydrogenase 65 or truncated glutamate dehydrogenase 65 and tyrosine phosphatase or truncated tyrosine phosphatase technology field. Background technique [0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia caused by absolute or relative insufficiency of insulin secretion, which is affected by various genetic and environmental factors. According to the 1997 American Diabetes Association and the 1999 World Health Organization (WHO) classification and diagnostic criteria for diabetes, diabetes can be divided into type I, type II, special type diabetes and gestational diabetes. [0003] During the immune destruction of type 1 diabetes, various autoantibodies against islet cell self-antigens are produced in patients. Since the 1970s, a variety of insulin autoantibodies have been discovered, such as...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N9/06G01N33/68G01N33/564C12N15/62
CPCC07K19/00C12N9/0016C12N9/16C12Y104/01002C12Y301/03048
Inventor 何海华戚永跃任印玲吴森仁袁锦云李武
Owner SHENZHEN NEW INDS BIOMEDICAL ENG