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Direct Application of Recombinant Fusion Proteins in Rapid Diagnostic Tests

A technology of fusion protein and protein, applied in the field of bioengineering, can solve problems such as water solubility decline, and achieve the effect of convenient purification

Inactive Publication Date: 2011-11-30
戴国祯 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But what is interesting is that after the fusion protein sequence is removed, the water solubility of these natural sequence proteins often decreases, so that people have to use the above-mentioned high-concentration urea or nitrosoguanidine and other solubilizing treatments to restore their solubility.
[0007] Although fusion protein technology has been widely used in many basic biological research and biotechnology fields, its application in the field of rapid detection is currently limited to the fusion of short tags such as His-tag tags on antigen proteins to facilitate protein purification.

Method used

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  • Direct Application of Recombinant Fusion Proteins in Rapid Diagnostic Tests
  • Direct Application of Recombinant Fusion Proteins in Rapid Diagnostic Tests
  • Direct Application of Recombinant Fusion Proteins in Rapid Diagnostic Tests

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Directly using the purified His-MBP-HIV fusion protein for rapid detection of HIV planar flow type

[0036] (1) Construction of His-HIV and His-MBP-HIV fusion genes

[0037] Different cDNA fragments of HIV virus (gp41 of HIV-1 and gp36 of HIV-2) were amplified by PCR and cloned into Escherichia coli expression vectors pET-28 and pET-32. Such as Figure 4 As shown, HIV recombinant genes A and B contain the same HIV viral gene sequence, and recombinant genes C and D contain the same HIV viral gene sequence, but the N-terminals of A and C only carry His-tag, while the N-terminals of B and D Carry His-tag and MBP fusion gene.

[0038] (2) His-MBP-HIV recombinant fusion protein is improved in water solubility and purity than His-HIV recombinant protein

[0039] The recombinant expression plasmids containing the above-mentioned A, B, C and D were transformed into Escherichia coli strain BL-21, and the expression was induced by IPTG to produce the recombinant pro...

Embodiment 2

[0047]Example 2: Directly using the purified His-MBP-HCV fusion protein for rapid detection of HCV planar flow type

[0048] (1) Construction of His-HCV and His-MBP-HCV fusion genes

[0049] Different HCV viral cDNA fragments (HCV genotypes I and II; gene fragments encoding core antigen, NS3, NS4 and NS5 nonstructural proteins) were amplified by PCR and cloned into E. coli expression vectors pET-28 and pET-32. Such as Figure 6 As shown, HCV recombinant genes A and B contain the same HCV viral gene sequence, and recombinant genes C and D contain the same HCV viral gene sequence, but the N-terminals of A and C only carry His-tag, while the N-terminals of B and D Carry His-tag and MBP fusion gene.

[0050] (2) His-MBP-HCV recombinant fusion protein is improved in water solubility and purity than His-HCV recombinant protein

[0051] The above-mentioned expression plasmids encoding HCV recombinant proteins A, B, C and D were transformed into Escherichia coli strain BL-21, and t...

Embodiment 3

[0059] Example 3: Using the purified His-MBP-HIV and His-MBP-HCV recombinant fusion protein for double rapid detection of planar flow type HIV / HCV

[0060] (1) Preparation of biotin-labeled His-MBP-HIV and His-MBP-HCV recombinant fusion protein and BSA

[0061] The HIV recombinant fusion protein B (His-MBP-gp36-gp41) in Example 1 was labeled with Sulfo-LC-NHS-biotin using the method described in Example 1 (3) and Example 2 (3) respectively and implemented The HCV recombinant fusion protein D (His-MBP-NS5-NS4-NS3-C8 asp) in Example 2 was used as the detection antigen. In the same principle, BSA was labeled with Sulfo-LC-NHS-biotin to make a quality control line.

[0062] (2) Preparation of gold-labeled streptavidin: colloidal gold suspension was prepared according to the method described in Example 1 (4), and colloidal gold was used to label streptavidin.

[0063] (3) Prepare detection test strips with His-MBP-HIV and His-MBP-HCV recombinant fusion protein

[0064] Use the H...

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Abstract

The invention belongs to the field of bioengineering technology. Based on the common problems in the field of rapid antibody detection at home and abroad, such as poor antigen water solubility, weak detection signal, low sensitivity, and poor specificity, the invention proposes to directly use the overall fusion protein carrying a fusion label to detect the analyte Rapid detection of planar flow type. 1. The main points of the present invention are: constructing the recombinant gene of the fusion protein; expressing and purifying the recombinant fusion protein; preparing detection reagents and capture reagents with the recombinant fusion protein. 2. The present invention shows that the fusion protein has the advantages of good water solubility, easy purification, natural configuration, high activity, and high labeling efficiency, and the rapid detection reagent made from it has high sensitivity and specificity. 3. The rapid detection reagent constructed according to the present invention can detect one or two analytes simultaneously. 4. The present invention has broad application prospects in the field of application and basic research such as disease diagnosis, epidemiological survey, medical identification, new drug development, agriculture, animal husbandry and veterinary medicine.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to rapid diagnosis and detection by directly adopting an integral fusion protein carrying a fusion label. Background technique [0002] Detecting the content of specific protein factors in human and other biological samples is of great significance for basic research and practical applications in the fields of medicine, biology, agriculture and animal husbandry. For example, detecting the presence of antibodies specific to pathogenic viruses such as hepatitis C in human blood is currently the main means of diagnosing these infectious diseases. Similarly, the determination of changes in the levels of early biomarker protein factors for cancer and cardiovascular diseases is extremely important for early detection and treatment of these diseases and observation of curative effects. With the continuous development of the fields of medicine and biology, various protei...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12N15/62C12N15/63C07K19/00
Inventor 戴国祯孙东旭
Owner 戴国祯
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