Here is the translated Chinese patent application showing Graphene-Oxide used in a Covid-19 recombinant vaccine for a carrier element.

(19) State Intellectual Property Office of the People's Republic of China
(12) Invention patent application
(10) Application publication number
(43) Application announcement date
(21) Application number 202011031367 .1
(22) Application date 2020 .09 .27
(71) Applicant Shanghai National Engineering Research on Nanotechnology and Application
Center Co., Ltd.
Address 201109 No.468 Jianchuan Road, Minhang District, Shanghai
(72) Inventor Cui Daxiang Gao Ang Liang Hui Tian Jing
Li Xueling Shen Qi
(74) Patent Agency Shanghai East Asia Patent and Trademark Agency Co., Ltd.
Company 31208
Agent Dong Mei
(51)Int.Cl.
A61K 39/215 (2006 .01)
A61K 39/39 (2006 .01)
A61K 39/385 (2006 .01)
A61P 31/14 (2006 .01)
C07K 14/165(2006 .01)
C07K 17/14 (2006 .01)
(54) Title of Invention
Recombination of nano-coronavirus with graphene oxide as carrier
vaccine
(57) Abstract
The invention belongs to the field of nanomaterials and biomedicine, and relates to
A vaccine, specifically, involves the 2019‑nCoV coronavirus nuclear
Development of recombinant nano-vaccine. The present invention also includes the preparation of the vaccine
Preparation methods and application in animal experiments. COVID-19
The vaccine contains graphene oxide, carnosine, CpG, and new coronavirus RBD;
The backbone of graphene oxide binds carnosine, CpG and new coronavirus
RBD; the coding sequence of the CpG is shown in SEQ ID NO 1;
The new coronavirus RBD refers to the new coronavirus protein receptor
The binding region can produce high titer against RBD in mice
Specific antibodies, which provide a strong
Strong support.
Claims 1 page, instructions 5 pages
Sequence table on page 2 and attached picture on page 1
CN 112220919 A
2021.01.15
CN 112220919 A
1. A coronavirus vaccine, characterized in that the coronavirus contains graphene oxide, carnosine, CpG and new coronavirus
Virus receptor binding area; binding to carnosine, CpG and new coronavirus receptor binding area on the framework of graphene oxide;
The coding sequence of CpG is shown in SEQ ID NO 1; the new coronavirus receptor binding region refers to the new coronavirus S
Protein receptor binding area.
2. The coronavirus according to claim 1, wherein the coronavirus passes through the activated graphite oxide
It is obtained by connecting carnosine, CpG and coronavirus receptor binding regions on the ene.
3. The preparation method of coronavirus according to claim 1, characterized in that the preparation method comprises the following steps
Step:
Obtain CpG, receptor binding region recombinant protein and carnosine, the coding sequence of CpG is shown in SEQ ID NO 1;
Add the graphene oxide freeze-dried powder to the phosphate buffer solution and ultrasonic treatment;
Add EDC and NHS to activate the graphene oxide solution, remove excess EDC/sulfo-NHS in the reaction solution by ultrafiltration, and adjust the pH of the reaction solution to neutral;
Add carnosine, CpG and receptor binding region recombinant protein to the reaction solution and incubate with activated graphene oxide;
Remove excess uncoupled protein from the reaction solution, sterilize, and set aside.
4. The preparation method of claim 3, wherein the duration of the ultrasound is 2-3 hours.
5. The preparation method of claim 3, wherein the pH of the phosphate buffer is 6.8-7.6.
6. The preparation method of claim 3, wherein the excess EDC/sulfo-NHS is removed or the
The method of coupling proteins is ultrafiltration.
7. The preparation method of claim 3, wherein the amount of carnosine is more than 1.5 times that of graphene oxide.
The amount of body binding area is 2-10 times that of CpG, and the amount of CpG is one ten thousandth of graphene oxide, mass ratio.
8. The preparation method of any one of claims 3-7, wherein the reaction temperature is 20-28
℃.
9. The application of the coronavirus according to claim 1, wherein the new coronavirus vaccine is used in the preparation of the prevention of coronavirus
Application of toxic drugs.
10. The application according to claim 9, wherein the coronavirus causes the body to produce receptor binding
Recombinant protein antibody.
Kwon Lee, want, ask, book 1/1 page
2
CN 112220919 A
2
Nano-Coronavirus Recombinant Vaccine Using Graphene Oxide as Carrier
Technical field
[0001] The present invention belongs to the field of nanomaterials and biomedicine, and relates to the development of a vaccine development platform. in particular,
It involves the development of 2019-nCoV coronavirus nuclear recombinant nano-vaccine. The invention also includes the application of the vaccine in animal experiments
use.
technical background
[0003] Vaccines are the ultimate weapon to eliminate major infectious diseases. Compared with other therapies, vaccines have the lowest cost and are more advanced.
The advantage of fighting the enemy has undoubtedly become the hope of the public. Through vaccination, mankind has eliminated smallpox and polio.
The number of cases has also been reduced by 99%. The incidence of infectious diseases such as diphtheria is rare, and the incidence of diseases such as measles and neonatal tetanus has dropped significantly. Epidemic
The impact of vaccines on human health cannot be overstated. Every new vaccine is born as a human being to overcome an infectious disease.
Great victory! So far, no medical measure can be as important and lasting as a vaccine for human health.
And far-reaching impact; there is no therapeutic drug that can eliminate a certain disease at an extremely low cost like a vaccine.
Wiped out on earth.
[0004] Soon after the SARS-CoV-2 epidemic occurred, different laboratories in China have completed the isolation of the virus strains.
Miao’s research and development has taken a big step forward. I believe we will soon have the ultimate weapon to eliminate SARS-CoV-2. However, until now
So far, there has not been an approved vaccine or drug for the treatment of CoV infection, so it is very urgent to develop an effective
Drug treatment or prevention of coronavirus infection and outbreak.
[0005] According to the research of coronavirus vaccines such as SARS and MERS, the main target of the coronavirus vaccine is currently
It is the S protein of coronavirus. Vaccines not only need to induce humoral and cellular immune responses, but also need to induce mucosal immune responses.
And with the help of adjuvants to induce a balanced Th1 and Th2 pathway, a truly effective vaccine can be produced. More SARS and
MERS vaccine research mainly focuses on viral vector vaccines and subunit vaccines. A large number of studies have shown the difficulty of SARS and MERS.
The point is that long-term memory B cells cannot be stimulated. In cured SARS and MERS patients, long-term memory cells can only hold
After 2-3 years, the immune memory cannot be generated, resulting in the failure of vaccine development. Up to now, only 6 potential coronavirus vaccines have been introduced.
Entering the clinical research stage, but no effective vaccine has been approved for marketing.
Summary of the invention
[0006] The purpose of the present invention is to provide a recombinant coronavirus vaccine.
[0007] Another object of the present invention is to provide a method for preparing the virus recombinant vaccine.
[0008] Another object of the present invention is to provide the application of the virus recombinant vaccine.
[0009] In view of the various problems existing in the current traditional vaccines, how to change the existing problems of the existing vaccines and enhance the immune response?
It should be a question we have been thinking about. In order to improve the immune activity of immunogens and strengthen the body’s immune response, the most basic
This method is to mix the immunogen with an adjuvant, which is a type of adjuvant that can strengthen the body’s immune response to the immunogen.
Agent. CpG oligodeoxynucleotide (oligodeoxynucleotide, ODN) is a very promising
Adjuvant. CpG ODN has been proved to have good adjuvant activity in vivo, in vitro and clinical studies in animals. The most fully studied is
CpG7909 and CpG1018. On November 9, 2017, the US FDA approved Dynavax Technologies to use CpG1018
The adjuvant hepatitis B vaccine is on the market. This vaccine is the world’s first approved CpG ODN adjuvant vaccine for the prevention of 18-year-old and
Speaking 　明　 book 1/5 page
3
CN 112220919 A
3
The above-mentioned adult HBV infection, and a number of different types of CpG ODN as adjuvants have been used in a number of clinical trials. CpG
By combining with TLR9, immature pDC cells are activated to induce natural and adaptive immune responses, but a single CpG node
The structure has limited activating effect on immune cells, and it is easily hydrolyzed by exonuclease, which makes its stability in the body insufficient.
Can cause side effects; CpG oligodeoxynucleotides (ODN) synthesized in the sequence can also enhance the stimulating effect and combine CpG with antigen
When combined with other proteins, it has a very significant immune activation effect.
[0010] Graphene is a two-dimensional carbon nanomaterial with a hexagonal honeycomb lattice composed of carbon atoms and sp² hybrid orbitals.
material. Its basic structural unit is the most stable benzene six-membered ring among organic materials, which is currently the most ideal two-dimensional material. oxidised graphite
Graphene oxide (GO), as a derivative of graphene, is the exfoliation of graphite oxide. Because of its uniqueness
SP2 hybrid and perfect two-dimensional structure and the characteristics of high reactivity at the edge make the design and development based on it
The treatment platform can be used as an ideal load and graft carrier for nano-drug delivery systems, biological detection, and tumor treatment.
And cell imaging plays an important role.
[0011] The present invention is completed on the basis of the above-mentioned research.
[0012] The present invention is based on graphene oxide materials as the backbone to load CpG molecules and recombinant proteins, and developed a new
Vaccine development method. Based on this technology platform, a recombinant protein combining the RBD region of the Spike protein of SAR-CoV-2 was prepared.
A new nano-coronal vaccine. The prepared nano-coronal vaccine has shown strong immunogenicity in mouse experiments and can produce
Produce high titer antibodies.
[0013] In one aspect, the present invention provides a coronavirus vaccine containing graphene oxide, carnosine, CpG, and RBD. In this
In a preferred embodiment of the invention, it is called GO-Car-carnosine-CpG-RBD vaccine.
[0014] Graphene oxide (GO, graphene oxide) is the oxide of graphene. After being oxidized, it contains oxygen functions.
The increase in clusters makes the properties more active than graphene. For example, randomly distribute hydroxyl and epoxy groups on a single graphene oxide sheet, and
Carboxyl and carbonyl groups are introduced at the edge of the monolith. Common graphene oxide commercially available products are powder, flake and solution
Shaped, brown-yellow in color.
[0015] Carnosine, the scientific name β-alanyl-L-histidine, is composed of two amino acid groups consisting of β-alanine and L-histidine
Dipeptide into a crystalline solid. Carnosine has a strong antioxidant capacity, which can remove the damage of cell membranes during oxidative stress.
Active oxygen free radicals (ROS) and α-β unsaturated aldehydes formed by excessive oxidation of fatty acids.
[0016] The CpG motif has the effect of activating the body's immune system and can be used as an adjuvant. Preferably, the coding of the CpG
The sequence is shown in SEQ ID NO 1.
[0017] RBD (spike receptor binding domain) is the receptor binding domain, and RBD in the present invention refers specifically to crown
The receptor binding domain (RBD) of the virus protein (S protein). For example, you can select an RBD protein with the following sequence:
PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFV
IRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGS
TPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAP (SEQ ID NO 2).
[0018] The coronavirus vaccine of the present invention combines carnosine, CpG and new coronavirus RBD on activated graphene oxide
And get.
[0019] In the coronavirus vaccine of the present invention, GO is used as the basis of the backbone, and the amount is usually excessive, and the amount of carnosine can be GO
About twice as much. CpG and new coronavirus RBD are biological macromolecules, and their dosage is small, and the dosage of both is usually ten thousand points of GO.
One, the quality ratio. The dosage of RBD is more than twice that of CpG, for example, CpG:RBD=1:2-10, preferably, the dosage of RBD is
3-6 times of CpG.
[0020] On the other hand, the present invention provides a preparation method of the coronavirus vaccine, and the preparation method includes
Speaking 　明　 Book 2/5 pages
4
CN 112220919 A
4
The following steps:
Obtain CpG, RBD recombinant protein and carnosine;
Add the GO freeze-dried powder to the phosphate buffer solution and ultrasonic treatment;
Add EDC and NHS to activate the GO solution, remove the excess EDC/sulfo-NHS in the reaction solution by ultrafiltration, and make the reaction
Adjust the pH of the solution to neutral;
Add carnosine, CpG and RBD recombinant proteins to the reaction solution and incubate with activated GO;
Remove excess uncoupled protein from the reaction solution, sterilize, and set aside.
[0021] Preferably, the duration of ultrasound is 2-3 hours. The ultrasonic conditions are 200 W, 40 kHz.
[0022] Preferably, the pH value of the phosphate buffer is neutral, for example, 6.8 to 7.6, more preferably 7.0 to 7.4, or
7.2.
[0023] Preferably, the method for removing excess EDC/sulfo-NHS or uncoupled protein is ultrafiltration.
[0024] In a preferred embodiment of the present invention, the ratio of graphene oxide, carnosine, CpG, and RBD is: 26mg:40mg:
1.2μg: (3-6)μg.
[0025] Preferably, the reaction temperature is 20-28°C. For example, use room temperature.
[0026] In a preferred embodiment of the present invention, the preparation method of GO-Car-carnosine-CpG-RBD vaccine is:
The improved method of the EDC-NHS reaction couples GO with carnosine and adds 26 mg of GO lyophilized powder to 5.20 mL of phosphate
Buffer (P

Source: https://patentimages.storage.googleapis.com/ef/0c/d4/2ed14dae3576f1/CN112220919A.pdf

Mirror:
https://web.archive.org/web/20210708093520/https://patentimages.storage.googleapis.com/ef/0c/d4/2ed14dae3576f1/CN112220919A.pdf