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His-Vx3-eGFP protein-nano aluminum covalent coupling ubiquitinated protein containing carbon-nitrogen bonds as well as extraction method and application thereof

A technology of his-vx3-egfp and ubiquitinated protein, which is applied in the field of tumor vaccines, can solve the problems of complicated product verification methods after coupling, high reaction temperature, and difficulty in effectively controlling the reaction temperature, so as to achieve easy control of the reaction process and high reaction efficiency. The effect of mild conditions and stable properties

Active Publication Date: 2016-07-20
SOUTHEAST UNIV
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AI Technical Summary

Problems solved by technology

[0007] At present, although there have been studies on ubiquitinated proteins in the prior art, there are still the following problems to be solved, such as the reaction temperature of His-Vx3-eGFP coupled to nano-aluminum is relatively high (90 ° C), and the reaction temperature is difficult Effective control; the proof method of the coupled product is complicated, and confocal microscopy is required to prove its coupling effect; the ability to recruit tumor cell ubiquitinated proteins needs to be further improved

Method used

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  • His-Vx3-eGFP protein-nano aluminum covalent coupling ubiquitinated protein containing carbon-nitrogen bonds as well as extraction method and application thereof
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  • His-Vx3-eGFP protein-nano aluminum covalent coupling ubiquitinated protein containing carbon-nitrogen bonds as well as extraction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: Modification of nano aluminum particles

[0062] 1. Weigh dry nano-aluminum (α-Al 2 o 3 ) 400mg, placed in a 50ml flat-bottomed test tube, added 1329.39mg triethoxysilane and 26.58mL absolute ethanol, stirred at room temperature for 12h;

[0063] 2. Centrifuge the above reaction product at 15000g for 10min, discard the supernatant, and wash the precipitate three times with a large amount of absolute ethanol;

[0064] 3. Add 1.1784 mL of glutaraldehyde (25%) and 10.605 mL of ultrapure water to the precipitate from the previous step, and stir at room temperature for 2 hours;

[0065] 4. Centrifuge the above reaction product at 15,000 g for 10 minutes, discard the supernatant, wash the precipitate with a large amount of ultrapure water three times, and take the precipitate for later use;

[0066] 5. Take an appropriate amount of the product obtained in the above steps 2 and 4 and α-Al 2 o 3 After drying in vacuum at high temperature overnight, the absorp...

Embodiment 2

[0068] Example 2: His-Vx3-eGFP protein covalently coupled to nano-aluminum particles

[0069] 1. Resuscitate Escherichia coli transformed with the His-Vx3-eGFP expression plasmid, smear it on an LB plate containing 50 μg / ml ampicillin, pick a single clone after 14 hours, and culture it in the LB medium containing ampicillin at 37°C for 6 hours with shaking After that, draw 1ml and inoculate it into 100ml LB culture medium, and add IPTG with a final concentration of 100mM to induce at low temperature for 16h when it grows to the logarithmic phase;

[0070] 2. Centrifuge the bacterial solution obtained from the upper part at low temperature and high speed to obtain bacteria, wash with PBS three times, resuspend in buffer, add 100 μg / ml lysozyme to act on ice for 20 minutes, then act on ultrasonic breaker for 20 minutes, and centrifuge at high speed and low temperature to obtain supernatant;

[0071] 3. Add the above supernatant to a nickel ion affinity chromatography column and ...

Embodiment 3

[0075] Example 3: 4T1 cells express the highest condition of ubiquitinated protein

[0076] 1. Tumor cell culture:

[0077] Thaw and freeze 4T1 cells (mouse breast cancer cells), add RPMI-1640 medium containing 10% (V / V) fetal bovine serum, 100U / ml penicillin and 100U / ml streptomycin immediately after thawing in a water bath at 40°C, Resuspend cells to 1 x 10 6 cells / ml, and transfer it into a 500ml cell culture flask, at 37°C, 5% (V / V) CO 2 cultured in a constant temperature incubator, the medium was changed every 1 to 2 days, and conventionally digested with 0.25% (g / mL) trypsin;

[0078] 2. Treatment of tumor cells:

[0079] 1) Cultivate the cells according to step 1 until the cells grow to 80%, add Velcade and ammonium chloride in amounts of 100nmol / L and 20mmol / L, intervene the tumor cells for 3, 6, 9, and 12 hours, and inhibit the expression of ubiquitinated proteins. Degradation; CCK-8 method to detect cell activity. Such as image 3 Shown: with the increase of th...

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Abstract

The invention discloses a His-Vx3-eGFP protein-nano aluminum covalent coupling ubiquitinated protein containing carbon-nitrogen bonds as well as an extraction method and application thereof. The ubiquitinated protein is prepared through the following steps of: forming a conjugate of His-Vx3-eGFP protein and modified nano aluminum by virtue of carbon-nitrogen double bonds and incubating the conjugate and a treated tumor cell lysis solution. Compared with the prior art, obtained alpha-Al2O3-His-Vx3-eGFP is stable in property, is capable of greatly improving the ubiquitinated protein collection effect and simplifying experiment operation; in addition, the defect that adjuvants are added in use of an existing ubiquitinated protein vaccine is overcome.

Description

technical field [0001] The invention relates to a His-Vx3-eGFP protein containing a carbon-nitrogen bond-nano-aluminum covalently coupled ubiquitinated protein and an extraction method thereof, belonging to the technical field of tumor vaccines. Background technique [0002] Tumors seriously threaten human health. Curative resection, chemotherapy, and radiotherapy are currently the preferred treatment methods for cancer patients. Clinical data show that most cancer patients are not sensitive to chemotherapy, radiotherapy and other treatments. Therefore, the clinical treatment of tumors urgently needs new methods and means. As a new tumor treatment strategy, tumor biotherapy has the advantages of strong specificity and less toxic side effects, and has attracted more and more attention. Among them, tumor vaccines based on dendritic cells (DC) have become a research hotspot in tumor biotherapy. [0003] In the process of tumor development, the ineffective presentation of tum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K1/107C07K1/14A61K39/00A61K39/39
CPCA61K39/0011A61K39/39A61K2039/55505C07K14/00
Inventor 王立新沈艳飞赵金金
Owner SOUTHEAST UNIV
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