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High affinity immune globulin binding molecule and method for preparation

A technology that combines molecules and affinity, applied in the fields of molecular biology and immunodiagnostics

Inactive Publication Date: 2007-06-06
SHANGHAI NATURE STANDARD R&D & BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • High affinity immune globulin binding molecule and method for preparation
  • High affinity immune globulin binding molecule and method for preparation
  • High affinity immune globulin binding molecule and method for preparation

Examples

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Embodiment 1

[0082] Example 1 Construction of Recombinant Ig Binding Molecule Library

[0083] 1. Synthesize primers

[0084] 1.1 For protein A, protein L, protein G single domain gene amplification

[0085] (1) The upstream primers for amplifying the sequence of Protein A-A, Protein A-B / C, Protein A-D, and Protein A-E from Mu-protein A / pGEM-T easy plasmid are:

[0086] PA-uA: 5’-cacgagctcGCTGACAACAATTTCAAC-3’

[0087] PA-uB: 5’-cgcgagctcGCGGATAACAAATTCAAC-3’

[0088] PA-uC: 5’-cgcgagctc GCTGACAACAAATTCAAC-3’

[0089] PA-uD: 5’-tccgagctcGCTGATGCGCAACAAAAT-3’

[0090] PA-uE: 5’-cgcgagctcGCTCAACAAAATGCTTTT-3’

[0091] The downstream primers are:

[0092] PA-dA: 5’-tctgagctcTTTCGGTGCTTGAGATTC-3’

[0093]PA-dB: 5’-tctgagctcTTTTGGTGCTTGTGCATC-3’

[0094] PA-dC: 5’-acggagctcTTTTGGTGCTTGAGCATC-3’

[0095] PA-dD: 5’-tcggagctcAAAGCCACGAACTCTAAG-3’

[0096] PA-dE: 5’-agagagctcTTTTGGAGCTTGAGAGTC-3’

[0097] (2) Amplify the B1-B5 sequence of Protein L from the Protein L / pGEM-T easy plasmid

[0098] The up...

Embodiment 2

[0131] Example 2. Phage display of recombinant Ig binding molecule library

[0132] 1. Preparation and linearization of phagemid vector pCANTAB5S DNA:

[0133] The pCANTAB5S phagemid DNA was extracted with the plasmid recovery kit of Shenneng Group, and proceeded according to the instructions. Quantification by agarose electrophoresis. After Sac I digestion, the alkaline phosphatase (CIAP) is dephosphorylated, and the linearized vector is recovered from the solution by the kit, and quantified by agarose electrophoresis.

[0134] 2. The Ig binding molecule library is connected to the phagemid vector:

[0135] The 12 kinds of recovered protein A fragments with single binding domains A, B, C, D, E, B1, B2, B3, B4, B single binding domain fragments of protein L, G1, G2 single binding domains of Protein G 80μl (5μg) of the mixture of binding domain digested fragments plus 5μl (25U) of T4 DNA ligase, 1.5μl of 10×Buffer, and a total reaction volume of 100μl. Divide 100μl into 20μl / branch...

Embodiment 3

[0142] Example 3. In vitro directed molecular evolution of Ig to screen high-affinity Ig binding molecules

[0143] 1. Ig affinity screening of recombinant phage library:

[0144] Coat 200μl of pH 9.6 carbonate buffer with human Ig (final concentration of 10μg / ml) on the enzyme-labeled strip plate at 37°C for 2 hours, and block the strip with 150μl of blocking solution (PBS+10% skimmed milk) Plate for 1 hour and store at 4°C. After washing 5 times with washing solution (PBS+0.05% Tween20), it was used for affinity screening of phage library. Add 100μl of blocking solution and 100μl of PALGn recombinant phage to each well of the slatted plate, react at 37°C for 3 hours, wash with washing solution (0.25% Tris+0.05% Tween20) 30 times, add 100μl of E. coli TG1 in logarithmic growth phase, react at 37°C for 1 After collecting, take 10μl, 1μl, 0.1μl coated with LB (containing ampicillin 100ng / ml) plates and count them overnight at 37°C to evaluate the binding of the phage library to Ig ...

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Abstract

This invention relates to high appetency immune globin combination molecule and its process method, wherein the method uses the structure units from the following fields: A, B, C, D, E fields of staphylococcus A protein; B1, B2, B3, B4, B5 structure fields of large digest streptococcus protein L; G1, G2 structure units of C and G streptococcus protein. The method connects the above structure units by random to recreate Ig combination molecule database expressed on the surface of the bacteriophage plaque. The immune globulin expresses the combination molecule to process appetency filtering for three to four times to get the aim combination molecule.

Description

Technical field [0001] The invention relates to the fields of molecular biology and immunodiagnostics, in particular to a recombinant immunoglobulin binding molecule library, its preparation and application. Background technique [0002] Enzyme-linked immunosorbent test (ELISA method) to detect antibodies has been widely used in the diagnosis of various clinical diseases, especially the immunodiagnosis of infectious diseases such as hepatitis and HIV infection, blood donor screening and epidemiological investigation. However, the current detection sensitivity of the ELISA method is not high, which often results in missed detection and affects the early diagnosis of the disease, delays the disease or causes post-transfusion hepatitis C and HIV infection, and seriously endangers people's health. Judging from the anti-HCV and anti-HIV ELISA reagents used for blood donors to screen, the latest detection reagents (third-generation reagents) are still missed. Among them, a large part o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C07K14/31C07K14/315C12N15/62G01N33/535G01N33/53C07K16/16C12N15/12
Inventor 潘卫徐容王金华谢天培李云华王德贵沈毅珺扬华王锦红许浩坤
Owner SHANGHAI NATURE STANDARD R&D & BIOTECH
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