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NMDAR recombinant protein related to autoimmune encephalitis as well as coding sequence, preparation method and application of NMDAR recombinant protein

An autoimmune and recombinant protein technology, applied in the field of NMDAR recombinant protein, can solve the problems of difficulty in automation, high cost, and limited detection range

Pending Publication Date: 2021-09-10
四川携光生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the most common methods for laboratory testing are western blotting and indirect immunofluorescence (IFA). The detection kits are produced by European and American manufacturers, and the cost is high. In clinical practice, the detection range is limited and it is difficult to realize automation. In addition, the above-mentioned research methods belong to the category of qualitative detection, which cannot truly realize the real-time and large-scale detection of autoantibody levels in the early onset, treatment and recovery stages.

Method used

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  • NMDAR recombinant protein related to autoimmune encephalitis as well as coding sequence, preparation method and application of NMDAR recombinant protein
  • NMDAR recombinant protein related to autoimmune encephalitis as well as coding sequence, preparation method and application of NMDAR recombinant protein
  • NMDAR recombinant protein related to autoimmune encephalitis as well as coding sequence, preparation method and application of NMDAR recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] This embodiment discloses the preparation method of the NMDAR recombinant protein of the present invention, specifically:

[0034] 1. Optimizing the nucleotide sequence encoding the recombinant protein

[0035] In order to increase the expression of recombinant protein in Escherichia coli, under the premise that the amino acid sequence of the recombinant protein remains unchanged, the nucleotide sequence encoding the recombinant protein is converted into the corresponding nucleotide sequence according to the preferred codons of Escherichia coli, and the optimized The sequence shown in SEQ ID NO.9. The CAI value of the nucleotide sequence before optimization was 0.84, and the CAI value of the optimized nucleotide sequence was 0.94. The results are attached Figure 5 shown.

[0036] 2. Use the gene sequence shown in SEQ ID NO.9 after codon preference optimization, connect the signal peptide, His tag and KozAk sequence, and synthesize the target gene sequence shown in S...

Embodiment 2

[0066] This example discloses the use of the NMDAR recombinant protein prepared in Example 1 to prepare a quantitative detection kit for anti-NMDAR antibodies in human blood (magnetic particle chemiluminescence method) and to detect the immunoreactivity of the antigen.

[0067] Coupling NMDAR recombinant protein with magnetic particles, the procedure is to take 2mL of Tosyl magnetic bead stock solution, wash with coating buffer three times, then add coating buffer 10mL, NMDAR recombinant protein 500μL, 3M ammonium sulfate 5.25mL, at 37℃ Reaction 16h. After the reaction, the magnetic beads were taken out, the supernatant was removed, and 15.75 mL of blocking solution was added to react at 37°C for 9 hours. Finally, wash twice with blocking solution, wash once with magnetic bead protection solution, and then dilute to volume with magnetic bead protection solution.

[0068] Use NMDAR antibody to dilute the small sample according to the ratio of 1 / 100, 1 / 400, 1 / 1600, and 1 / 32000,...

Embodiment 3

[0074] According to the method of Example 2, the NMDAR recombinant protein was coupled with magnetic particles to prepare a quantitative detection kit for anti-NMDAR antibodies in human blood (magnetic particle chemiluminescence method), and the performance of the reagent was evaluated to verify the minimum detection limit of the detection reagent. , linearity and precision.

[0075] 1. Minimum detection limit verification

[0076] Using the zero concentration calibrator and the adjacent concentration calibrator, the minimum detection limit was back-calculated according to the calculation software, and the results are shown in the following table:

[0077] Table 2

[0078]

[0079] The measurement results showed that the minimum detection limit was 0.0295RU / mL, which was in line with the industry regulation not higher than 0.50RU / mL.

[0080] 2. Linearity Verification

[0081] Use clinical high-value samples to gradiently dilute, verify the linear correlation, and calcul...

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Abstract

The invention discloses an NMDAR recombinant protein related to autoimmune encephalitis as well as a coding sequence, a preparation method and application of the NMDAR recombinant protein, and belongs to the technical field of biology. The coding sequence of the NMDAR recombinant protein related to the autoimmune encephalitis is as shown in SEQ ID NO. 1; and the amino acid sequence of the recombinant protein is as shown in SEQ ID NO. 2. The preparation method of the recombinant protein comprises the following steps of connecting a coding sequence as shown in SEQ ID NO.1 to a recombinant vector to construct a recombinant protein expression vector; transforming into competent cells of escherichia coli, and culturing to obtain a recombinant protein expression strain; extracting plasmids, and transfecting the plasmids into CHO-S cells for culture; and collecting a cell supernatant, and purifying to obtain the recombinant protein. The invention also provides the application of the recombinant protein in preparation of an anti-NMDAR autoantibody detection material, and an anti-NMDAR autoantibody detection kit. The NMDAR recombinant protein can be recognized by an anti-NMDAR autoantibody in the blood of a patient through heterologous recombinant expression of NR1-ATD, and is expressed in vitro in quantity, so that the production cost is reduced, and quantitative detection can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to NMDAR recombinant protein related to autoimmune encephalitis, its coding sequence, preparation method and application. Background technique [0002] Autoimmune encephalitis (AE) is an inflammatory disease of the nervous system mediated by autoimmune mechanisms, which can affect the limbic system, brainstem and cerebellum, with acute or subacute onset. Typical clinical manifestations include memory loss, seizures, mental abnormalities, and cognitive dysfunction. The initial symptoms of autoimmune encephalitis are not obvious, but family members often see a doctor in the psychiatry department because of their strange behavior, which misses the best early diagnosis. In addition, there are many similarities and indistinguishable differences between the disease and infectious encephalitis in terms of etiology, clinical manifestations, and diagnostic criteria. The biggest diffe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/85C07K14/705G01N33/68G01N33/543
CPCC07K14/70571C12N15/85G01N33/6893G01N33/6872G01N33/54326C12N2800/22G01N2333/70571G01N2800/28
Inventor 许敏黄敬双秦枫潘敬梅段继龙王健潘进海张伟曹长春赵婷
Owner 四川携光生物技术有限公司
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