Polymer microsphere and preparation and application thereof

A technology of polymers and microspheres, which is applied in the preparation methods of peptides, alkali metal compounds, biochemical equipment and methods, etc., can solve problems such as difficulty in expression, large molecular weight of Protein A, and limited processing capacity of chromatographic operations, so as to solve the problem of formulating Expensive base, simple operation, and cheap raw materials

Active Publication Date: 2021-09-07
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large molecular weight of Protein A, it is difficult to express, and the expressed Protein A still needs a series of processes such as separation and purification, grafting modification, etc., which makes the market price of Protein A affinity filler relatively expensive, and it is currently produced on a large scale in China. There are very few companies with Protein A affinity fillers. In addition, the scale of domestic antibody culture and expression is small, and the processing capacity of chromatographic operations is limited. Therefore, the production cost remains high and lacks market competitiveness.

Method used

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  • Polymer microsphere and preparation and application thereof
  • Polymer microsphere and preparation and application thereof
  • Polymer microsphere and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Construction and induced expression of embodiment 1 recombinant strain

[0036] 1. Construction of recombinant strains

[0037] The amino acid sequence of the ZZ domain is (SEQ ID NO.1):

[0038] RQHDEAVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAN.

[0039] The amino acid sequence of the peptide connecting the ZZ domain and the LppOmpA protein is: GIPG.

[0040] Ankyrin (LppOmpA) amino acid sequence is (SEQ ID NO.3):

[0041] MKATKLVLGAVILGSTLLAGCSSNAKIDQGINPYVGFEMGYD7WLGRMPYKGSVENGAYKAQGVQLTAKLGYPITDDLDIYTRLGGMVWRADTKSNVYGKNHDTGVSPVFAGGVEYAITPEIATRLEYQWTNNIGDAHTIGTRPDN.

[0042] (1) pET28a-LppOmpA-ZZ plasmid

[0043] According to the coding sequence of the ZZ domain gene (AAB00807.1, nucleotide sequence such as SEQ ID NO.2), it is fused with the anchor protein (LppOmpA) through the connecting peptide (GIPG) and provided by Sangon Bioengineering (Shanghai) Co., Ltd. The company carried out c...

Embodiment 2

[0057] Embodiment 2, preparation and characterization of polymer microspheres

[0058] (1) Add 0.6g of polyvinyl alcohol particles (PVA1799 type), 0.1g of sodium alginate (SA) powder and 9.3mL of double distilled water into a 25ml eggplant-shaped bottle, and stir at 95°C until the solution is clear and transparent to obtain PVA / SA mixed solution (wherein PVA mass concentration 6.0wt%, SA mass concentration 1.0wt%); Add the recombinant bacteria E.coli BL21 (DE3) / pET-28a-LppOmpA-ZZ bacterium that embodiment 1 prepares after being cooled to 40 ℃ 0.5ml solution (wet cell content: 5.8mg / mL), stirred evenly at room temperature (20-25°C) for 30min to obtain 10.5mL cell mixture solution as the water phase.

[0059] (2) In a 100mL round bottom flask, add 20ml liquid paraffin, 1.5ml Span 80 as a surfactant, mechanically stir at a speed of 300r / min for 30min, then use it as the oil phase, slowly add 20.0mL of the water phase prepared in step (1) drop by drop , stirred in a water bath a...

Embodiment 3

[0062] Embodiment 3, performance and application of polymer microsphere

[0063] 1. The binding properties of polymer microspheres:

[0064] The polymer microspheres prepared in Example 2 were washed three times with Tris-HCl buffer solution with pH=7.2, 0.02mol / L, 0.1g was put into a 1.5mL centrifuge tube, and 0.5mL was added with pH=7.2, 0.02mol / L FITC-labeled rabbit anti-mouse IgG solution (H+L, purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) with a concentration of 0.2 mg / mL prepared in Tris-HCl buffer solution, and incubated in a 25°C incubator After 3 hours, they were quickly washed 2-3 times with Tris-HCl buffer solution of pH = 7.2 and 0.02 mol / L, and then examined by fluorescence microscope as the experimental group.

[0065] Under the same conditions, no FITC-IgG antibody solution was used as the control group. The polymer microspheres were replaced by the control polymer microspheres of Example 2 as a blank control group.

[0066] The results of fluore...

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Abstract

The invention discloses a polymer microsphere and preparation thereof and application thereof in preparation of antibody purification filler. The polymer microsphere is prepared by the following steps of: taking polyvinyl alcohol and sodium alginate as raw materials, adding water to dissolve at 80-100 DEG C, cooling to 40 DEG C, and adding a recombinant escherichia coli solution containing ZZ fusion protein to prepare a water phase; and mixing liquid paraffin and a surfactant to form an oil phase, slowly dropwise adding the water phase into the oil phase, stirring at 30-70 DEG C to form emulsion, and adding a saturated boric acid calcium chloride aqueous solution to prepare the polymer microsphere, wherein the ZZ fusion protein is formed by fusing ZZ protein and ankyrin (LppOmpA) through a connecting peptide. The diameter of the polymer microsphere prepared by the method is 50-250nm, the ultra-large pore diameter is 1-2 microns, the raw materials are cheap, the cost is low, the operation is simple, the preparation processes of expression, purification, grafting and the like of conventional ligands are omitted, and the problems of expensive ligands and the like are solved.

Description

[0001] (1) Technical field [0002] The invention relates to a polymer microsphere and its preparation and application. [0003] (2) Background technology [0004] Antibody drugs are widely used in the diagnosis, treatment, and immunity of diseases because of their strong targeting, high specificity, and low toxicity and side effects, and have broad application prospects. However, the technical threshold for antibody drug production is relatively high, involving core technologies such as antibody screening, antibody recombination, high-expression cell line construction, large-scale suspension culture, and downstream purification. With the rapid development of molecular biology technology, antibody screening, recombination There are not too many obstacles in upstream technologies such as humanization and humanization. The current difficulties are mainly concentrated in the large-scale production in the midstream and purification in the downstream. Currently, about 80% of the do...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/084C12N11/10C07K16/00C07K1/22B01J20/26B01J20/28B01J20/30B01D15/38C12R1/19
CPCC12N11/084C12N11/10C07K16/00C07K1/22B01J20/24B01J20/267B01J20/28019B01D15/3804
Inventor 易喻岑灵照应国清梅建凤陈建澍张彦璐王旭东
Owner ZHEJIANG UNIV OF TECH
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