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Composite material containing graphene oxide and recombinant streptococcal protein G, and preparation and application thereof
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A technology of recombinant streptococcus and composite materials, which is applied in the field of chemical synthesis, can solve the problem of low antibody adsorption rate, and achieve the effects of good reproducibility, easy large-scale production, and easy promotion
Active Publication Date: 2016-02-03
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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At present, most commercial reagents for antibody enrichment use agarose and trehalose as carriers to immobilize recombinant streptococcal protein G for absorbing antibodies. Due to the limitation of its own immobilization capacity, its antibody adsorption rate is low, and the adsorption capacity is 27mg / mL Vector left and right (GE Healthcare Life Sciences, 28-9768-01AA)
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Embodiment 1
[0029] (1) Activation of graphene oxide (GO)
[0030] 1. 1 mg GO was resuspended in 1 mL of deionized water, ultrasonicated at room temperature for 3 hours to fully disperse;
[0031] 2. Add 500mM (pH6.1) MES500μL, then simultaneously 30mg / mlNHS500μL and 10mg / mlEDC600uL, mix well, and stir rapidly at room temperature for 30min;
[0032] 3. The material was washed repeatedly with 500 mM (pH 6.1) MES to remove residual NHS and EDC, with a final volume of 1 mL.
[0033] (2) PEI modification of the activated graphene oxide material
[0034] 1. Add 15mgPEI to the above materials, shake at room temperature for 1h;
[0035] 2. After centrifugation at 16400×g / 10min, remove the supernatant, and the precipitate is GOPEI.
[0036] (3) Activation of recombinant streptococcal protein G
[0037] 1. Dissolve 500ugPG in 500mM (pH6.1) MES1mL;
[0038] 2. Then add 1.5mg NHS and 3.5mg EDC at the same time, mix well, and stir rapidly for 30min;
[0039] 3. The solution is activated PG.
[...
Embodiment 2
[0048] In the activation scheme of graphene oxide (GO), the concentration of NHS was changed to 50 mg / ml, and the others were the same as in Example 1, so that GO-PG composite material could be prepared.
Embodiment 3
[0050] In the activation scheme of graphene oxide (GO), the concentration of EDC was changed to 20mg / ml, and the others were the same as in Example 1, so that GO-PG composite material could be prepared.
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Abstract
The invention relates to a composite material containing grapheneoxide and a recombinant streptococcal protein G, and a preparation method and application thereof. According to the invention, by utilizing the characteristic of high specific surface area of the grapheneoxide, through a covalent binding method, the recombinant streptococcusprotein G is immobilized onto the grapheneoxide so as to prepare a high-capacity antibody enrichment material with antibody adsorption biological activity. The prepared composite material containing the graphene oxide and the recombinant streptococcal protein G can be applied in the fields of antibody purification, antibody enrichment, pathogen detection, pretreatment of biological samples, etc.
Description
technical field [0001] The invention belongs to the technical field of chemical synthesis, and relates to the preparation technology of graphene composite material and its application in protein enrichment, in particular to the preparation and functional application of a composite material based on graphene oxide-recombinant streptococcal protein G. Background technique [0002] Infectious disease pathogens themselves as antigens can produce corresponding specific antibodies in biological organisms. Most of the existing detection methods detect pathogens through the specific recognition between antigens and antibodies, such as enzyme-linked immunosorbent assay, immunofluorescence and immunocolloidal gold technology. wait. These immunological techniques have the characteristics of simple operation and easy analysis of results. However, the sensitivity of immunological detection methods is relatively low, which may easily cause false negative results. In order to improve the ...
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