CN111166869A - Quantum dot and recombinant human endostatin conjugate and preparation process thereof - Google Patents
Quantum dot and recombinant human endostatin conjugate and preparation process thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
Abstract
The invention provides a quantum dot and recombinant human vascular endothelial inhibin conjugate and a preparation process thereof, wherein quantum dots are adopted to react with target protein recombinant human vascular endothelial inhibin by a carbodiimide method, and the coupling of the quantum dots and the recombinant human vascular endothelial inhibin is realized by adjusting the coupling molar ratio, the coupling environment and the like so as to achieve the highest coupling efficiency; the preparation method has low production cost and simple preparation process, and is suitable for large-scale industrial production.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a quantum dot and recombinant human endostatin conjugate and a preparation process thereof.
Background
The recombinant human endostatin rhEndostatin, rh-ES, is widely used clinically at present, but only common injection is on the market, so the stability is poor, and frequent administration is needed. In the traditional coupling preparation process such as electrostatic adsorption, although the operation is simple and other reaction reagents are not required to be added, the stability of the compound prepared by the method is poor, the coupling efficiency is low, the compound is difficult to store for a long time, the activity of the compound is seriously influenced, the compound is difficult to realize into a medicine property, and the target evaluation is difficult. Therefore, a novel mild preparation process is needed, the influence on the activity of the protein is reduced, and meanwhile, the construction of a protein administration system and the evaluation of the targeting of the protein administration system are all the key points of the research on novel dosage forms of the recombinant human endostatin.
Disclosure of Invention
Aiming at the technical problems, the invention provides a quantum dot and recombinant human vascular endothelial inhibin conjugate and a preparation process thereof, wherein quantum dots QDs are taken as imaging materials, and the QDs and the recombinant human vascular endothelial inhibin rh-ES are covalently combined to obtain a compound combining protein and the quantum dots, so that the compound has the fluorescence characteristic of the quantum dots and the bioactivity of protein drugs. The covalent bonding is that the carboxyl of the quantum dot and the amino of the protein form a covalent bond through condensation reaction, the bonding force is strong, and the obtained compound has good stability and better optical performance. The invention couples the recombinant human vascular endothelial inhibin and the tracer such as quantum dots and the like by a carbodiimide method, has mild reaction conditions, does not influence the activity of protein, and is favorable for later-stage targeting evaluation. The preparation process of the quantum dot and recombinant human endostatin conjugate provided by the invention has high coupling efficiency.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation process of a quantum dot and recombinant human vascular endothelial inhibin conjugate comprises the following steps:
the conjugate takes quantum dots as a tracer and recombinant human vascular endothelial inhibin as a model drug, and the quantum dots after activation and the recombinant human vascular endothelial inhibin protein are subjected to coupling reaction to prepare the quantum dot and recombinant human vascular endothelial inhibin conjugate.
In the above scheme, the activating step of the quantum dot specifically comprises:
taking a quantum dot QDs solution, diluting, and respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxy thiosuccinimide NHS to activate carboxyl on the surface of the quantum dot to obtain a solution A;
the molar ratio of the quantum dots to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC is 1: 500-5000;
the coupling molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC to the N-hydroxythiosuccinimide NHS is 1: 1.
in the scheme, the step of performing coupling reaction on the quantum dots QDs and the recombinant human vascular endothelial inhibin protein specifically comprises the following steps:
and adding a recombinant human vascular endothelial inhibin protein solution into the solution A to perform coupling reaction, and separating and purifying the labeled protein and the unlabeled protein to obtain a solution B.
Further, the concentration of the recombinant human vascular endothelial inhibin protein solution is 5 mg/mL.
Furthermore, the molar ratio of the quantum dots QDs to the recombinant human vascular endothelial inhibin is 1: 1-2.5.
Further, the coupling reaction time is 1-4 h.
Further, the quantum dots are water-soluble quantum dots.
In the above scheme, the method further comprises the following steps:
adding 2% by volume and mass of bovine serum albumin BSA solution into the solution B, and vortexing for 2min to close carboxyl on the surface of the quantum dot which is not coupled with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxy thiosuccinimide NHS.
A quantum dot and recombinant human vascular endothelial inhibin conjugate is prepared by a preparation process of the quantum dot and recombinant human vascular endothelial inhibin conjugate.
Compared with the prior art, the invention has the beneficial effects that: the invention couples the quantum dots on the recombinant human vascular endothelial inhibin by a carbodiimide method, which is not only beneficial to the targeted evaluation of protein drugs but also provides more possibility for the construction of a protein drug delivery system on the basis of not influencing the biological activity of the protein drugs and the optical performance of the quantum dots. The conjugate prepared under the preparation process condition has good stability, the reaction is mild, simple and convenient, and the operation is easy.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is the agarose gel electrophoresis picture of the quantum dot and recombinant human endostatin conjugate and recombinant human endostatin prepared in example 2;
FIG. 2 is a circular dichroism spectrum of the quantum dot and recombinant human endostatin conjugate and recombinant human endostatin prepared in example 2.
Detailed Description
Embodiments of the present invention are described in detail below with reference to the drawings, but the scope of the present invention is not limited thereto.
The invention relates to a quantum dot and recombinant human vascular endothelial inhibin conjugate and a preparation process thereof, wherein quantum dots QDs are used as imaging materials, and are covalently bonded with recombinant human vascular endothelial inhibin rh-ES to obtain a protein and quantum dot bonded compound, so that the compound has the fluorescence characteristics of the quantum dots and the biological activity of protein drugs. The preparation process of the quantum dot and recombinant human endostatin conjugate provided by the invention has high coupling efficiency.
The quantum dot QDs and recombinant human vascular endothelial inhibin conjugate is prepared by providing carboxyl functional groups by quantum dots and amino functional groups by protein through a carbodiimide method; the coupling molar ratio of the quantum dots to the EDC is 1: 500-5000, the coupling molar ratio of the quantum dots to the recombinant human vascular endothelial inhibin is 1: 1-2.5, the coupling reaction time is 1-4 h, the quantum dots are coupled on the recombinant human vascular endothelial inhibin by a carbodiimide method under the preparation process conditions, and on the basis of not influencing the biological activity of the protein drug and the optical performance of the quantum dots, the targeted evaluation on the protein drug is facilitated, and more possibilities are provided for the construction of a protein drug delivery system. The conjugate prepared by the method has good stability, and the reaction is mild, simple and convenient and is easy to operate.
The preparation method of the quantum dot and recombinant human vascular endothelial inhibin conjugate specifically comprises the following steps:
activating the quantum dots, namely taking a QDs solution with the concentration of 8 mu M/L, diluting the QDs solution by four times with BBS with the pH value of 7.4, respectively adding EDC and NHS, wherein the EDC solution and the NHS solution need to be prepared in situ, and vortexing for 15min to activate carboxyl on the surfaces of the quantum dots to obtain an activated quantum dot solution A;
and (3) carrying out coupling reaction on the quantum dots QDs and the recombinant human vascular endothelial inhibin protein, adding 5mg/mL of the recombinant human vascular endothelial inhibin protein solution into the solution A, continuing to vortex for a moment, and then carrying out oscillation reaction at the constant temperature and the normal temperature under a constant temperature oscillator at the vibration speed of 250 r/min. After the reaction is finished, removing redundant EDC and NHS through ultrafiltration, and then performing high-speed centrifugation for 8min at 5000r/min and 4 ℃ to realize separation and purification of labeled protein and unlabeled protein to obtain a quantum dot and recombinant human endostatin protein conjugate solution B;
and (3) blocking to improve the coupling efficiency, adding a BSA solution with the volume mass percent of 2% into the solution B, swirling for 2min to close carboxyl on the surfaces of the quantum dots which are not coupled with EDC and NHS so as to avoid self-aggregation reaction of the conjugate, reducing the coupling efficiency, and blocking light after the reaction is completed and storing in a refrigerator cold storage layer for later use.
Preferably, the coupling molar ratio of the quantum dots to EDC in the activation step of the quantum dots is 1: 500-1: 5000; the reaction environment is boric acid buffer solution with pH 7.4.
In the activation step of the quantum dots, the reaction molar ratio of EDC to NHS is 1: 1.
the activation time in the activation step of the quantum dots is 15 min.
The coupling molar ratio of the quantum dots to the recombinant human vascular endothelial inhibin in the step of coupling reaction of the quantum dots QDs and the recombinant human vascular endothelial inhibin protein is 1: 1-1: 2.5; the reaction time of the reaction under a constant-temperature oscillator is 1-4 h; after the reaction is finished, redundant EDC and NHS are removed through ultrafiltration, and the separation and purification of the labeled protein and the unlabeled protein are realized through a high-speed centrifugation method.
The invention also provides a quantum dot and recombinant human vascular endothelial inhibin conjugate, which is prepared by utilizing the preparation process of the quantum dot and recombinant human vascular endothelial inhibin conjugate.
The quantum dots are adopted to react with the target protein recombinant human vascular endothelial inhibin by a carbodiimide method, and the coupling of the quantum dots and the recombinant human vascular endothelial inhibin and the achievement of the highest coupling efficiency are realized by adjusting the coupling molar ratio, the coupling environment and the like; the preparation method has low production cost and simple preparation process, and is suitable for large-scale industrial production.
Example 1: preparation of quantum dot and recombinant human vascular endothelial inhibin conjugate
(1) Activating the quantum dots, namely taking 10 mu L of 8 mu M/L QDs solution, diluting the solution by four times with BBS with the pH value of 7.4, respectively adding 3.08 mu L of 2.5mg/mL EDC solution and 3.48 mu L of 2.5mg/mL NHS solution, preparing the EDC solution and the NHS solution in situ, and vortexing for 15min to activate carboxyl on the surfaces of the quantum dots;
(2) coupling the activated quantum dots with protein, adding 4.2 mu L of recombinant human endostatin protein solution of 5mg/mL, continuing to vortex for 15min, and oscillating at normal temperature for reaction for 2 h;
(3) blocking to improve coupling efficiency, and finally adding 10 μ L of 2% BSA solution to vortex for 2min to close the carboxyl on the surface of the quantum dot which is not coupled with EDC and NHS.
Example 2: preparation of quantum dot and recombinant human endostatin conjugate
(1) Activating quantum dots, namely taking 10 mu L of 8 mu M/L QDs solution, diluting the solution by four times with BBS with the pH value of 7.4, respectively adding 3.08 mu L of 5mg/mL EDC solution and 3.48 mu L of 5mg/mL NHS solution, preparing the EDC solution and the NHS solution in situ, and vortexing for 15min to activate carboxyl on the surfaces of the quantum dots;
(2) coupling the activated quantum dots with protein, adding 4.2 mu L of recombinant human endostatin protein solution of 5mg/mL, continuing to vortex for 15min, and oscillating at normal temperature for reaction for 2 h;
(3) blocking to improve coupling efficiency, and finally adding 10 μ L of 2% BSA solution to vortex for 2min to close the carboxyl on the surface of the quantum dot which is not coupled with EDC and NHS.
Example 3: preparation of quantum dot and recombinant human endostatin conjugate
(1) Activating quantum dots, namely taking 10 mu L of 8 mu M/L QDs solution, diluting the solution by four times with BBS with the pH value of 7.4, respectively adding 3.08 mu L of 10mg/mL EDC solution and 3.48 mu L of 10mg/mLNHS solution, preparing the EDC solution and the NHS solution in situ, and vortexing for 15min to activate carboxyl on the surfaces of the quantum dots;
(2) coupling the activated quantum dots with protein, adding 4.2 mu L of recombinant human endostatin protein solution of 5mg/mL, continuing to vortex for 15min, and oscillating at normal temperature for reaction for 2 h;
(3) blocking to improve coupling efficiency, and finally adding 10 μ L of 2% BSA solution to vortex for 2min to close the carboxyl on the surface of the quantum dot which is not coupled with EDC and NHS.
Example 4: preparation of quantum dot and recombinant human endostatin conjugate
(1) Activating quantum dots, namely taking 10 mu L of 8 mu M/L QDs solution, diluting the solution by four times with BBS with the pH value of 7.4, respectively adding 3.08 mu L of 25mg/mL EDC solution and 3.48 mu L of 25mg/mLNHS solution, preparing the EDC solution and the NHS solution in situ, and vortexing for 15min to activate carboxyl on the surfaces of the quantum dots;
(2) coupling the activated quantum dots with protein, adding 4.2 mu L of recombinant human endostatin protein solution of 5mg/mL, continuing to vortex for 15min, and oscillating at normal temperature for reaction for 2 h;
(3) blocking to improve coupling efficiency, and finally adding 10 μ L of 2% BSA solution to vortex for 2min to close the carboxyl on the surface of the quantum dot which is not coupled with EDC and NHS.
TABLE 1 Effect of different ratios of QDs to EDC on coupling products
Note: more + indicates better stability and fluorescence intensity
As can be seen from the results of table 1, in examples 1, 2, 3, and 4, an excess amount of EDC causes fluorescence quenching of quantum dots, agglomeration of QDs, reduction of coupling efficiency, and influence on optical properties thereof. Since the amount of EDC is insufficient and the activation of carboxyl groups on the surface of the quantum dot is incomplete, the coupling efficiency is lowered and the fluorescence intensity is lowered, it is necessary to select an appropriate coupling ratio. From the results, it can be seen that the quantum dots have a sedimentation phenomenon and the fluorescence of the quantum dots is partially quenched at molar ratios of 1:5000 and 1:2000, the coupling efficiency is low at a molar ratio of 1:500, indicating that the amount of EDC is insufficient, the stability of the complex is good at a molar ratio of 1:1000, the fluorescence intensity after centrifugation is good, and finally the molar ratio of the quantum dots to EDC is determined to be 1: 1000.
Example 5: preparation of quantum dot and recombinant human endostatin conjugate
(1) Activating quantum dots, namely taking 10 mu L of 8 mu M/L QDs solution, diluting the solution by four times with BBS with the pH value of 7.4, respectively adding 3.08 mu L of 5mg/mL EDC solution and 3.48 mu L of 5mg/mLNHS solution, preparing the EDC solution and the NHS solution in situ, and vortexing for 15min to activate carboxyl on the surfaces of the quantum dots;
(2) coupling the activated quantum dots with protein, adding 2.1 mu L of recombinant human endostatin protein solution of 5mg/mL, continuing to vortex for 15min, and oscillating at normal temperature for reaction for 2 h;
(3) blocking to improve coupling efficiency, and finally adding 10 μ L of 2% BSA solution to vortex for 2min to close the carboxyl on the surface of the quantum dot which is not coupled with EDC and NHS.
Example 6: preparation of quantum dot and recombinant human endostatin conjugate
(1) Activating quantum dots, namely taking 10 mu L of 8 mu M/L QDs solution, diluting the solution by four times with BBS with the pH value of 7.4, respectively adding 3.08 mu L of 5mg/mL EDC solution and 3.48 mu L of 5mg/mLNHS solution, preparing the EDC solution and the NHS solution in situ, and vortexing for 15min to activate carboxyl on the surfaces of the quantum dots;
(2) coupling the activated quantum dots with protein, adding 5mg/mL recombinant human endostatin protein solution of 5.25 muL, continuing to vortex for 15min, and oscillating at normal temperature for reaction for 2 h;
(3) blocking to improve coupling efficiency, and finally adding 10 μ L of 2% BSA solution to vortex for 2min to close the carboxyl on the surface of the quantum dot which is not coupled with EDC and NHS.
TABLE 2 Effect of different ratios of Quantum dots to protein on coupling products
As can be seen from the results in table 2, in example 2, example 5 and example 6, the coupling efficiency increases with the increase of the molar ratio of the quantum dots to the protein, and when the molar ratio is too large, the coupling efficiency decreases, probably due to the effect of steric hindrance on the surface of the quantum dots, so the coupling ratio is selected to be 1: 2.
Example 7: preparation of quantum dot and recombinant human endostatin conjugate
(1) Activating quantum dots, namely taking 10 mu L of 8 mu M/L QDs solution, diluting the solution by four times with BBS with the pH value of 7.4, respectively adding 3.08 mu L of 5mg/mL EDC solution and 3.48 mu L of 5mg/mLNHS solution, preparing the EDC solution and the NHS solution in situ, and vortexing for 15min to activate carboxyl on the surfaces of the quantum dots;
(2) coupling the activated quantum dots with protein, adding 4.2 mu L of recombinant human endostatin protein solution of 5mg/mL, continuing to vortex for 15min, and oscillating at normal temperature for reaction for 1 h;
(3) blocking to improve coupling efficiency, and finally adding 10 μ L of 2% BSA solution to vortex for 2min to close the carboxyl on the surface of the quantum dot which is not coupled with EDC and NHS.
Example 8: preparation of quantum dot and recombinant human endostatin conjugate
(1) Activating quantum dots, namely taking 10 mu L of 8 mu M/L QDs solution, diluting the solution by four times with BBS with the pH value of 7.4, respectively adding 3.08 mu L of 5mg/mL EDC solution and 3.48 mu L of 5mg/mLNHS solution, preparing the EDC solution and the NHS solution in situ, and vortexing for 15min to activate carboxyl on the surfaces of the quantum dots;
(2) coupling the activated quantum dots with protein, adding 4.2 mu L of recombinant human endostatin protein solution of 5mg/mL, continuing to vortex for 15min, and oscillating at normal temperature for reaction for 4 h;
(3) blocking to improve coupling efficiency, and finally adding 10 μ L of 2% BSA solution to vortex for 2min to close the carboxyl on the surface of the quantum dot which is not coupled with EDC and NHS.
TABLE 3 Effect of different reaction times on the coupling products
As can be seen from the results in Table 3, the reaction in examples 2, 7 and 8 is almost complete in 2-4h, and the time has little influence on the fluorescence intensity and stability of the complex, but the reaction time is finally selected to be 2h in consideration of the protein activity, experimental operation and other factors.
Example 9: agarose gel electrophoresis experiment of quantum dots and conjugates
The conjugate and the quantum dot in the example 2 are respectively subjected to agarose gel electrophoresis experiments to prove that the conjugate exists, and the specific experimental processes are as follows: the gel plate is prepared by 1% agarose, the electrophoresis buffer is 1xTBE, 1xTBE plus 25% glycerol is used as a loading buffer, and the loading buffer and the sample solution are mixed in a proportion of 1: 4, mixing uniformly, taking 10 mu L of sample, and carrying out electrophoresis for 1h under the condition of 80V. After electrophoresis is finished, the picture is taken under an ultraviolet lamp, and the difference between QDs and rh-ES-QDs is judged according to the position of the strip.
As can be seen from the agarose gel electrophoresis result of FIG. 1, when A is labeled and B is labeled, the particle size and the potential of the labeled quantum dot will be increased, the migration on the agarose gel electrophoresis will also be changed, and the band of the labeled quantum dot complex is delayed obviously, indicating that the quantum dot is successfully labeled with protein.
Example 10: secondary structure analysis experiment of recombinant human vascular endothelial inhibin and conjugate
The secondary structure analysis experiment of protein was carried out on the recombinant human endostatin conjugate and the recombinant human endostatin in example 2, respectively, to prove the integrity of the secondary structure of the protein, and the specific experimental process was as follows: circular dichroism spectrograms are used for characterizing the secondary structure of the protein and the protein labeling compound. The specific operation is as follows: preparing a rh-ES solution with the concentration of 5 mu g/mL as a reference substance, preparing a rh-ES-QDs solution (containing rh-ES with the concentration of 5 mu g/mL), scanning an extreme ultraviolet region (195-250 nm) in a scanning range, scanning at a speed of 50nm/min and response time of 1s at room temperature, and performing spectrogram scanning.
As can be seen from FIG. 2, there is no significant difference between the two-color circular spectra of rh-ES-QDs solution with the same concentration and the two-color circular spectra of the recombinant human endostatin solution with the same concentration. From this, it is presumed that the secondary structure of rh-ES bound to the quantum dot is not significantly changed.
The above examples are only for illustrating the technical idea and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered in the protection scope of the present invention.
Claims (8)
1. A preparation process of a quantum dot and recombinant human vascular endothelial inhibin conjugate is characterized by comprising the following steps:
the conjugate takes quantum dots as a tracer and recombinant human vascular endothelial inhibin as a model drug, and the quantum dots after activation and the recombinant human vascular endothelial inhibin protein are subjected to coupling reaction to prepare the quantum dot and recombinant human vascular endothelial inhibin conjugate.
2. The preparation process of the quantum dot and recombinant human vascular endothelial inhibin conjugate according to claim 1,
the activation steps of the quantum dots are as follows:
taking a quantum dot QDs solution, diluting, and respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxy thiosuccinimide NHS to activate carboxyl on the surface of the quantum dot to obtain a solution A;
the molar ratio of the quantum dots to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC is 1: 500-5000;
the coupling molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC to the N-hydroxythiosuccinimide NHS is 1: 1.
3. the preparation process of the quantum dot and recombinant human vascular endothelial inhibin conjugate according to claim 2, wherein the step of performing the coupling reaction of the quantum dot QDs and the recombinant human vascular endothelial inhibin protein is as follows:
and adding a recombinant human vascular endothelial inhibin protein solution into the solution A to perform coupling reaction, and separating and purifying the labeled protein and the unlabeled protein to obtain a solution B.
4. The preparation process of the quantum dot and recombinant human vascular endothelial inhibin conjugate according to claim 3,
the concentration of the recombinant human vascular endothelial inhibin protein solution is 5 mg/mL.
5. The preparation process of the quantum dot and recombinant human vascular endothelial inhibin conjugate according to claim 3,
the molar ratio of the quantum dots QDs to the recombinant human vascular endothelial inhibin is 1: 1-2.5.
6. The preparation process of the quantum dot and recombinant human vascular endothelial inhibin conjugate according to claim 3,
the coupling reaction time is 1-4 h.
7. The preparation process of the quantum dot and recombinant human endostatin conjugate according to claim 3, characterized by further comprising the following steps:
adding 2% by volume and mass of bovine serum albumin BSA solution into the solution B, and vortexing for 2min to close carboxyl on the surface of the quantum dot which is not coupled with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxy thiosuccinimide NHS.
8. A quantum dot and recombinant human endostatin conjugate, which is prepared by the preparation process of the quantum dot and recombinant human endostatin conjugate according to any one of claims 1 to 7.
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