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PR3, MPO and GBM fusion protein as well as construction method and application thereof

A fusion protein and construction method technology, applied in the field of MPO, GBM fusion protein and its construction, PR3, can solve the problems of poor detection sensitivity and specificity, missed detection of patients' diseases, and inability to achieve simultaneous detection, etc., to reduce costs and time, reduce the cost of repeated testing, and save costs

Pending Publication Date: 2021-01-22
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

[0006] At present, the antigens of the three ANCA items are all single antigens, which cannot be detected simultaneously during the development of in vitro diagnostic reagents. The detection sensitivity and specificity are poor, resulting in high reagent costs, cumbersome detection processes, missed and wrong detection of patient diseases, and competitiveness. Lack of insufficiency

Method used

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  • PR3, MPO and GBM fusion protein as well as construction method and application thereof
  • PR3, MPO and GBM fusion protein as well as construction method and application thereof
  • PR3, MPO and GBM fusion protein as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Synthesis of the whole gene sequence of the fusion gene (target gene):

[0039] S1. Connect the nucleotide sequences of PR3, MPO, and GBM protein segments in sequence. According to the sequence information published by GenBank, remove the stop codon and start codon between the two gene segments, and add 5 amino acid residues. The flexible peptide gene sequence (Linker sequence) of base (GGGGS); Bee venom signal peptide sequence is added at N end, and 6*His tag sequence is added at C end; Restriction endonuclease site BamH I is added at sequence upstream, in its Downstream, an EcoR I restriction site is added to synthesize the whole gene. The order of arrangement of the sequences during the whole gene synthesis is: eukaryotic KOZAK-signal peptide-PR3-Linker-MPO-Linker-GBM-6*His, the nucleotide sequence of the target gene (fusion gene) such as SEQ ID No. 4.

[0040] like figure 1 As shown, the multiple cloning site is an artificially synthesized DNA fragment contained ...

Embodiment 2

[0042] Construction of recombinant plasmids containing fusion genes:

[0043] 2.1 The complete gene sequence obtained in Example 1 and the plasmid vector pFastBac 1 were double digested with BamH I and EcoR I, the digested product was recovered by the kit, and the digested product was ligated by T4 ligase.

[0044] 2.2 Transform the ligation product (recombinant plasmid) into competent Escherichia coli DH5α, the volume does not exceed 5% of the competent cells, gently swirl several times to mix the contents, and put the tube in a 42°C water bath for 60 seconds For heat shock, quickly transfer the tube to an ice bath for 120 seconds; add 400 μL LB medium to each tube, shake slowly at 37°C for 60 minutes, centrifuge at low speed for 2 minutes, remove the supernatant, leave about 100 μL of medium in the centrifuge tube, and resuspend the bacteria , Spread the bacteria solution evenly on the agar plate with a glass spreader.

[0045] 2.3 Place the plate upside down in a constant ...

Embodiment 3

[0047] Expression of fusion protein

[0048] 3.1 Transform the recombinant plasmid obtained in Example 2 into Escherichia coli DH10 Bac competent cells, the volume of which should not exceed 5% of the competent cells, gently swirl several times to mix the contents, and place in ice bath for 30 minutes.

[0049]3.2 Put the tubes in a water bath at 42°C, heat shock for 90 seconds, and quickly transfer the tubes to an ice bath for 120 seconds; add 800 μL LB medium to each tube, shake slowly for 4 hours at 37°C, to recover the bacteria and express the antibiotics encoded by the plasmids Marker gene; spread 30 μL of the bacterial solution on the resistant Kan+Gent+Tet agar plate evenly with a glass spreader; place the plate upside down in a 37°C constant temperature incubator, and blue-white colonies will appear after 30-48 hours.

[0050] 3.3 Pick a single positive white spot colony and inoculate 5mL of resistant LB, shake slowly for 12-16 hours, take the bacterial liquid for PCR ...

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Abstract

The invention discloses PR3, MPO and GBM fusion protein as well as a construction method and application thereof. The construction method comprises the following steps of S1, recombining a whole-genesynthesis sequence: sequentially connecting nucleotide sequences of PR3, MPO and GBM protein sections to obtain a target gene; S2, constructing recombinant plasmids: constructing the recombinant whole-gene synthesis sequence obtained in the step S1 to an expression vector so as to obtain the recombinant plasmids; S3, performing primary transfection: transfecting host cells with the recombinant plasmids in the step S2 to obtain recombinant bacmids; S4, performing secondary transfection: transfecting the host cells with the recombinant bacmids in the step S3 again to obtain first-generation baculoviruses; S5, performing expression: expressing the first-generation baculoviruses in the step S4; and S6, obtaining the fusion protein: screening and purifying a cell culture obtained by expressionin the step S5 to obtain the fusion protein. The fusion protein constructed by the method provided by the invention can improve the sensitivity and specificity of a detection kit and reduce missing detection and wrong detection.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to PR3, MPO, GBM fusion proteins and their construction methods and applications. Background technique [0002] ANCA (anti-neutrophil cytoplasmic antibody) is an antibody that uses neutrophil and monocyte cytoplasmic components as target antigens, which can cause primary systemic vasculitis, also known as ANCA-associated vasculitis. The disease mainly affects small and medium blood vessels. Wegener's granulomatosis, acute progressive glomerulonephritis, polyarteritis, ulcerative colitis and primary sclerosing cholangitis and other primary systemic vasculitis are closely related to ANCA. The detection can also greatly improve the early diagnosis rate of renal vasculitis. The detection of anti-PR3 (protease 3) antibody, anti-MPO (myeloperoxidase) antibody and anti-GBM (glomerular basement membrane) antibody has become a highly sensitive and specific serological detect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/866C12N15/70C12N15/85
CPCC12N9/50C12N9/0065C12Y111/02002C07K14/47C12N15/86C12N15/70C12N15/85C07K2319/00
Inventor 秦枫李娜张朋飞任伟超张小飞颜松周玥黄敬双张伟鲜静吴斌
Owner 四川携光生物技术有限公司
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