[Beta]2-GP1 modified protein as well as construction method and application thereof

A technology of 2-GP1 and its construction method, which is applied in the field of β2-GP1 modified protein and its construction, can solve the problems of large differences in sensitivity and specificity, missed detection of patients' diseases, and cumbersome detection process, etc., to achieve improved detection rate, improve detection sensitivity and specificity, and reduce the effect of missed detection and false detection

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

[0004] However, at present, in the three laboratory standard testing methods, there are methodological defects, namely, high cost, cumbersome testing process, large differences in sensitivity and specificity, and single antibody detection, which is prone to patient disease leakage. check

Method used

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  • [Beta]2-GP1 modified protein as well as construction method and application thereof
  • [Beta]2-GP1 modified protein as well as construction method and application thereof
  • [Beta]2-GP1 modified protein as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Synthesis of the whole gene sequence of the modified gene (target gene):

[0046] S1. Connect the nucleotide sequences of domain 1, domain 4, and domain 5 protein segments sequentially to obtain the target gene. According to the sequence information published by NCBI, remove the stop codon and start between the two gene segments codon, and add a flexible peptide gene sequence (Linker sequence) of 5 amino acid residues (GGGGS); connect an Avi-tag on the domain 5, and add a 6*His tag sequence at the C-terminal; add restrictions upstream of the sequence The endonuclease site BamH I, and an EcoRI restriction site is added downstream of it, and according to the codon preference of mammals, the sequence of the sequences during gene synthesis is eukaryotic KOZAK-signal peptide-D1- Linker-D4-Linker-D5-Avi-6*His, the nucleotide sequence of the target gene (modified gene) is shown in SEQ ID No.2.

Embodiment 2

[0048] Construction of recombinant plasmids containing modified genes:

[0049] 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.

[0050] 2.2 Transform the ligation product into competent Escherichia coli DH5α, and the volume should not exceed 10% of the competent cells. Gently swirl several times to mix the contents, and place the tube in an ice bath for 30 minutes; Transfer the tube to an ice bath for 120 seconds to cool the cells, add 400 μL LB medium to each tube, and shake slowly at 37°C for 60 minutes to revive the bacteria and express the antibiotic resistance marker gene encoded by the plasmid, centrifuge at low speed for 2 minutes, and remove the supernatant. Leave about 100 μL of medium in the centrifuge tube, resuspend the bacteria, and spread the bacteria evenly on the agar plate with a ...

Embodiment 3

[0053] Expression of the modified protein

[0054] 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.

[0055] 3.2 Put the tube in a water bath at 42°C, heat shock for 90 seconds at regular intervals, and quickly transfer the tube to an ice bath for 120 seconds to cool the cells; add 800 μL LB medium to each tube, shake slowly at 37°C for 4 hours, to recover the bacteria and express The antibiotic marker gene encoded by the plasmid; 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 constant temperature incubator at 37°C, blue and white spots will appear after 30-48 hours colony.

[0056] 3.3 Pick a single positive white spot colony and inoculate 5mL of resistant LB, shake slowly f...

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Abstract

The invention discloses [beta]2-GP1 modified protein as well as a construction method and application thereof. The construction method comprises the following steps of S1, recombining a whole-gene synthesis sequence: sequentially connecting nucleotide sequences of protein sections of a structural domain 1, a structural domain 4 and a structural domain 5 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, preforming 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 modified protein: purifyinga cell culture obtained by expression in the step S5 to obtain the modified protein. The modified 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 a β2-GP1 restructured protein and its construction method and application. Background technique [0002] Antiphospholipid syndrome (APS) is a disease characterized by recurrent arterial or venous thrombosis, morbid pregnancy, and persistent positive antiphospholipid antibodies (APL). Antiphospholipid antibodies (APL) can lead to multi-organ vascular involvement at the same time, manifested as malignant hypertension, mild hemiplegia, dyspnea, hepatic infarction, cardiac artery thrombosis and other symptoms. Due to long-term diagnosis difficulties and lack of effective drugs, the mortality rate of patients is as high as 75%. [0003] Classification of antiphospholipid syndrome (APS) must meet at least one clinical (vascular thrombosis or pregnancy comorbidity) and one laboratory (anticardiolipin antibody, lupus anticoagulation or anti-β2-GP1 antibody positive) indic...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C07K14/775C12N15/12C12N15/866G01N33/543G01N33/535
CPCC07K14/775C12N15/86G01N33/54326G01N33/535C12N2710/14043
Inventor秦枫姚俊腾季纯宇任伟超张小飞颜松周玥黄敬双张伟万文琴鲜静
Owner四川携光生物技术有限公司