CN113248581A - Novel corona S antigen for generating neutralizing antibody of novel corona virus and preparation method thereof - Google Patents

Abstract

The invention discloses a novel corona S antigen for generating a novel corona virus neutralizing antibody and a preparation method thereof, wherein the novel corona S antigen is provided with a novel corona S antigen tripolymer combined with a cell membrane based on a Flag label, and the novel corona S antigen can generate a high-titer neutralizing antibody so as to more effectively immunize animals.

Description

Novel corona S antigen for generating neutralizing antibody of novel corona virus and preparation method thereof

Technical Field

The application relates to the technical field of antigen preparation, in particular to a novel corona S antigen for generating a novel corona virus neutralizing antibody and a preparation method thereof.

Background

The new coronavirus (2019-nCoV) can be widely spread among interpersonals, causes serious respiratory diseases and death of patients, has no specific medicine for treatment, and mainly depends on improving the immunity of the patients to resist the viruses for infectious diseases caused by the viruses.

The immunity can be divided into active immunity and passive immunity, the development of vaccines as the main treatment method of the active immunity needs a long time, exogenous antibodies as the passive immunity can be obtained by the following way, the fastest way is to extract from serum of a rehabilitee, the method obtains the most abundant types of antibodies aiming at the new coronavirus, and the antibodies show good clinical effects, but the method has the technical problems that firstly, the collection of a large amount of serum of the rehabilitee is difficult, particularly, under the condition that the number of patients is limited in the early stage of disease prevalence, secondly, the patients have individual difference, the antibody level difference is large, the standardization operation is difficult, and products derived from human blood have potential risks of other viruses.

The development and production of monoclonal antibodies against the new coronavirus can also be carried out using modern genetic engineering techniques, but this method has some technical difficulties and high costs, and requires a long time to be approved for marketing. The method for obtaining the heterologous antibody by immunizing the non-human animal with the antigen can quickly obtain the specific antibody, has important significance for the early epidemic stage and strategic reserve of diseases, has low cost and mature technical route, plays an important role in historically dealing with infectious diseases, and is still an important product in the current market for anti-tetanus, anti-rabies and anti-snake venom serum. The candidate animal, preferably a large animal such as horse, can obtain more serum once, also select the immune chicken to obtain the yolk antibody or the immune cow to obtain the milk containing the antibody, and for the antigen, the inactivated virus and the existing vaccine or recombinant antigen can be selected, the analysis of the effect that the inactivated virus is adopted as the antigen is the first choice because the antigen is rich in types and maintains the natural structure, but for the virulent infectious diseases such as the neocoronaviruses, the cultured virus is a great risk, the preparation of the recombinant antigen by adopting the genetic engineering mode is a good technical scheme, the results of the recent clinical vaccine experiments show that the inactivated vaccine has good protection rate, and the protection power of the protein antigen vaccine and the adenovirus vector vaccine is not good because the S antigen which is taken as the main antigen on the inactivated vaccine is a natural tripolymer structure, this structure is also important for the binding of the virus to the receptor ACE2, so maintaining its trimeric structure for the antigen is very important for the neutralizing effect of the produced antibody, so for recombinant antigens, even if high titers of antibodies against the S antigen are produced, it is not necessary to neutralize the true virus.

Therefore, how to provide a new corona S antigen for generating new corona virus neutralizing antibodies with high neutralizing activity, and thus more effectively immunize animals, is a technical problem to be solved at present.

Disclosure of Invention

The invention provides a novel corona S antigen for generating a novel corona virus neutralizing antibody, which is used for solving the technical problem that the antigen in the prior art cannot generate a novel corona virus neutralizing antibody with high neutralizing activity, and further cannot effectively immunize animals.

The novel crown S antigen is provided with a novel crown S antigen trimer combined with a cell membrane based on a Flag tag.

Accordingly, the present invention also proposes a process for the preparation of an immunogen comprising the novel corona S antigen as described above, said process comprising:

coding a transmembrane motif and a 3xFLAG tag of a full-length S protein based on a codon optimized cDNA, and obtaining a pseudovirus with a new crown S protein gene based on the transmembrane motif and the 3xFLAG tag;

infecting ID8 cells based on the pseudovirus and culturing up to a predetermined number of ID8 cells with S antigen;

based on the ID8 cells with the S antigen and an adjuvant, an immunogen containing the neo-corona S antigen was prepared.

Preferably, the pseudovirus carrying the new crown-S protein gene is obtained based on the transmembrane motif and the 3xFLAG tag, and specifically comprises:

cloning the transmembrane motif and the 3xFLAG tag into a derivative vector of a plasmid pRRL by using restriction enzymes XbaI or XmaI, constructing a plasmid pRRL-19S-FLAG-BSD, and obtaining a plasmid SARS-CoV-2S based on the plasmid pRRL-19S-FLAG-BSD;

transfecting 293FT cells by lentivirus prepared based on plasmid SARS-CoV-2S and a vector containing a packaging element, replacing a primary culture medium 12-16 hours after transfection, collecting pseudovirus LV-19S from supernatant, and filtering by a 0.45 mu m membrane for later use;

wherein the vector containing the packaging element comprises pLP-1-Gag/Pol, pLP-2-Rev, and pLP-VSVG.

Preferably, ID8 cells are infected based on the pseudovirus and up to a predetermined number of ID8 cells with S antigen are cultured, specifically:

inoculating ID8 cells into an initial culture medium containing DMEM +10% FBS at a density of 10000 cells/well, incubating overnight in a 6-well culture plate, adding 1ml LV-19S and a solvent control in the morning the next day, continuing to culture for 24 hours, then replacing the initial culture medium with a screening culture medium containing DMEM +10% FBS and 10 mug/ml blasticidin S HCl, replacing the screening medium once every 3 days until non-infected cells disappear on the 7 th day, continuing to culture infected cells, and storing a stable cell line in a DMEM +10% FBS complete culture medium containing 8 mug/ml blasticidin S HCl;

the ID8 cells with S antigen in complete medium were subcultured every 3 days, trypsinized when the total number of cells reached 3E +8, washed 5 times with 1xPBS, removed of pancreatin and FBS residues, resuspended in 20ml PBS, and frozen to-80 ℃ in a refrigerator for future use.

Preferably, after the stable cell line is preserved in DMEM +10% FBS complete medium containing 8 μ g/ml blasticidin S HCl, the preparation method further comprises:

ID8 cells bearing the S antigen were stained with anti-FLAG-FITC or human ACE2-mFc followed by anti-mouse IgG-FITC secondary antibody and FACS analysis to verify S protein expression, structure and function.

Preferably, based on the ID8 cells with S antigen and an adjuvant, an immunogen containing the neo-corona S antigen is prepared, specifically:

the S antigen bearing ID8 cells were subjected to cell disruption and mixed with adjuvant at volume 1:1 preparing the immunogen.

Preferably, the ID8 cells carrying the S antigen are disrupted on the basis of repeated freezing and thawing or sonication or high-pressure homogenization.

Preferably, the adjuvant comprises paraffin oil and lanolin.

Accordingly, the present invention also provides a process for the preparation of novel coronavirus neutralizing antibodies, said neutralizing antibodies being generated based on an immunogen as defined above, said process comprising:

completing immunization of non-human animal sources according to a preset immunization program based on the immunogen, and collecting and obtaining plasma of the non-human animal sources;

obtaining the neutralizing antibodies based on the plasma of non-human animal origin.

Preferably, the non-human animal source is a female horse aged 4-5 years old, the immunization of the non-human animal source is completed according to a preset immunization program based on the immunogen, and the plasma of the non-human animal source is collected and obtained, specifically:

adopt 1ml to the horse for the first time after 1 month's quarantine the immunogen carries out the immunity, adopt 2ml after 10 days the immunogen carries out the immunity for the second time, adopt 3ml after 10 days again the immunogen carries out the immunity for the third time, adopt 4ml after 8 days again the immunogen carries out the immunity for the fourth time, accomplish basic immunity, then adopt 4ml after 41 days rest the immunogen carries out the immunity, adopt 5ml after 7 days the immunogen carries out the immunity, adopt 6ml after 7 days again the immunogen carries out the immunity, adopt 8ml after 7 days again the immunogen carries out the immunity, adopt 10ml after 7 days again the immunogen carries out the immunity, accomplish the hyperimmunization procedure, gather horse blood plasma after 36 days again, add 0.2% m-cresol as the preservative, put 4-8 ℃ freezer for later use.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts pseudovirus to infect ID8 cells in the process of simulating virus budding, forms a natural tripolymer structure of the new coronavirus on the cell surface, enables the new corona S protein to maintain a natural structure, simultaneously, the broken ID8 cells can be used as an adjuvant to play a role in enhancing the immune effect, and the immune animal adopting the antigen can enable the animal to generate an antibody with high neutralizing activity, thereby more effectively immunizing the animal.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 shows a schematic representation of the expression of the novel crown S antigen trimer on ID8 cells according to an embodiment of the present invention;

FIG. 2 is a graph showing the results of detecting S protein expressed on ID8 cells using flow cytometry in accordance with an embodiment of the present invention;

FIG. 3 shows a graph of the activity of neutralizing antibodies determined using pseudoviruses in an embodiment of the invention.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The implementation of the invention comprises the acquisition of pseudovirus with a new crown S protein gene, the expression of S protein, immunogen preparation, animal immunization, serum treatment, antibody detection, non-human animal sources including but not limited to horses, pigs, chickens, cattle, sheep, alpacas, rabbits and the like, preferably, healthy horses about 4 years old are selected in animal selection and quarantine is carried out.

Example 1

Obtaining pseudovirus with new crown S protein gene, firstly constructing a codon optimized cDNA (GenBank: NC-045512.2), encoding Transmembrane Motif (TM) and 3xFLAG tag of full-length S protein based on the cDNA (shown in figure 1), cloning into plasmid pRRL derivative vector (R48) by restriction enzyme XbaI or XmaI to obtain constructed plasmid pRRL-19S-FLAG-BSD, and obtaining plasmid SARS-CoV-2S based on the plasmid pRRL-19S-FLAG-BSD. Lentiviruses prepared from the plasmid SARS-CoV-2 and other vectors containing packaging elements (pLP-1-Gag/Pol, pLP-2-Rev and pLP-VSVG) were transfected into 293FT cells. The medium was changed 12-16 hours after transfection. A false virus stock (designated LV-19S) was collected from the supernatant and was filtered through a 0.45 μm membrane for future use.

Example 2

Expression of S protein to establish a cell line stably expressing Covid-19S, ID8 cells (donated by university of Fujian medical science, immunotherapy research institute) were seeded at a density of 10000 cells/well in a starting medium containing DMEM (Gibco) +10% FBS (GeminiBio) and incubated overnight in 6-well culture plates. The next morning, 1ml LV-19S (and vehicle control) was added and incubation was continued for 24 hours before replacing the initial medium with selection medium (DMEM +10% FBS containing 10 μ g/ml blasticidin S HCl (Invitrogen)). The selection medium was changed every 3 days until all non-infected cells disappeared by day 7. The culture of infected cells, i.e., stable cell lines, was continued in DMEM +10% FBS complete medium containing 8 μ g/ml blasticidin S HCl. To verify the expression, structure and function of the spinous process proteins, ID8 cells were stained with anti-FLAG-FITC (left panel in fig. 2) or human ACE2-mFc, then with anti-mouse IgG-FITC secondary antibody (right panel in fig. 2), and analyzed by FACS (Fluorescence activated Cell Sorting). The results show that both anti-FLAG-FITC and anti-mouse IgG-FITC secondary stains show a significant right shift in the ID8 population (right peak on left and right panels in fig. 2), validating the ability of ID8 to bind ACE2, indicating that the spike is in a trimeric state, similar to the function of Covid-19 peak.

Example 3

Immunogen preparation, ID8 cells bearing the S antigen (ID 8/19-nCovS) were cultured in DMEM +10% FBS complete medium and the cells were sub-expanded every 3 days. Trypsinization was performed in multiple dishes to a total of 3E +8, 1xPBS washed 5 times, pancreatin and FBS residues removed, cells resuspended in 20ml PBS, and frozen to-80 ℃ in a freezer for use.

The cells of ID8 with S antigen were repeatedly frozen and thawed three times, and then mixed with an adjuvant consisting of liquid paraffin and lanolin in a volume of 1:1, preparation. Thus, the immunogen comprises neocorona S antigen, host cell components, paraffin oil and lanolin.

Example 4

Immunizing animals, selecting female horses of 4-5 years old, immunizing according to the following immunization program after quarantine for 1 month, immunizing by 1ml of immunogen for the first time, immunizing by 2ml of immunogen for the second time after 10 days, immunizing by 3ml of immunogen for the third time after 10 days, immunizing by 4ml of immunogen for the fourth time after 8 days, completing basic immunization, performing hyperimmunization program by the following process after rest for 41 days, immunizing by 4ml of immunogen for the first time, immunizing by 5ml of immunogen after 7 days, immunizing by 6ml of immunogen after 7 days, immunizing by 8ml of immunogen after 7 days, immunizing by 10ml of immunogen after 7 days, completing the hyperimmunization program, collecting horse blood plasma after 36 days, adding 0.2% of m-cresol as a preservative, storing in a refrigerator at 4-8 deg.C for use.

The neutralizing antibody can be obtained based on collected blood plasma, and can be used as a raw material of a medicine or a diagnostic reagent after being processed and purified.

Example 5

The titer and the neutralizing activity are measured, the antibody titer of the collected plasma is measured by ELISA to be 1:16000, the neutralizing titer of the collected plasma is measured by virus neutralization test to be 1:512, the neutralizing antibody titer is measured to be about 1:5000 by a pseudovirus measuring method, and the activity graph of the neutralizing antibody measured by the pseudovirus in the embodiment of the invention is shown in figure 3.

By applying the technical scheme, the ID8 cells are infected by adopting the pseudovirus in the process of simulating virus budding, a natural tripolymer structure of the new corona virus is formed on the cell surface, the new corona S protein maintains a natural structure, meanwhile, the broken ID8 cells can be used as an adjuvant to play a role in enhancing the immune effect, the impure antigen is widely used as a vaccine in animal vaccines, for example, a porcine circovirus vaccine of a Germany company BI adopts the mode, the horse is immunized by adopting the antigen, the horse can generate an antibody with high neutralizing activity, and the titer of the neutralizing antibody is far higher than the level of the neutralizing antibody (generally more than 100) in a convalescent patient.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit the same; although the present application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not necessarily depart from the spirit and scope of the corresponding technical solutions in the embodiments of the present application.

Claims (10)

1. Novel corona S antigen for generating neutralizing antibodies against novel corona viruses, characterized in that said novel corona S antigen is provided with a trimer of novel corona S antigen bound to the cell membrane based on a Flag tag.

2. A method for the preparation of an immunogen comprising the neocorona S antigen of claim 1, comprising:

coding a transmembrane motif and a 3xFLAG tag of a full-length S protein based on a codon optimized cDNA, and obtaining a pseudovirus with a new crown S protein gene based on the transmembrane motif and the 3xFLAG tag;

infecting ID8 cells based on the pseudovirus and culturing up to a predetermined number of ID8 cells with S antigen;

based on the ID8 cells with the S antigen and an adjuvant, an immunogen containing the neo-corona S antigen was prepared.

3. The method of claim 2, wherein obtaining a pseudovirus carrying a novel crown-S protein gene based on the transmembrane motif and the 3xFLAG tag comprises:

cloning the transmembrane motif and the 3xFLAG tag into a derivative vector of a plasmid pRRL by using restriction enzymes XbaI or XmaI, constructing a plasmid pRRL-19S-FLAG-BSD, and obtaining a plasmid SARS-CoV-2S based on the plasmid pRRL-19S-FLAG-BSD;

transfecting 293FT cells by lentivirus prepared based on plasmid SARS-CoV-2S and a vector containing a packaging element, replacing a primary culture medium 12-16 hours after transfection, collecting pseudovirus LV-19S from supernatant, and filtering by a 0.45 mu m membrane for later use;

wherein the vector containing the packaging element comprises pLP-1-Gag/Pol, pLP-2-Rev, and pLP-VSVG.

4. The method according to claim 3, wherein ID8 cells are infected based on the pseudovirus and up to a predetermined number of ID8 cells with S antigen are cultured, specifically:

inoculating ID8 cells into an initial culture medium containing DMEM +10% FBS at a density of 10000 cells/well, incubating overnight in a 6-well culture plate, adding 1ml LV-19S and a solvent control in the morning the next day, continuing to culture for 24 hours, then replacing the initial culture medium with a screening culture medium containing DMEM +10% FBS and 10 mug/ml blasticidin S HCl, replacing the screening medium once every 3 days until non-infected cells disappear on the 7 th day, continuing to culture infected cells, and storing a stable cell line in a DMEM +10% FBS complete culture medium containing 8 mug/ml blasticidin S HCl;

the ID8 cells with S antigen in complete medium were subcultured every 3 days, trypsinized when the total number of cells reached 3E +8, washed 5 times with 1xPBS, removed of pancreatin and FBS residues, resuspended in 20ml PBS, and frozen to-80 ℃ in a refrigerator for future use.

5. The preparation method of claim 4, further comprising, after storing the stable cell line in DMEM +10% FBS complete medium containing 8 μ g/ml blasticidin S HCl:

ID8 cells bearing the S antigen were stained with anti-FLAG-FITC or human ACE2-mFc followed by anti-mouse IgG-FITC secondary antibody and FACS analysis to verify S protein expression, structure and function.

6. The method of claim 4, wherein the immunogen comprising the neocorona S antigen is prepared based on the ID8 cells with the S antigen and an adjuvant, and in particular:

the S antigen bearing ID8 cells were subjected to cell disruption and mixed with adjuvant at volume 1:1 preparing the immunogen.

7. The method of claim 6, wherein the cells of ID8 harboring the S antigen are disrupted by repeated freezing and thawing, or by ultrasonication, or by high-pressure homogenization.

8. The method of claim 6, wherein the adjuvant comprises paraffin oil and lanolin.

9. A method for producing neutralizing antibodies against a novel coronavirus, said neutralizing antibodies being produced based on the immunogen of claim 2, said method comprising:

completing immunization of non-human animal sources according to a preset immunization program based on the immunogen, and collecting and obtaining plasma of the non-human animal sources;

obtaining the neutralizing antibodies based on the plasma of non-human animal origin.

10. The method according to claim 9, wherein the non-human animal source is a female horse aged 4-5 years old, the immunization of the non-human animal source is completed according to a preset immunization program based on the immunogen, and the plasma of the non-human animal source is collected and obtained, specifically:

adopt 1ml to the horse for the first time after 1 month's quarantine the immunogen carries out the immunity, adopt 2ml after 10 days the immunogen carries out the immunity for the second time, adopt 3ml after 10 days again the immunogen carries out the immunity for the third time, adopt 4ml after 8 days again the immunogen carries out the immunity for the fourth time, accomplish basic immunity, then adopt 4ml after 41 days rest the immunogen carries out the immunity, adopt 5ml after 7 days the immunogen carries out the immunity, adopt 6ml after 7 days again the immunogen carries out the immunity, adopt 8ml after 7 days again the immunogen carries out the immunity, adopt 10ml after 7 days again the immunogen carries out the immunity, accomplish the hyperimmunization procedure, gather horse blood plasma after 36 days again, add 0.2% m-cresol as the preservative, put 4-8 ℃ freezer for later use.

Images