CN106868025B

CN106868025B - Method for preparing tripolymer Ebola virus glycoprotein mutant by using yeast

Info

Publication number: CN106868025B
Application number: CN201710145936.7A
Authority: CN
Inventors: 吴军; 吴慕胜; 刘波
Original assignee: Institute of Bioengineering Chinese Academy of Military Medical Sciences
Current assignee: Institute of Bioengineering Chinese Academy of Military Medical Sciences
Priority date: 2017-03-13
Filing date: 2017-03-13
Publication date: 2020-01-21
Anticipated expiration: 2037-03-13
Also published as: CN106868025A

Abstract

The invention discloses a method for preparing a tripolymer Ebola virus glycoprotein mutant by using yeast. The method comprises the following steps: 1) introducing a gene encoding an ebola virus glycoprotein mutant (a borra virus glycoprotein with a mucin-like region and a C-terminal transmembrane region deleted) into a recipient yeast cell to obtain a recombinant yeast cell; 2) and culturing and crushing the recombinant yeast cells in sequence to obtain the boragana virus glycoprotein mutant from the crushed product. The vaccine for preventing the ebola hemorrhagic fever prepared by the ebola virus glycoprotein mutant prepared by the invention can induce a mouse to generate obvious antibody titer aiming at the ebola virus glycoprotein. The invention has the typical characteristics of engineering strain construction, short culture period, simple culture conditions, low cost, suitability for large-scale fermentation, safety, non-toxin production and capability of performing modification processing on the proteins after translation, and is suitable for efficient research and development and large-scale production of vaccines under emergency conditions such as epidemic situations.

Description

Method for preparing tripolymer Ebola virus glycoprotein mutant by using yeast

Technical Field

The invention belongs to the technical field of biology, and relates to a method for preparing an Ebola virus glycoprotein mutant with a tripolymer structure by using saccharomycetes.

Background

The envelope Glycoprotein (GP) of the Ebola virus is the only surface protein on the surface of the Ebola virus, exists in a form of tripolymer as a structural unit, plays an important role in the virus invasion process and is also the main action target of a neutralizing antibody, thereby being the main component of the Ebola hemorrhagic fever prevention vaccine.

Vaccination is the simplest and most effective means of prevention and treatment, plays an important role in the prevention of viral epidemics, and in the development of ebola vaccines, the existing research contents are roughly divided into the following categories: the first category includes inactivated vaccines, reverse genetically modified vaccines and DNA vaccines, which have proven to be unsuitable as the vaccine type of ebola vaccine in current studies on the immunogenicity, safety and efficacy of such vaccines; the second type of vaccine is called as vector vaccine, the vaccine is a vaccine form which is developed by inserting partial antigen genes of Ebola virus into Adenovirus (Adenoviral) vectors, Vesicular Stomatitis Virus (VSV) vectors, vaccinia virus vectors, human 3-type parainfluenza virus vectors, Newcastle disease virus vectors, rabies virus vectors and the like, the vaccine is proved to have good protection and good safety, but the vaccine as the vector vaccine has the defects of high requirements on pre-existing immunity and storage and transportation of the vaccine; the third type is protein vaccines, which are vaccines with viral protein components as main components as the name implies, and virus-like particle Vaccines (VLPs) and subunit vaccines are mainly studied. Research shows that GP protein and VP40 protein are co-expressed in mammalian cells or baculovirus expression systems, or GP protein, VP40 protein and NP protein can be self-assembled to form virus-like particles, 100% protection can be achieved for cynomolgus monkeys after challenge after immunization by VLPs, and obvious humoral and cellular immune responses are generated by vaccines induced animals. Subunit vaccines prepared with GP fused to human IgG1Fc fragment recombinant protein as the protective antigen have also been shown to confer one hundred percent protection to mice and guinea pigs.

In a natural state, GP protein is embedded on a virus membrane by taking a trimer as a structural unit, and the maintenance of the trimer plays an important role in maintaining the function and the immunogenicity of the EBOV-GP protein. The GP protein is a highly glycosylated protein, and two highly glycosylated regions, namely a Glycan Cap (Glycan Cap) and a mucin-like domain (MLD), exist on GP 1. During the virus invasion process, two highly glycosylated sugar caps of Glycancap and mucin like domain are cut off by cathepsin B/L cleavage, so that RBSs are combined with NCP1 receptors on host cells, fusion loop on GP2 is stimulated to carry out allosteric transformation, and the virus invasion is mediated.

Studies have shown that the glycosylation modification region of EBOV-GP protein is also closely associated with viral adsorption, protein folding, and maintenance of spatial conformation, and often involved in epitope formation, and for example, N-glycosylation modification at N563, after mutation of N-glycosylation modification site at N563, binding to neutralizing antibody KZ52 can be completely released. The glycosylation modification of Ebola envelope protein GP is the same as that of general glycosylation modified protein, the glycosylation modification mainly comprises two types of N-linked glycosylation modification and O-linked glycosylation modification, the glycosylation modification of GP protein is almost totally concentrated in two highly glycosylated regions of Glycan Cap and mucin like domain, the glycosylation modification of Glycan Cap is totally N-linked glycosylation modification, while the mucin like domain contains a large number of O-linked glycosylation modification sites besides a plurality of N-glycosylation sites, and the molecular weight and the space volume size of the mucin like domain region after the complete glycosylation modification are both equivalent to those of GP protein lacking the mucin like domain region. Osvaldo Martinez et al found that The GP protein with The excision of The mucin Like Domain Induced The mice to produce significantly different Antibody titers than The full length GP protein, but The titers of The neutralizing antibodies in The serum were comparable, indicating that The mice produced mostly non-neutralizing antibodies against mucin Like Domain regions (Osvaldo M, Lee T, Nirupa M, et al, impact of Ebola Mucin-Like Domain on neutralizing antibodies Induced by antibodies in mice [ J ] The Journal of Infectious Diseases,2011,204: S825-S832).

The characteristic feature of glycoprotein is that it will produce glycosylation modification during the expression process, and the glycosylation modification of protein, especially the more complex glycosylation modification, is carried out in eukaryotic cells, so when selecting expression system, eukaryotic expression system should be selected for expression. The existing research on protein vaccines based on the GP protein of Ebola virus aiming at the vaccines for preventing the hemorrhagic fever of Ebola is carried out in a cell expression system or a prokaryotic expression system. Pichia pastoris, one of the simplest and most commonly used eukaryotic expression systems, has the typical advantages of short culture period, simple culture conditions, low cost, suitability for large-scale fermentation, safety, non-toxin production and capability of performing post-translational modification processing of proteins, but the secretion efficiency of the Pichia pastoris is lower than that of a cell platform particularly for proteins with larger molecular weights, and the Pichia pastoris has very thick and firm cell walls and has higher difficulty in purifying intracellular proteins than that of the cell platform. At present, no report on the preparation of Ebola virus glycoprotein with glycosylation modification and trimer structure by using yeast is found.

Disclosure of Invention

The invention aims to provide a method for preparing an Ebola virus tripolymer glycoprotein mutant which has a mature peptide structure and a tripolymer structure and is provided with glycosylation modification. The ebola virus glycoprotein provided by the invention is envelope Glycoprotein (GP) of the ebola virus.

The method for preparing the Ebola virus glycoprotein mutant provided by the invention can comprise the following steps:

(1) introducing a gene encoding an Ebola virus glycoprotein mutant into a receptor yeast cell to obtain a recombinant yeast cell expressing the gene;

(2) sequentially culturing and crushing the recombinant yeast cells, and obtaining the boragana virus glycoprotein mutant from a crushed product;

the mutant of the Bora virus glycoprotein is the Bora virus glycoprotein which is deleted with Mucin Like Domain (MLD) and C-terminal transmembrane region (TM).

The method for obtaining the gene for coding the mutant of the boragana virus glycoprotein can be a whole gene synthesis method, a PCR fusion method or a fragment fusion method after fragment deletion.

Wherein the yeast can be Pichia pastoris, Saccharomyces cerevisiae, Hansenula or Kluyveromyces lactis. In one embodiment of the invention, the yeast is specifically pichia pastoris GS 115.

Wherein the Bora virus may be Sudan Ebola virus (Sudan Ebola virus), Zaire Ebola virus (Zaire Ebola virus), CotedDewa Ebola virus (coded' Ivore Ebola virus), Bundbury Ebola virus (Bundbugyo Ebola virus), or Reston Ebola virus (Reston Ebola virus).

Of course, it is within the scope of the invention to use the methods of the present invention for Marburg virus (Marburgvirus) which is closely related to Ebola virus.

In the step (2), the culture is carried out, and the step of adding 0.5% by volume of methanol into the culture system is further included, so as to induce the expression of the blera virus glycoprotein mutant. Methanol was added every 12 hours for up to 72 hours.

In step (2), the method for performing the disruption may be a physical method, a biological method or a chemical method.

Wherein the physical method can be a high-pressure homogenizing method, a glass bead oscillation method or a ball milling method; the biological method can be specifically an enzyme digestion cracking method; the chemical process may specifically be an alkaline lysis process.

In step (2), the step of adding a detergent after the disruption is performed to obtain a crude extract containing the ebola virus glycoprotein mutants.

Wherein the detergent may be a chaotropic agent, a non-ionic detergent, a weakly ionic detergent or a zwitterionic detergent. The chaotropic agent can be urea or thiourea; the non-ionic detergent can be specifically Triton, Tween or ethylphenyl polyethylene glycol; the weak ionic detergent can be deoxycholate specifically; the zwitterionic detergent can be 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt or 3-1-alkane sulfonic acid.

In one embodiment of the invention, the following substances are added to the crushed system in the final concentration: 8M urea, 50mM pH8.0 phosphate buffer, 500mM NaCl, 10mM imidazole and 5% (volume percentage) glycerol.

In the step (2), the method further comprises a step of purifying the crude extract.

In the invention, the purification specifically comprises sequentially carrying out affinity chromatography, gel exclusion chromatography and ion exchange chromatography on the crude extract.

Wherein, the affinity chromatography medium can be specifically chemical Fast Flow or Ni-NTA; the gel exclusion chromatography medium can be Sephadex G25, Superdex200 or Superose6 gel pre-packed column; the ion exchange chromatography may be cation exchange chromatography or anion exchange chromatography; the cation exchange chromatography medium can be SOURCE30S, Sepharose Fast Flow SP or CM Fast Flow; the anion exchange chromatography medium can be SOURCE30Q Fast Flow.

In one embodiment of the invention, the affinity chromatography is performed using a medium, specifically a Chelatingfast Flow; the composition of the column equilibrium liquid (liquid A) used was as follows: 6M urea, 500mM NaCl, 50mM phosphate buffer solution with pH7.5, 10mM imidazole and 5% (volume percentage content) glycerol, and the balance of water; the eluent (solution B) used had the following composition: : 6M urea, 500mM NaCl, 50mM phosphate buffer pH7.5, 500mM imidazole and 5% (volume percentage content) glycerol, the balance being water. The elution gradient used was: (1) 5% of the liquid B and 95% of the liquid A, (2) 50% of the liquid B and 50% of the liquid A, and (3) 100% of the liquid B, wherein the% represents the volume percentage. The gradient elution of the target protein (Ebola virus glycoprotein mutant) is 50% B (namely 50% of the solution B and 50% of the solution A).

In one embodiment of the invention, the gel exclusion chromatography is performed using a medium specifically Sephadex G25; the mobile phase composition used was as follows: 20mM phosphate buffer pH6.2, 6M urea, 5% (volume percentage) glycerol, and the balance water.

In one embodiment of the invention, the ion exchange chromatography carried out is in particular cation exchange chromatography, the medium employed is in particular SOURCE 30S; the composition of the equilibrium liquid (liquid A) used was as follows: 20mM phosphate buffer solution with pH6.2, 6M urea, 5% (volume percentage content) glycerol and the balance of water; the eluent (solution B) used had the following composition: 20mM pH6.2 phosphate buffer, 6M urea, 5% (volume percentage content) glycerol, and 1M NaCl, the balance is water. The elution gradient used was: (1) 5% of the liquid B and 95% of the liquid A, (2) 15% of the liquid B and 85% of the liquid A, (3) 30% of the liquid B and 70% of the liquid A, (4) 50% of the liquid B and 50% of the liquid A, (5) 100% of the liquid B, (6) 100% of 0.5M NaOH,% representing volume percentage content. The gradient eluent in which the target protein (Ebola virus glycoprotein mutant) is mainly located is 15% B eluent (namely 15% of the solution B and 85% of the solution A).

In step (1), the gene encoding the ebola virus glycoprotein mutant can be specifically a DNA molecule as shown in any one of (b1) to (b 6):

(b1) DNA molecules shown in 29 th-1468 th site of a sequence 2 in a sequence table;

(b2) DNA molecules shown in 1 st-1468 th site of a sequence 2 in a sequence table;

(b3) DNA molecules shown at 29 th to 1507 th sites of a sequence 2 in a sequence table;

(b4) DNA molecules shown in 1 st-1507 th sites of a sequence 2 in a sequence table;

(b5) a DNA molecule that hybridizes under stringent conditions to a DNA molecule defined in any one of (b1) - (b4) and encodes said ebola virus glycoprotein mutant;

(b6) a DNA molecule having more than 90% homology with the DNA sequence defined in any one of (b1) - (b5) and encoding the Ebola virus glycoprotein mutant.

The full length of the sequence 2 is 1507 bp. Wherein, the CDS region is located at the 29-1507 th site, the coding sequence of the Ebola virus glycoprotein mutant is located at the 29-1486 th site, the coding sequence of five amino acids G added artificially is located at the 1469-1483 th site, the recognition sequence of the restriction enzyme SalI added artificially is located at the 1484-1489 th site, and the coding sequence of the 6His tag added artificially is located at the 1490-1507 th site.

In the invention, the gene shown in the sequence 2 is a gene subjected to codon optimization according to pichia pastoris preferred codons.

The Ebola virus glycoprotein mutant prepared by the method also belongs to the protection scope of the invention.

The Ebola virus glycoprotein mutant prepared by the method has a mature peptide structure and a tripolymer structure and has glycosylation modification.

The molecular weight of the Ebola virus glycoprotein mutant is more than 150 KD.

Further, the amino acid sequence of the ebola virus glycoprotein mutant prepared in the invention is specifically any one of the following (a1) or (a 2):

(a1) 33 th to 480 th bit of a sequence 3 in a sequence table;

(a2) 33 to 493 of the sequence 3 in the sequence table.

Wherein, the 1 st to 32 th positions of the sequence 3 are signal peptides; positions 33-480 are mature peptide sequences of the Ebola virus glycoprotein mutants; the 481-485 position is five amino acids G which are added manually; the 486-487 site is an amino acid coded by a recognition sequence of an added restriction enzyme SalI; position 488-493 was an added 6His tag.

The following (A) or (B) are also included in the scope of the present invention:

(A) the gene for coding the Ebola virus glycoprotein mutant and the application of yeast in preparing the Ebola virus glycoprotein mutant;

(B) The application of the ebola virus glycoprotein mutant in preparing the vaccine for preventing ebola hemorrhagic fever is provided.

The invention also provides a vaccine for preventing ebola hemorrhagic fever.

The active component of the vaccine for preventing ebola hemorrhagic fever provided by the invention is the ebola virus glycoprotein mutant.

The vaccine may or may not contain an adjuvant.

The adjuvant can be Freund's adjuvant or aluminum adjuvant, such as Al (OH)₃. The proportion of Freund's adjuvant in the vaccine can be 50%; the aluminum adjuvant (such as Al (OH)₃) The proportion in the vaccine may be 10%. Wherein,% represents volume percentage.

The invention also protects any one of the following biomaterials:

A) a protein, the amino acid sequence of which is any one of (a1) - (a5) as follows:

(a1) 33 th to 480 th bit of a sequence 3 in a sequence table;

(a2) 33 th to 493 th in a sequence 3 in a sequence table;

(a3) 1-480 th site of sequence 3 in the sequence table;

(a4) 1-493 of sequence 3 in the sequence table;

(a5) and (b) a sequence which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence defined in any one of (a1) - (a4) and has the same function.

B) A gene encoding the protein of A);

C) recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the genes described in II).

Further, the gene is a DNA molecule shown in any one of (b1) to (b6) below:

(b6) a DNA molecule having more than 90% homology with the DNA sequence defined in any one of (b1) - (b5) and encoding said mutant Ebola virus glycoprotein;

the application of the biological material in preparing the vaccine for preventing the ebola hemorrhagic fever also belongs to the protection scope of the invention.

Experiments prove that the vaccine for preventing the ebola hemorrhagic fever prepared by the ebola virus glycoprotein mutant prepared by the invention can induce a mouse to generate obvious antibody titer aiming at the GP glycoprotein of the ebola virus. The vaccine for preventing the ebola hemorrhagic fever prepared by the method has the typical characteristics of short construction period of engineering strains, short culture period, simple culture conditions, low cost, suitability for large-scale fermentation, safety, non-toxin production and capability of protein post-translational modification processing, and is very suitable for efficient research and development and large-scale production of the vaccine under emergency conditions such as epidemic situations and the like.

Drawings

FIG. 1 shows the acquisition of EBOV-GP. DELTA. MLD. DELTA.

TM gene fragments

1 and 2.

FIG. 2 is a chromatogram of Chelating FF Ni affinity chromatography crude purification EBOV-GP delta MLD delta TM protein

FIG. 3 is a graph showing the identification of Chelating FF Ni affinity chromatography crude purified EBOV-GP. DELTA. MLD. DELTA. TM protein. The left image is a Coomassie brilliant blue staining image of SDS-PAGE, and the right image is a Western blot result image. The arrow indicates the target protein.

FIG. 4 is a Sephadex G25 desalt chromatogram of a 50% B eluate from a Chelating FF Ni affinity chromatography.

FIG. 5 is a chromatogram of SOURCE30S cation exchange chromatography fine purification of EBOV-GP Δ MLD Δ TM protein.

FIG. 6 is SDS-PAGE Coomassie brilliant blue staining pattern of finely purified EBOV-GP Δ MLD Δ TM protein by cation exchange chromatography of SOURCE 30S.

FIG. 7 shows the EBOV-GP Δ MLD Δ TM protein PNGaseF cleavage analysis. The left image is a Coomassie brilliant blue staining image of SDS-PAGE, and the right image is a Western blot result image.

FIG. 8 shows the N-terminal sequencing analysis of the EBOV-GP Δ MLD Δ TM recombinant protein. A. An EBOV-GP delta MLD delta TM protein N-terminal sequencing result graph; B. 1-50aa encoded by the EBOV-GP Δ MLD Δ TM gene.

FIG. 9 shows the EBOV-GP. DELTA. MLD. DELTA. TM protein Superdex200 gel column separation. A. Western blot AntiGP; B. SDS-PAGE Coomassie blue staining.

FIG. 10 is a graph showing the effect of Superose6 gel separation. A. 669KD protein standard; B. a 440KD protein standard substance; C. human IgG (150 KD); D. BSA (67 KD); E. EBOV-GP Δ MLD Δ TM.

FIG. 11 is a lg Mr standard curve.

FIG. 12 shows the protein electrophoresis and blot analysis of Superose6 gel separation samples. A. Superose6 gel separation chromatogram; B. SDS-PAGE Coomassie blue staining; C. western blot Anti GP; D. western blot Anti HumanIgG. 1. 12-13 mL; 2. 13-14 mL; 3. 14-15 mL; 4. 15-16 mL; 5. 16-17 mL; 6. 17-18 mL; 7. 18-20 mL.

FIG. 13 shows EBOV-GP Δ MLD Δ TM protein-immunization antibody titers. P <0.05, P < 0.001. Freund's adjuvant: a Freund's adjuvant group; al (OH)₃: an aluminum hydroxide group; BLANK: and (4) a negative control group.

FIG. 14 shows EBOV-GP Δ MLD Δ TM protein diabody titers. P <0.05, P < 0.001. Freund's adjuvant: a Freund's adjuvant group; al (OH)₃: an aluminum hydroxide group; BLANK: and (4) a negative control group.

FIG. 15 shows EBOV-GP Δ MLD Δ TM protein triabodies titers. P <0.05, P < 0.001. Freund's adjuvant: a Freund's adjuvant group; al (OH)₃: an aluminum hydroxide group; BLANK: and (4) a negative control group.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The pPICZ-alpha A plasmid is a product of Invitrogen company; pichia pastoris GS115 is a product of Invitrogen corporation; EBOV-GP antibodys, Rabbit PAb is a product of Beijing Yi Qiao Shenzhou Biotechnology Limited; the Zaire type EBOV-GP protein expressed by the Human cells is a product of Beijing Yi Qian Shenzhou biotechnology limited; the Goat Anti Rabbit IgG (H + L) X-Adsorbed-HRP is a product of Sigma company; the Goat Anti Mouse IgG (H + L) X-Adsorbed-HRP is a product of Sigma company; the Goat pAb to Human IgG (H + L) HRP is a product of abcam company; restriction enzymes such as BstBI, BglII, BamHI, SalI, NotI and the like are products of NEB company; q5 hot start high fidelity DNA polymerase is a product of NEB company; T4-DNA ligase is a product of NEB company; alkaline phosphatase CIAP is a product of NEB company; the Ifusion connecting kit is a product of Clonetech company; PNGaseF peptide N-endoglycosidase F is a product of NEB corporation.

Example 1 construction of Ebola Virus glycoprotein mutant expression vector

First, acquisition of Ebola virus glycoprotein gene

By means of artificial synthesis, an EBOV-GP protein full-length gene (> KM034549| Zaireebola virus isolates Hsapiens-wt/SLE/2014/Manoriver-EM095B | Homo sapiens |01-Jun-2014) is synthesized, codon optimization is carried out according to pichia pastoris preferred codons, meanwhile, in order to synthesize the EBOV-GP gene to obtain the gene capable of expressing the full-length GP protein, an 'A' is artificially added at the position where RNA editing occurs, and the synthesis work is entrusted to Nanjing Jinruis Biotech limited company for synthesis, and the sequence is detailed as sequence 1 in a sequence table.

Second, construction of expression vector of GP glycoprotein mutant of Ebola virus

1. Design and synthesis of primers:

2. acquisition of EBOV-GP Delta MLD Delta TM Gene fragments 1 and 2

EBOV-GP. DELTA. MLD. DELTA. TM. gene fragment 1 was amplified by PCR using Q5 hot-start ultra-fidelity DNA polymerase with the plasmid returned from Nanjing Kinshire as a template (of course, sequence 1 may be used as a template) and GP-infu 5 and GP. DELTA. MLD-infu medium R as upstream and downstream primers. EBOV-GP. DELTA. MLD. DELTA. TM gene fragment 2 was amplified by PCR using Q5 hot-start ultra-fidelity DNA polymerase using the returned plasmid synthesized by Kinsley as a template (of course, sequence 1 may be used as a template) and GP. DELTA. MLD-infu medium F and GP. DELTA. TM-infu3 as upstream and downstream primers.

The product obtained by PCR amplification was separated by 1% agarose gel electrophoresis (FIG. 1), and the fragment was recovered by using a DNA fragment recovery kit of the centrifugal column type.

3. The pPICZ-alpha A plasmid is cut by restriction enzymes BstBI and SalI to obtain a linearized pPICZ-alpha A plasmid.

4. And (3) connecting the EBOV-GP delta MLD delta

TM gene segments

1 and 2 obtained in the step (2) and the linearized pPICZ-alpha A plasmid obtained in the step (3) by using an infusion kit to obtain a recombinant plasmid, and naming the recombinant plasmid as pPICZ-GP delta MLD delta TM.

Sequencing pPICZ-GP delta MLD delta TM, wherein the recombinant plasmid is a recombinant vector obtained after inserting a DNA molecule (EBOV-GP delta MLD delta TM) containing Ebola virus GP glycoprotein mutant shown in a sequence 2 in a sequence table into a pPICZ-alpha A vector AOX1 promoter.

Example 2 expression, purification and characterization of Ebola Virus GP glycoprotein mutants

Construction and screening of recombinant yeast

About 10. mu.g of the expression plasmid pPICZ-GP. DELTA. MLD. DELTA. TM constructed in example 1 was single-point linearized with the restriction enzyme BglII, as follows (50. mu.L): expression plasmid pPICZ-GP Δ MLD Δ TM 43 μ L; BglII 2. mu.L; 10 XNEB3.1buffer 5. mu.L.

After digestion for 1h at 37 ℃, samples were taken and separated by electrophoresis on a 1% agarose gel to analyze whether the plasmid was completely linearized. The separation result showed that the enzyme-digested product was completely linearized and subjected to fragment recovery using a DNA fragment recovery kit of the centrifugal column type, and finally the linearized plasmid was eluted with 30. mu.L of pure water.

The following procedures for preparing yeast electrotransformation competent cells are described in the Invitrogen company's Manual of operation and "Molecular Cloning, A Laboratory Manual (Fourth Edition)", 2012Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Pichia pastoris GS115 was streaked onto YPD plates for resuscitation and single clones isolated. Selecting recovered monoclonal antibody, inoculating into YPD liquid culture medium, culturing in test tube to logarithmic phase, taking 1ml, transferring into 100ml YPD shake flask, shake-culturing at 25 deg.C and 200rpm to OD_600nm1.3-1.5. Centrifuging the above culture solution at 1500g and 4 deg.C for 5min, discarding supernatant, re-suspending with equal volume of precooled distilled water, centrifuging at 1500g and 4 deg.C for 5min, discarding supernatant, and repeatingThis step was performed 3 times. Resuspend it with equal volume of precooled 1M sorbitol, centrifuge at 1500g 4 ℃ for 5min, discard the supernatant and repeat this step 3 times. The bacterial pellets washed with 3 times of distilled water and 3 times of sorbitol are added with a proper amount of 1M sorbitol to be just sucked up, and 100 mul of each is subpackaged into a sterile centrifuge tube for preservation at minus 80 ℃.

The linearized recombinant plasmid is transformed into yeast. Taking a prepared yeast competent cell, placing on ice for about 5min, after dissolving, taking 80 μ L of competent cell and 20 μ L of plasmid obtained by the last step of linearization, pre-mixing, transferring into a pre-cooled 0.2cm electric transfer cup, and placing on ice for 5 min. According to the requirements of yeast electrotransfer manual, 900 microliter of precooled 1M sorbitol is quickly added after 2kV voltage electric shock, and is transferred into a clean test tube and is placed in an incubator at 25 ℃ for standing for 2 hours. Then, 1ml of YPD liquid medium without antibiotic addition was added thereto, and shake-cultured at 25 ℃ and 200rpm for 3 to 4 hours. The bacterial liquid obtained by shaking culture is taken out, 300 mu L of the bacterial liquid is coated on a YPD plate with screening resistance of Zeocin, and the bacterial liquid is inversely cultured for 60 to 72 hours at the temperature of 25 ℃.

After the coated plate grows out of monoclone, randomly selecting 8 monoclonals to puncture and inoculate the monoclonals to a new YPD/Zeocin plate, and carrying out inverted culture at 25 ℃. After the colony of the puncture grows out, inoculating the colony into 3ml YPD liquid culture medium, culturing at 25 ℃ and 200rpm by shaking table, after the YPD bacterial liquid grows to be thick, transferring the bacterial liquid to 3ml BMGY (formula: 10g/L yeast extract, 20g/L tryptone, 100mM phosphate buffer solution with pH of 6.0, 13.4g/L YNB, 4X 10^-4g biotin/L, 1% glycerol), shake culturing at 25 deg.C and 200rpm in culture medium, and supplementing methanol (0.5% in addition, volume percentage content) every 12 hr after 48 hr. After the induction for 72 hours, the cells were collected at 12000rpm for 3 min.

And adding 1/5 volumes of water and a proper amount of glass beads into the thallus collected after the methanol induction for 72 hours, carrying out vortex oscillation and crushing, breaking the thallus liquid at 4000rpm, centrifuging for 2min, taking the supernatant, and screening out a positive clone capable of expressing the target protein by using a specific antibody western blot of the anti-EBOV-GP protein.

The Western Blot procedure is roughly as follows: (1) separating the sample by 10% SDS-PAGE gel; (2) transferring the sample on the SDS-PAGE gel to a PVDF membrane; (3) sealing and transferring PVDF (polyvinylidene fluoride) membrane of target protein by 5% milk sealing liquid at room temperature for 1 hour; (4) transfer to incubation with 5% milk at a dilution of 1:4000 primary Antibody (EBOV-GP Antibody, rabbitpab) for 2 hours; (5) PBST is washed for 5min and 5 times; (6) transfer to 5% milk with 1:10000 dilution of secondary antibody (Goat Anti Rabbit IgG (H + L) X-Adsorbed-HRP) incubation for 1 hours; (7) PBST is washed for 5min and 5 times; (8) pro-light HRP Chemiluminescent Kit was added and the mixture was developed. A specific band with the molecular weight of about 67KD is detected as a positive clone.

Expression and purification of recombinant Ebola virus glycoprotein GP mutant

1. Recombinant yeast culture expression

And (4) selecting the positive clone identified in the first step, inoculating the positive clone into a YPD liquid culture medium, and culturing at 25 ℃ and 200rpm for about 48 hours. Then transferred into 100mL YPD liquid shake flask culture medium with the inoculum size of 1 percent (volume percentage content), cultured at 25 ℃ and 200rpm until the bacterial density OD600_nmIs greater than 10. OD600 obtained by the above culture_nmThe bacterial liquid with the inoculation amount of more than 10 percent is transferred into a BMGY culture medium (the formula is the same as the above) with the inoculation amount of 5 percent (volume percentage content), cultured for 24 hours at 25 ℃, added with methanol with the volume percentage of 0.5 percent to induce the expression of the target protein, induced once every 12 hours, induced for 72 hours and centrifugally collected the thalli.

2. Purification of Ebola virus GP mutant glycoproteins

(1) Taking 1L of the bacterial liquid which is induced by methanol for 72 hours and cultured by shaking the flask, and centrifuging at 8000rpm for 20min to collect thalli. The cells were resuspended in 300ml of pure water, homogenized for 3 times at 600bar pressure in a high-pressure homogenizer, the homogenate thus obtained was supplemented with 8M urea, 50mM phosphate buffer solution pH8.0, 500mM NaCl, 10mM imidazole and 5% (by volume) glycerol (the concentration of each substance therein being the final concentration in the homogenization system), dissolved at room temperature with stirring for 3 hours, and centrifuged at 12000rpm for 20 minutes to obtain a supernatant.

(2) The above dissolved supernatant was adjusted to pH7.5 and then subjected to crude purification by Ni affinity chromatography using Chelating of chemical Fast Flow chromatography media. Column equilibrium used for purification (liquid a): 6M urea, 500mM NaCl, 50mM phosphate buffer solution with pH7.5, 10mM imidazole and 5% (volume percentage content) glycerol, and the balance of water; eluent (liquid B): 6M urea, 500mM NaCl, 50mM phosphate buffer solution with pH7.5, 500mM imidazole and 5% (volume percentage content) glycerol, and the balance of water. The eluent consists of a solution A and a solution B, the elution gradient is 5% B, 50% B and 100% B, and the% represents the volume percentage content.

As shown in fig. 2 and 3 (fig. 2 is a chromatogram, fig. 3 shows a reduced SDS-PAGE protein electrophoresis result on the left and a Western blot detection result using a specific antibody against EBOV-GP protein on the right), it was found that the gradient elution of the target protein (ebola virus glycoprotein GP mutant) was 50% B elution.

(3) The 50% B gradient elution sample enriched in the desired band detected in step (2) was desalted using Sephadex G25 using 20mM pH6.2 phosphate buffer, 6M urea, 5% (volume percent) glycerol, and the balance water as mobile phase. And collecting an elution peak. The chromatogram is shown in FIG. 4.

(4) The sample obtained after desalting treatment by Sephadex G25 was further purified by Source30S cation exchange chromatography. Column equilibrium used for purification (liquid a): 20mM phosphate buffer solution with pH6.2, 6M urea, 5% (volume percentage content) glycerol and the balance of water; eluent (liquid B): 20mM pH6.2 phosphate buffer, 6M urea, 5% (volume percentage content) glycerol and 1M NaCl, the balance is water. Gradient elution was performed according to the following gradient, using the elution gradient: (1) 5% of the liquid B and 95% of the liquid A, (2) 15% of the liquid B and 85% of the liquid A, (3) 30% of the liquid B and 70% of the liquid A, (4) 50% of the liquid B and 50% of the liquid A, (5) 100% of the liquid B, (6) 100% of 0.5M NaOH,% representing volume percentage content. As a result, as shown in FIGS. 5 and 6 (FIG. 5 is a chromatogram, and FIG. 6 is a reduced SDS-PAGE protein electrophoresis chart), the gradient elution in which the target protein (Ebola virus glycoprotein GP mutant) is mainly present was determined to be 15% B elution according to the molecular weight of the electrophoresis band.

3. Identification of GP mutant glycoprotein of Ebola virus

(1) Glycosylation modification identification

Analyzing the molecular weight change of the protein before and after glycosyl excision by PNGaseF enzyme digestion treatment to analyze the EBOV-GP mutant protein prepared in the step 2(4), and the specific steps are as follows: taking 90 mu L of glycoprotein sample purified in the step 2(4), adding 10 mu L of 10 times glycoprotein denaturation buffer solution, and boiling water bath for 10min to fully denature the glycoprotein. Then, the reaction was carried out in a1 XNEB GlycoBuffer 2 reaction buffer containing 1% (by volume) NP40 in a water bath at 37 ℃ for 2 hours while setting a control group without the enzyme.

The specific enzyme digestion system is as follows:

the reduction SDS-PAGE of the digested sample was performed, and the results are shown in FIG. 7 (Coomassie blue staining on the left, and Western blot detection on the right).

In FIG. 7, PNGaseF represents peptide N-glycosidase F; EBOV-GP delta MLD delta TM + PNGaseF represents that the EBOV-GP mutant protein prepared in the step 2(4) is cut by PNGaseF enzyme; EBOV-GP delta MLD delta TM represents the negative control that the EBOV-GP mutant protein prepared in the step 2(4) is not subjected to enzyme digestion treatment; m represents a protein molecular weight marker. FIG. 7 shows that the molecular weight of the prepared EBOV-GP mutant protein is about 67KD when the PNGaseF is not cut by enzyme, and the molecular weight is reduced to about 52KD after the PNGaseF is cut by enzyme, which is consistent with the theoretical molecular weight (51130Da) of the glycosylated EBOV-GP mutant mature protein. The EBOV-GP mutant protein is a glycoprotein modified by glycosylation.

(2) Identification of mature peptide sequence

And (3) analyzing the EBOV-GP mutant protein prepared in the step 2(4) by protein N-terminal sequencing. Whether the target protein is determined to be the EBOV-GP mutant protein or not is identified, and whether the signal peptide is processed and cut off in the translation process is also analyzed. The result is shown in figure 8 (A is a protein N-terminal sequencing result map, B is 1-50aa coded by the EBOV-GP mutant gene), and the 5 amino acid residues at the N-terminal of the EBOV-GP mutant protein prepared in the step (4) are IPLGV in sequence and are consistent with the theoretical mature peptide N-terminal sequence of the EBOV-GP mutant protein after the signal peptide is processed and cut off. The proteins prepared in step 2(4) are EBOV-GP mutant proteins with mature peptide sequences.

(3) Molecular size determination

The EBOV-GP mutant protein sample prepared in the step 2(4) is subjected to protein renaturation by desalting and urea removal by dialysis, wherein gradient dialysis is adopted to perform the steps as follows: normal saline containing 4M urea, 5% (volume percentage content) glycerol and 0.05% (volume percentage content) Tween 20; normal saline containing 3M urea, 5% (volume percentage content) glycerol, 0.05% (volume percentage content) Tween 20; normal saline containing 2M urea, 5% (volume percentage content) glycerol, 0.05% (volume percentage content) Tween 20; normal saline containing 1M urea, 5% (volume percentage content) glycerol, 0.05% (volume percentage content) Tween 20; finally dialyzed into normal saline without urea, containing 5% (volume percent) glycerol and 0.05% (volume percent) Tween 20.

In order to verify whether the target protein obtained above forms a trimer structure, a Superdex200 type gel column (. phi.1.0X 30cm) was selected for separation, analysis and identification of the purified sample. The mobile phase used was physiological saline containing 5% (volume percent) glycerol, 0.05% (volume percent) Tween 20. Human IgG standard and BSA standard were added as internal references in the isolation of EBOV-GP. DELTA. MLD. DELTA. TM. recombinant proteins. The eluate was collected from the 6 th mL fraction in 1mL fractions. The collected samples were analyzed by SDS-PAGE and Western blotting, and the results are shown in FIG. 9, which revealed that the target protein eluted at 8-13mL and concentrated mainly at 9-11mL (A and B in FIG. 9). Whereas the human IgG standard was predominantly eluted at 11-13mL (B in FIG. 9), the BSA standard was predominantly eluted at 13-15mL (B in FIG. 9). Thus, the molecular weight of the EBOV-GP. DELTA. MLD. DELTA. TM recombinant protein in the non-denatured state is larger than that of IgG having a molecular weight of 150 KD.

Based on the above results, it was preliminarily considered that the prepared EBOV-GP Δ MLD Δ TM recombinant protein exists in the form of a polymer. In order to achieve better separation effect, higher resolution more suitable for purifying large protein and protein complex is selectedThe analysis was carried out by gel filtration of Superose6 Increate in the form of a pre-packed column using a mobile phase of physiological saline containing 5% (by volume) glycerol and 0.05% (by volume) Tween 20. When the purified EBOV-GP. DELTA. MLD. DELTA. TM protein was analyzed on Superose6 gel, the elution volumes (Ve) were determined using 669KD protein standard, 440KD protein standard, human IgG (150KD) and BSA (67KD) as external standards, respectively. The results are shown in FIG. 10, which shows that the Ve of the EBOV-GP Δ MLD Δ TM recombinant protein is 15.57mL (E in FIG. 10), which is between 440KD standard (Ve 14.65mL, B in FIG. 10) and human IgG (Ve 16.11mL, C in FIG. 10). Drawing with Ve as abscissa lgMr as ordinate to obtain R²The lgMr standard curve y for Ve of 0.9184 is-0.234 x +8.9058 (fig. 11). The molecular weight of the EBOV-GP delta MLD delta TM recombinant protein is estimated to be about 185KD, which is about three times of that of the monomer 67 KD. In order to further verify the reliability of the result, a human IgG standard product with approximate molecular weight obtained by calculation is selected as an internal reference and is mixed with the EBOV-GP delta MLD delta TM recombinant protein, and then the mixture is separated by a Superose6 gel column, eluent is collected in sections and is stained by SDS-PAGE Coomassie brilliant blue, the result shows that the light and heavy chain sizes of the human IgG standard product after denaturation and reduction are respectively about 26KD and 55KD, the elution is concentrated in 16-18mL (figure 12, lane 5 and lane 6), the molecular weight of the EBOV-GP delta MLD delta TM recombinant protein after reduction and denaturation is about 67KD, the elution is concentrated in 14-16mL (figure 12, lane 3 and lane 4) and is slightly eluted earlier than the human IgG standard product, and the molecular weight of the EBOV-GP delta MLD delta TM recombinant protein in a non-denaturation state is slightly larger than that of human IgG. This result was confirmed by Western blot analysis using an antibody specific to GP protein and an antibody specific to anti-human IgG (C and D in FIG. 12).

The results show that the EBOV-GP delta MLD delta TM recombinant protein purified by the method mainly exists in the form of trimer.

Example 3 immunogenicity of EBOV-GP Δ MLD Δ TM recombinant proteins

The recombinant EBOV-GP mutant protein is prepared by respectively using Freund's adjuvant (complete Freund's adjuvant is used for the first immunization, incomplete Freund's adjuvant is used for the second and third immunizations, the complete Freund's adjuvant is matched with the target protein before each administration and ultrasonic emulsification is needed) and Al (OH)₃Adjuvant (12 hr before each administration, the recombinant protein is mixed and adsorbed sufficiently) is injected subcutaneously into 7 week-old BalB/C mice according to 5 μ g recombinant protein/mouse, the buffer system is physiological saline, and the administration dosage of each mouse is controlled to 100 μ L (wherein the adjuvant accounts for 50% of Freund's adjuvant, and Al (OH))₃Adjuvant group adjuvant is 10%, and% represents volume percentage content), each adjuvant group has 10 mice, and negative control group which only injects adjuvant with equal dose is set at the same time. Three immunization administrations were performed on days 0, 14 and 28, and orbital bleeding was performed on days 10 (10 days after the primary immunization), 24 (10 days after the secondary immunization) and 38 (10 days after the tertiary immunization), respectively, and blood samples of the mice collected above were centrifuged at 12000rpm for 5min at room temperature after being left in a 37 ℃ incubator for two hours to separate sera, and the sera of the mice separated were carefully aspirated and then the antibody titer induced after immunization was measured by ELISA.

The ELISA method for measuring the antibody titer comprises the following steps:

1. coating antigen: the antigen used for coating was GP protein of Zaire-type ebola virus expressed using human cells (beijing yi qiao shenzhou biotechnology limited). Preparing the coating antigen into antigen coating solution with the concentration of 1 mu g/mL by using the coating solution, placing 100 mu L of the antigen coating solution in each hole of an enzyme linked plate, and incubating for 2 hours at 37 ℃;

2. pouring out the antigen coating solution in the ELISA plate, and washing the ELISA plate for 5 times by using PBST (basic molecular weight assay) with 300 mu L/hole after the solution is completely removed, wherein each time is 5 minutes;

3. pouring out PBST washing liquor in the enzyme-linked plate, adding 300 mu L of sealing liquid (5% milk) into each hole after the PBST washing liquor is completely removed, and incubating and sealing for 1 hour at 37 ℃;

4. pouring the blocking solution in the enzyme-linked plate, and washing the enzyme-linked plate for 5 times by using PBST with 300 mu L/hole after the blocking solution is completely removed, wherein each time is 5 minutes;

5. diluting the immunized mouse serum with 5% milk gradient as primary antibody;

6. pouring out the PBST washing liquor in the enzyme-linked plate in the step 4, adding 100 mu L of primary antibody prepared in the step 5 after the PBST washing liquor is completely removed, and incubating for 1 hour in an incubator at 37 ℃;

7. pouring out the primary antibody solution in the enzyme-linked plate, and washing the enzyme-linked plate for 5 times by using PBST with 300 mu L/hole after the solution is completely removed, wherein each time is 5 minutes;

8. pouring the PBST washing liquor in the enzyme-linked plate in the step 7, adding 100 mu L of goat anti-mouse IgG H + L HRP (secondary antibody) solution prepared by 5% milk according to the dilution ratio of 1:5000 after the PBST washing liquor is completely removed, and incubating for 1 hour at 37 ℃ in an incubator;

9. removing the secondary antibody, and washing the enzyme-linked plate 5 times by PBST (Poly-N-phenyleneisophthalamide) with 300 mu L/hole after the secondary antibody is completely removed, wherein each time is 5 minutes;

10. pouring the PBST washing solution in the enzyme-linked plate in the step 9, and adding 100 mu L of color development liquid into each hole for developing after the PBST washing solution is completely removed, wherein the standard color development time is 15 minutes;

11. after the development was complete, 50. mu.L of stop solution (2M H) was added to each well₂SO₄) Terminating the color development;

12. after the color development is stopped, the OD value is measured by reading with an enzyme-linked immunosorbent assay (ELIASA) by using double wavelengths of 490nm and 630 nm.

Read results, when analyzed, OD of initial dilution gradient with negative control injected with the same dose of adjuvant only_490nm/630nmValue as base, OD of initial dilution gradient of 2.1-fold or more negative control_490nm/630nmThe value is positive standard, each tested mouse is more than or equal to 2.1 times of negative control initial dilution gradient OD_490nm/630nmThe maximum dilution of the value is the titer of the antibody produced by the corresponding subject. In the experimental process, the OD value of the negative control obtained by the research is close to 0, and in order to ensure the reliability of experimental data, the OD is introduced into the research_490nmMore than or equal to 0.1 as an additional positive judgment criterion, and the maximum dilution at that time as the antibody titer produced by the corresponding subject.

The first immunization was given, and day 0 was recorded, and orbital bleeds were performed on day 10 to detect antibody titers. The ELISA results showed that the aluminum hydroxide adjuvant group failed to detect antibody production after immunization, and the Freund's adjuvant group produced better antibody titers with a ratio of 1:100(p <0.05, CV 0.88), but the positive rate was only 60% (FIG. 13).

A second immunization was given on day 14 at the same dose as the first and orbital bleeds were tested for antibody titers on day 24. The ELISA results showed that antibody production was detectable in both the aluminum hydroxide adjuvant group and the freund adjuvant group after two immunizations, with 100% positive rate, and the freund adjuvant group produced antibody titers of 1:16800(p <0.001, CV0.096) that were significantly higher than the aluminum hydroxide adjuvant group of 1:260(p <0.001, CV 0.236) (fig. 14).

A third immunization was given on day 28 at the same dose as the first two and antibody titers were measured by orbital bleeds on day 38. The ELISA result shows that the aluminum hydroxide adjuvant group and the Freund adjuvant group can detect the generation of the antibody after three times of immunization, the positive rate is 100%, and the titer of the generated antibody is obviously increased. The antibody titer produced by the Freund's adjuvant group was 1:58800(p <0.001, CV 0.088) and the antibody titer of the aluminum hydroxide adjuvant group was 1:800(p <0.001, CV 0.109) (FIG. 15).

<110> institute of bioengineering of military medical science institute of people's liberation force of China

<120> method for preparing trimeric ebola virus glycoprotein mutant by using yeast

<130>GNCLN170584

<160>3

<170>PatentIn version 3.5

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Met Gly Val Thr Gly Ile Leu Gln Leu Pro Arg Asp Arg Phe Lys Arg

1 5 10 15

Thr Ser Phe Phe Leu Trp Val Ile Ile Leu Phe Gln Arg Thr Phe Ser

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Ile Pro Leu Gly Val Ile His Asn Ser Thr Leu Gln Val Ser Asp Val

35 40 45

Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Asn Gln Leu Arg

50 55 60

Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro

65 70 75 80

Ser Val Thr Lys Arg Trp Gly Phe Arg Ser Gly Val Pro Pro Lys Val

85 90 95

Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Glu

100 105 110

Ile Lys Lys Pro Asp Gly Ser Glu Cys Leu Pro Ala Ala Pro Asp Gly

115120 125

Ile Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr

130 135 140

Gly Pro Cys Ala Gly Asp Phe Ala Phe His Lys Glu Gly Ala Phe Phe

145 150 155 160

Leu Tyr Asp Arg Leu Ala Ser Thr Val Ile Tyr Arg Gly Thr Thr Phe

165 170 175

Ala Glu Gly Val Val Ala Phe Leu Ile Leu Pro Gln Ala Lys Lys Asp

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Phe Phe Ser Ser His Pro Leu Arg Glu Pro Val Asn Ala Thr Glu Asp

195 200 205

Pro Ser Ser Gly Tyr Tyr Ser Thr Thr Ile Arg Tyr Gln Ala Thr Gly

210 215 220

Phe Gly Thr Asn Glu Thr Glu Tyr Leu Phe Glu Val Asp Asn Leu Thr

225 230 235 240

Tyr Val Gln Leu Glu Ser Arg Phe Thr Pro Gln Phe Leu Leu Gln Leu

245 250 255

Asn Glu Thr Ile Tyr Ala Ser Gly Lys Arg Ser Asn Thr Thr Gly Lys

260 265 270

Leu Ile Trp Lys Val Asn Pro Glu Ile Asp Thr Thr Ile Gly Glu Trp

275280 285

Ala Phe Trp Glu Thr Lys Lys Asn Leu Thr Arg Lys Ile Arg Ser Glu

290 295 300

Glu Leu Ser Phe Thr Ala Val Ser His His Gln Asp Thr Gly Glu Glu

305 310 315 320

Ser Ala Ser Ser Gly Lys Leu Gly Leu Ile Thr Asn Thr Ile Ala Gly

325 330 335

Val Ala Gly Leu Ile Thr Gly Gly Arg Arg Thr Arg Arg Glu Val Ile

340 345 350

Val Asn Ala Gln Pro Lys Cys Asn Pro Asn Leu His Tyr Trp Thr Thr

355 360 365

Gln Asp Glu Gly Ala Ala Ile Gly Leu Ala Trp Ile Pro Tyr Phe Gly

370 375 380

Pro Ala Ala Glu Gly Ile Tyr Thr Glu Gly Leu Met His Asn Gln Asp

385 390 395 400

Gly Leu Ile Cys Gly Leu Arg Gln Leu Ala Asn Glu Thr Thr Gln Ala

405 410 415

Leu Gln Leu Phe Leu Arg Ala Thr Thr Glu Leu Arg Thr Phe Ser Ile

420 425 430

Leu Asn Arg Lys Ala Ile Asp Phe Leu Leu Gln Arg Trp Gly Gly Thr

435 440445

Cys His Ile Leu Gly Pro Asp Cys Cys Ile Glu Pro His Asp Trp Thr

450 455 460

Lys Asn Ile Thr Asp Lys Ile Asp Gln Ile Ile His Asp Phe Val Asp

465 470 475 480

Gly Gly Gly Gly Gly Val Asp His His His His His His

485 490

Claims

1. A method of making an ebola virus glycoprotein mutant, comprising the steps of:

(1) introducing a gene for coding the Ebola virus glycoprotein mutant into a Pichia pastoris cell to obtain a recombinant yeast cell for expressing the gene;

(2) sequentially culturing and crushing the recombinant yeast cells, and obtaining the Ebola virus glycoprotein mutant from a crushed product;

the ebola virus glycoprotein mutant is an ebola virus glycoprotein with a mucin-like region and a C-terminal transmembrane region deleted;

in the step (1), the gene encoding the ebola virus glycoprotein mutant is a DNA molecule shown in any one of (b1) to (b 4):

in the step (2), the crushing method is to homogenize for 3 times by a high-pressure homogenizer at a pressure of 600 bar;

in the step (2), the crushing further comprises the step of adding the following substances in final concentration into the crushed system: 8M urea, 50mM phosphate buffer solution with pH8.0, 500mM NaCl, 10mM imidazole and 5% by volume of glycerol to obtain a crude extract containing the Ebola virus glycoprotein mutant;

in the step (2), the method further comprises a step of purifying the crude extract;

the purification is to carry out affinity chromatography, gel exclusion chromatography and ion exchange chromatography on the crude extract in sequence;

when the affinity chromatography is carried out, the adopted medium is a chemical Fast Flow; the column equilibrium liquid adopted is marked as liquid A, and the composition of the liquid A is as follows: 6M urea, 500mM NaCl, 50mM phosphate buffer solution with pH7.5, 10mM imidazole, 5% glycerol by volume percentage and the balance of water; the eluent adopted is marked as B liquid, and the B liquid comprises the following components: 6M urea, 500mM NaCl, 50mM phosphate buffer solution with pH7.5, 500mM imidazole, 5 percent of glycerol by volume percentage and the balance of water; the elution gradient used was: (1) 5% of the liquid B and 95% of the liquid A, (2) 50% of the liquid B and 50% of the liquid A, (3) 100% of the liquid B,% represents volume percentage; said ebola virus glycoprotein mutant is present in said "(2) 50% of said B fluid and 50% of said a fluid" eluate;

when the gel exclusion chromatography is carried out, the adopted medium is Sephadex G25; the mobile phase composition used was as follows: 20mM pH6.2 phosphate buffer solution, 6M urea, 5% glycerol by volume percentage, and the balance of water;

the ion exchange chromatography is cation exchange chromatography, and the adopted medium is SOURCE 30S; the equilibrium liquid adopted is marked as A 'liquid, and the A' liquid comprises the following components: 20mM phosphate buffer solution with pH6.2, 6M urea, 5% glycerol by volume percentage, and the balance of water; the eluent adopted is marked as B 'liquid, and the B' liquid comprises the following components: 20mM phosphate buffer solution with pH6.2, 6M urea, 5 percent of glycerol and 1M NaCl by volume percentage, and the balance of water; the elution gradient used was: (1) 5% of said B ' solution and 95% of said a ' solution, (2) 15% of said B ' solution and 85% of said a ' solution, (3) 30% of said B ' solution and 70% of said a ' solution, (4) 50% of said B ' solution and 50% of said a ' solution, (5) 100% of said B ' solution, (6) 100% of 0.5M NaOH,% representing volume percentage; the ebola virus glycoprotein mutant is predominantly present in the "15% of the B 'solution and 85% of the a' solution" eluate.

2. The method of claim 1, wherein: in the step (2), the culture process includes a step of adding 0.5% by volume of methanol to the culture system.