PLA2R-THSD7A fusion protein as well as application and kit thereof

A technology of fusion protein and diagnostic kit, applied in the field of PLA2R-THSD7A fusion protein and its application and kit, to achieve good specificity, simple method, and improved sensitivity

Active Publication Date: 2017-02-22
SHENZHEN BLOT BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] PLA2R and THSD7A are two autoantigens of spontaneous membranous nephropathy, the detection rates of spontaneous MN are 70%-75% and 5%-10%, respectively, and there is a certain miss rate when used alone

Method used

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  • PLA2R-THSD7A fusion protein as well as application and kit thereof
  • PLA2R-THSD7A fusion protein as well as application and kit thereof
  • PLA2R-THSD7A fusion protein as well as application and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Preparation of embodiment 1 fusion protein

[0072] The preparation of this fusion protein comprises the following steps:

[0073] a. Trizol method to extract human tissue total RNA;

[0074] b. Prepare cDNA by using universal primers;

[0075] c. References, use computer and primer design software to design PCR primers and amplify the full-length fragments of PLA2R and THSD7A respectively;

[0076] d. Linking the two full-length sequences into PLA2R-THSD7A and THSD7A-PLA2R two recombinant gene coding sequences respectively through the additional restriction sites of the primers;

[0077] e. Transfer the above recombinant gene sequence into pET SUMO and pcDNA3.1 expression vectors, and perform prokaryotic and eukaryotic expression respectively;

[0078] f. SDS-PAGE electrophoresis and anti-tagged protein antibody WESTERN-BLOT detection of expression products;

[0079] g. Then use the positive antiserum preliminary test to identify;

[0080] h. Amplify, express and ...

Embodiment 2

[0264] Example 2 Application of PT protein in diagnosis of spontaneous MN

[0265] ELISA method for detecting spontaneous MN-related autoantibodies in subjects using the complex protein PT as an antigen:

[0266] Using PT complex protein to detect spontaneous MN-associated autoantibodies by ELISA method includes: the coating buffer is 0.05M carbonate buffer at pH 9.6 (where Na 2 CO 3 1.59g / L, NaHCO 3 2.93g / L), blocking solution (BSA 10g / L, sucrose 50g / L), washing solution is TBS containing Tween-20, pH7.6 (NaCl 8.7g / L, Tris 2.4g / L), sample diluent (Tris 1.21g / L, BSA 10g / L, Tween-20 0.05%), the chromogenic solution is TMB (3,3",5,5"-tetramethylbenzidine), and the stop solution is 2M sulfuric acid. Secondary antibody diluent (Tris 1.2083g / L, BSA 10g / L, Tween-20 0.05%), positive control and negative control were serum from IMN patients and healthy people from non-IMN patients, respectively.

[0267] The 96-well plate was a Nuc96-well plate, and BSA was purchased from Sigma R...

Embodiment 3

[0285] Embodiment 3 clinical detection application

[0286] The method in Example 2 was used to detect the sera of patients with spontaneous MN, patients with secondary MN, patients with other diseases and normal people, respectively. For 41 cases of clinical spontaneous MN patients, 10 cases of secondary MN patients, 10 cases of other disease patients, and 40 cases of normal human serum detection results are shown in Table 6, Figure 12 .

[0287] At the same time, PLA2R and THSD7A were used as controls.

[0288] Table 6 Comparison of fusion protein of the present invention with PLA2R and THSD7A on test results of subjects

[0289]

[0290] The results show that the detection rate of the fusion protein of the present invention is 78.1% for spontaneous MN patients, and the detection rate for secondary MN patients, patients with other diseases and normal human serum is 0%. The results show that the sensitivity of the fusion protein of the present invention to spontaneous ...

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Abstract

The invention relates to the medical field and particularly relates to a PLA2R-THSD7A fusion protein as well as application and kit thereof. The fusion protein comprises a first region and a second region, wherein the first region has at least 85% of sequence homology with a phospholipase A2 receptor (PLA2R), and the second region has at least 85% of sequence homology with 1-type thrombospondin 7A domain (THSD7A). The fusion protein prepared by virtue of the preparation method integrates epitopes of two antibodies including PLA2R and THSD7A and simultaneously has the immunoreactivities of the two antibodies, and the detection ratio of spontaneity MN-relevant autoantibodies is increased and can reach 78.1%.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a PLA2R-THSD7A fusion protein and its application and kit. Background technique [0002] Primary membranous glomerulonephritis (pMGN), also known as idiopathic membranous nephropathy (IMN), is a chronic glomerular inflammatory disease and one of the common types of nephropathy in nephrotic syndrome. The typical feature of the disease is proteinuria, with the increase of urinary protein content, there is a tendency to develop renal failure. Most pMGN patients present with nephrotic syndrome, some with edema, weight gain, and decreased urination. pMGN differs from secondary membranous glomerulonephritis (sMGN), which is a secondary or concurrent disease. Drug treatment, substance abuse, infectious diseases, other autoimmune diseases, and tumors may all lead to the occurrence of sMGN. At the same time, sMGN can improve with the treatment of the underlying disease. The "gold standard" for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00G01N33/68G01N33/564
CPCC07K14/47C07K14/705C07K2319/00G01N33/564G01N33/6854G01N33/6893G01N2800/347G01N2800/52
Inventor 熊祖应王洪涛林俊刘尧张永顶张大准张巧马伟民
Owner SHENZHEN BLOT BIOTECH
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