WO2006136084A1 - Proteines de fusion de proteines structurales recombinantes du coronavirus sars, leur production et leurs utilisations - Google Patents

Proteines de fusion de proteines structurales recombinantes du coronavirus sars, leur production et leurs utilisations Download PDF

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WO2006136084A1
WO2006136084A1 PCT/CN2006/001293 CN2006001293W WO2006136084A1 WO 2006136084 A1 WO2006136084 A1 WO 2006136084A1 CN 2006001293 W CN2006001293 W CN 2006001293W WO 2006136084 A1 WO2006136084 A1 WO 2006136084A1
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protein
sars
fusion protein
cov
amino acid
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PCT/CN2006/001293
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French (fr)
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WO2006136084A8 (fr
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Chengyu Jiang
Feng Guo
Shuan Rao
Bing Guan
Yi Huan
Peng Yang
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Chinese Academy Of Medical Sciences, Institute Of Basic Medical Sciences
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Priority to US11/993,176 priority Critical patent/US20100150923A1/en
Publication of WO2006136084A1 publication Critical patent/WO2006136084A1/zh
Publication of WO2006136084A8 publication Critical patent/WO2006136084A8/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a fusion protein of a SARS-CoV viral structural protein and high expression thereof in mammalian cells; use of the fusion protein for the preparation of genetically engineered vaccines and medicaments for controlling SARS-CoV virus infection; The use of the fusion protein in the preparation of a kit for detecting SARS-CoV virus infection.
  • the present invention also relates to the discovery of toxic fragments of the SARS-CoV viral structural protein S; and various vaccines designed to prevent SARS-CoV infection.
  • SARS-CoV Severe Acute Respiratory Syndrome
  • SARS-CoV virus vaccine Since the SARS-CoV virus vaccine is applied to humans, the stability and safety of the vaccine is the most basic and important requirement. The research on genetic engineering vaccines is relatively mature, which is most consistent with this requirement.
  • the SARS-CoV virus has four structural proteins, S, M, N and E.
  • S and N proteins have strong immunogenicity and are the main antigens for vaccine research.
  • M and E also have certain immunogenicity and are likely to be effective vaccines.
  • S protein is the most likely to be an effective vaccine.
  • S protein Due to the large number of post-translational modification sites in the s protein, mainly glycosylation sites, proteins expressed by prokaryotic cells or expressed by yeast will not fold correctly, affecting their biological activities. Only when expressed in mammalian cells, S protein can be correctly modified, folded and processed, and closer to the natural state, otherwise it will seriously affect the immune effect of the vaccine. However, the S protein encoded by the S virus originally has a low expression level in mammalian cell expression systems and is difficult to apply in practice.
  • the SARS-CoV virus S protein is efficiently expressed in mammalian cells, and the appropriate conditions are selected as much as possible during the purification process.
  • the protein can be effectively separated from the host protein and DNA to ensure that the structure of the viral protein, especially the steric structure, is not destroyed; the second is to increase the expression yield of the S protein and make the production of the vaccine more economical.
  • the safety of vaccines limited research on the pathogenesis of SARS-CoV virus, how SARS-CoV virus causes acute lung injury, heart failure, and the path of immune system collapse are not clear.
  • Preparation of prior art SARS vaccine There are security risks in it. And the prior art failed to solve this problem, because Therefore, the preparation of a safe and effective SARS-CoV virus vaccine requires further inventive research. Summary of the invention
  • a second object of the present invention is to highly express and purify the structural proteins of SARS virus and their truncated forms of fusion proteins in mammalian cell expression systems.
  • a third object of the present invention is to prepare a genetically engineered vaccine for preventing SARS-CoV infection by using a fusion protein of a structural protein of the SARS virus, including a fusion protein of S protein; our study found that S protein and its receptor The combination of ACE2 can cause or exacerbate the body's acute respiratory distress syndrome, and the development of a safe and effective vaccine requires the deletion or modification of a fragment of the S protein that binds to its receptor ACE2.
  • a fourth object of the present invention is to prepare a kit for detecting SARS-CoV virus infection using the fusion protein of the structural protein of the obtained SARS-CoV virus.
  • a fifth object of the present invention is to prepare and screen a medicament for treating SARS-CoV virus infection using the obtained S protein fusion protein.
  • a sixth object of the present invention is to prepare a vaccine for preventing SARS-CoV infection by removing, mutating, or modifying a fragment of the amino acid 318-510 of the SARS-CoV virus S protein and preventing the S protein from binding to its receptor ACE2. , DNA vaccines, protein vaccines, and viral vector vaccines.
  • the amino acid from the a-th amino acid to the b-position amino acid of the full-length S protein of the wild-type SARS-CoV virus is the amino acid from the a-th amino acid to the b-position amino acid of the full-length S protein of the wild-type SARS-CoV virus.
  • S318-510 means that the protein expressed by the gene fragment is 318 to 510 amino acids of the entire SARS-CoV virus S protein
  • S511 means that the protein expressed by the gene fragment is the entire SARS-CoV virus S protein 1 to 511 amino acid
  • S685 means that the protein expressed by this gene fragment is 1 to 685 amino acids of the entire SARS-CoV virus S protein, and so on.
  • a fusion protein of SARS-CoV viral structural protein the structure of which is: X-Y-Z,
  • X comprises the SARS-CoV viral structural protein S or M or E or N, or any truncated form of the above structural protein, the SARS-CoV viral structural protein S comprising removing, modifying or mutating amino acid 318 to amino acid 510 Any amino acid fragment, or remove, modify or mutate amino acid 318 to amino acid 510;
  • Y is a linking moiety consisting of 0-20 any amino acids; Is Z a person with a hinge region, CH 2 , CH 3 domain? 0 and its variant or protein label.
  • the protein tag includes, but is not limited to, a hexahistidine (6XHis) tag, a polyethylene glycol (PEG) tag, and a human serum albumin (HS A) tag.
  • the SARS-CoV viral structural protein S comprises the full length of the protein S or any truncated form thereof.
  • the SARS-CoV viral structural protein S is a protein that does not bind to the receptor ACE2 or reduces the ability to bind to ACE2.
  • the Y is preferably two amino acids, and the amino acids are lysine and arginine.
  • the present invention also relates to a SARS-CoV virus S protein gene capable of being expressed in a mammalian cell line, characterized in that the gene is SEQ ID NO: 1.
  • the invention further relates to a recombinant expression plasmid comprising SEQ ID NO: 1, said plasmid comprising a eukaryotic PEAK series.
  • the present invention still further relates to a mammalian cell strain comprising the fusion protein of said SARS-CoV viral structural protein capable of expressing a SARS-CoV virus S protein gene.
  • the mammalian cell line includes CHO, 293 and Vero cell lines and derived cell lines thereof.
  • the invention also relates to a method for preparing a fusion protein of the SARS-CoV viral structural protein, comprising:
  • the mammalian cell expression strain produces 10 ⁇ ⁇ or more of the recombinant protein in its medium per million cells per 24 hours under normal growth;
  • the recombinant expression plasmid contains a leader sequence of a fusion protein which is a leader sequence of a CD5 protein.
  • the recombinant expression plasmid, the coding gene of the structural protein is artificially synthesized, and the codon sequence in the viral gene of the same amino acid is replaced by a common or preferred codon in the human cell, and the structural protein of the virus is subjected to human codon optimization.
  • the fusion protein of a structural protein expressing the SARS-CoV virus which is a S protein gene of SARS-CoV which is commonly used or preferentially synthesized by human cells, is represented by SEQ ID NO: 1.
  • the screening drug used to construct the mammalian cell expression strain preferably includes puromycin and methotrexate.
  • per 24 hours per million cells or more than 30 ⁇ ⁇ produce recombinant proteins in their culture medium.
  • the invention also relates to the use of the fusion protein in the preparation of a vaccine for preventing SARS-CoV virus; and the use in the preparation of a SARS-CoV virus detection kit; in preparing or screening for anti-SARS-CoV disease Use in toxic infectious drugs; use in the preparation of antibodies against SA S-CoV virus infection.
  • the invention particularly relates to a method for removing, modifying or mutating an amino acid fragment of amino acid 318 to amino acid 510 of the SARS-CoV viral structural protein S, or to remove, modify or mutate the expression.
  • the DNA sequence of the SARS-CoV viral structural protein S 318 amino acid to 510 amino acid fragment sequence and the amino acid expressed by this DNA DNA.
  • the invention further relates to the use of said DNA sequence or said DNA-expressed amino acid for the preparation of a vaccine against SARS-CoV virus, said vaccine comprising a DNA vector vaccine, a protein vaccine, a viral vector vaccine.
  • BRIEF DESCRIPTION OF THE DRAWINGS - Figure 1 is a Western Blotting method for determining the expression of optimized expression of E, M, N and S in fusion protein host cells. From left to right are E-Fc, M-Fc, N-Fc and S-Fc, respectively. The results showed that the four structural proteins of SARS-CoV virus can be well expressed in host cells.
  • Fig. 2 is a result of agarose gel electrophoresis of the S1190 gene fragment inserted into an expression vector.
  • the three lanes from left to right are ⁇ -Hind III Marker, S1190, DL2000 Marker.
  • ⁇ -Hind ⁇ Marker from small to large (from bottom to top) is 564bp (harder to distinguish from the picture), 2027bp, 2322bp, 4361bp, 6557bp, 9416bp, 23130bp ; DL2000 Marker from small to large (from bottom to top)
  • DL2000 Marker from small to large (from bottom to top)
  • Figure 3 shows the expression of the fusion protein S1190-Fc in host cells by Western Blotting. It is proved that the fusion protein S1190-FC can be expressed well in host cells, and the expressed protein size is about 185KD.
  • Figure 4 shows the results of Coomassie blue staining of the purified fusion protein S1190-FC polyacrylamide gel, demonstrating that a relatively pure fusion protein S1190-Fc can be obtained.
  • Figure 5 is a flow chart of cell fusion of fusion protein S1190-FC at 4 °C in combination with Vero E6 cells. From the results, the fusion protein S1190-FC was bound to Vero E6 cells. According to internationally published literature, the receptor for ACE protein ACE2 exists on the surface of Vera E6 cells, and the region of ACE2 that binds to S protein is 318 to 510 amino acids. Therefore, with the cell adhesion fusion protein S1190-Fc, the activity of the expressed fusion protein S1190-FC can be detected. The blank peak region in the figure is the negative control (PBS buffer), and the shaded portion is the experimental result, demonstrating that the fusion protein S1190-Fc adheres to Vero E6 cells.
  • PBS buffer negative control
  • shaded portion is the experimental result, demonstrating that the fusion protein S1190-Fc adheres to Vero E6 cells.
  • Figure 6 is a flow chart of cell binding of fusion protein S1190-FC to 293E cells transfected with human ACE2 (hACE2).
  • 293E cells transfected with hACE2 bind to the fusion protein S1190-Fc at 4 °C Thereafter, it was bound to FITC-labeled anti-Fc antibody, and untransfected 293E cells were bound to the Fc antibody as a negative control (blank-shaped region), and then detected by flow.
  • the results showed that the 293E cells transfected with hACE2 bound to the fusion protein S1190-FC, and a significant shift (shaded portion) occurred.
  • Figure 7 is a flow chart of cell binding of fusion protein S1190-FC to 293E cells transfected with murine ACE2 (mACE2).
  • mACE2 murine ACE2
  • 293E cells transfected with mACE2 bind to the fusion protein S1190-Fc at 4 ° C and then bind to FITC-labeled anti-Fc antibody, and untransfected 293E cells bind to Fc antibody as a negative control (blank-shaped region) And then use streaming to detect.
  • the results showed that 293E cells transfected with mACE2 bound to the fusion protein S1190-FC with significant shift (shaded portion).
  • the left panel of Figure 8 is a photograph of cell fusion after 293ET cells were transfected with ACE2 and S1190 genes respectively (magnification 100 times); the right panel is a photograph of cell fusion after 293ET cells were transfected with CD4 and S1190 genes, respectively ( Zoom in 100 times). Prove that protein S1190 binds to ACE2 and can cause cell fusion.
  • FIG. 9 shows the results of co-immunoprecipitation (IP) of S1190-FC with the ACE2 receptor.
  • S1190-FC and ACE2 were transfected separately, and cells of control Fc and ACE2 were lysed and then detected by Western Blotting.
  • the first band on the left is the cell lysate transfected with control Fc and ACE2 as a control, and the second band is the IP result of the cell lysate transfected with control Fc and ACE2, and the third band is transfected.
  • Cell lysates stained with S1190-FC and ACE2 served as controls, and the fourth band was IP results transfected with S1190-Fc and ACE2.
  • Figure 10 shows the results of experiments with cultured cells for down-regulation of ACE2 receptors. Put Vero E6 cells in 4
  • the °C (basket line) and 37°C (red line) were fully activated with the fusion protein S1190-FC, and Fc was used as a control (black line), and then detected with an anti-Fc antibody.
  • Figure 11 shows the results of experiments with cultured cells for down-regulation of ACE2 receptors. Place Vero E6 cells in 4 respectively. C (basket line) and 37 ⁇ (red line) were fully functional with the fusion protein S1190-FC, and Fc was used as a control (black line), and then detected with an antibody against ACE2. The results demonstrated that at 37 °C, the fusion protein S1190-FC interacts with the receptor ACE2 and causes the ACE2 receptor to down-regulate.
  • Figure 12 is a graph showing changes in lung rebound rate of wild-type mouse lung acid or salt perfusion and addition of fusion protein S1190-FC.
  • the mice were divided into 4 groups, 5 to 7 in each group. The two groups were subjected to acid perfusion, and the fusion protein S1190-FC and control Fc were added. The two groups were subjected to salt perfusion, and the fusion protein S1190-FC and control Fc were also added. Each mouse was perfused at a dose of 5.5 nmol/kg fusion protein S1190-Fc or 5.5 nmol/kg control Fc.
  • Figure 13 is a pathological section of mouse lung tissue.
  • the lung tissue of the mouse treated in the same manner as in the drawing of Fig. 11 was subjected to pathological section, and the results were in agreement with the results described in the description of Fig. 11.
  • lung tissue showed significant edema and acute lung injury.
  • the addition of the fusion protein S1190-FC significantly aggravated the degree of acute lung injury.
  • Figure 14 shows the results of lung injury scores. This result confirms the results of Figure 11 and Figure 12, demonstrating that acute lung injury occurs in lung tissue under acid perfusion conditions, and the addition of the fusion protein S1190-FC significantly aggravates the extent of acute lung injury compared with the control Fc. There is a significant difference ( ⁇ ⁇ ⁇ . ⁇ ).
  • Figure 15 is the result of the wet-dry lung tissue weight ratio. This result confirms the results of Figures 11, 12, and 13. It was demonstrated that in the case of acid-induced acute lung injury, the degree of pulmonary edema caused by the addition of the fusion protein S1190-FC group was more serious than that of the control Fc group, and the wet-dry lung tissue weight ratio was larger, compared with the control Fc group. The ratio was significantly different (p ⁇ 0.05).
  • Figure 16 Wild type mouse lung acid or salt perfusion and spiked with fusion protein S1190-FC or fusion protein
  • mice were divided into 5 groups, 5 to 7 in each group, 3 of which were subjected to acid perfusion, and the fusion protein S1190-Fc, fusion protein S318-510-FC and control Fc were added respectively, and the two groups were subjected to salt perfusion, and The fusion protein S318-510-FC and control Fc were separately injected at a dose of 5.5 nmol/kg of fusion protein or control Fc per dose.
  • the results showed that the wild-type mouse acid perfusion fusion protein S1190-Fc group or fusion protein S318-510-FC group was significantly different from the acid perfusion plus control Fc group in all measurement time. Difference (p ⁇ 0.05). It was demonstrated that both S1190-FC and S318-510-FC can aggravate acute lung injury under acid perfusion conditions.
  • Figure 17 shows the results of changes in lung rebound rate in the lung acid or salt perfusion of the ACE2 knockout mice and the fusion protein S1190-FC.
  • the process and grouping can be referred to the description of FIG.
  • the results showed that in the ACE2 knockout mice, there was no significant difference in the rebound rate of lung tissue between S1190-FC and control Fc under acid perfusion conditions.
  • FIG. 18 S1190-Fc was detected in lung tissue homogenate after intraperitoneal injection of the fusion protein S1190-FC.
  • the fusion protein S1190-Fc was detected by Western blotting and protein G agarose using an anti-Fc antibody, and the control Fc of the control was not detected.
  • Figure 19 is the result of detecting lung immunohistochemistry of the fusion protein S1190-FC. Fusion protein
  • S1190-FC accumulates in bronchial epithelial cells (left column, magnified 100 times), inflammatory secretory cells (middle column, magnified 200 times) and alveolar cells (right column, magnified 200 times), which is also the site of acute lung injury , It was demonstrated that S1190-Fc first accumulated in the site of acute lung injury.
  • Figure 20 shows the expression of ACE2 protein in lung tissue in wild-type mice treated with fusion protein S1190-FC. Wild type mice were treated with fusion protein S1190-FC and control Fc protein, respectively, and then detected by Western blotting using an antibody against ACE2. The results demonstrated that S1190-Fc-treated mice caused a decrease in the expression of ACE2 protein in lung tissue.
  • Figure 21 is a graph showing the results of detecting Angll levels in lung tissues of wild-type mice. Wild type mice were perfused with lung tissue with acid or salt, and the fusion protein S1190-FC or control Fc protein was added, and after 3 hours, the level of Angll in lung tissue was measured by EIA. The results showed that in the acid-treated group, the level of Angll infused with the fusion protein S1190-FC and the control Fc protein was significantly different (p ⁇ 0.05), and the acid-treated group was supplemented with the wild-type mouse lung tissue of the fusion protein S1190-Fc. The level of Ang ll was significantly increased, overlooking the level of Angll in the lung tissue of mice treated with acid and supplemented with Fc protein.
  • Figure 22 is a titer (orange) of neutralizing antibodies produced in vivo after immunization of mice with S1190-FC.
  • Five-week-old female b al b/e mice were divided into two groups, 5 in each group, one group was injected with 50 ⁇ g of S1190-Fc plus adjuvant at 0, 2, and 4 weeks, and the other group was injected.
  • the same dose of Fc plus adjuvant was used as a control (blue), and serum was collected from the sixth week.
  • the heat inactivated serum was subjected to a microneutralization assay to detect the presence of neutralizing antibodies. The results showed that there was a significant difference in antibody titer between the S1190-FC group and the control group.
  • the mice immunized with S1190-FC were able to produce a large number of effective neutralizing antibodies, which could effectively prevent SARS-CoV infection.
  • Figure 23 is an electropherogram showing the results of restriction enzyme digestion of the S gene and its fragment after insertion into an expression vector. From left to right are ⁇ -Hind III Marker, S317, S318-510, S318-1190, S511-1190, S685, S900, S1148, S1190, DL2000 Marker.
  • the ⁇ -Hind III Marker is 564 bp from the bottom to the top (the bottom band is difficult to distinguish from the figure), 2027 bp, 2322 bp, 4361 bp, 6557 bp, 9416 bp, 23130 bp ; DL2000 Marker from small to large (from the bottom)
  • the end-to-end end is 100 bp, 250 bp, 500 bp, 750 bp, lOOOObp, 2000 bp. It can be confirmed from this figure that the S protein gene and its fragment are correctly inserted into the expression vector.
  • Figure 24 shows the results of Western Blotting to determine the expression of the optimized S fusion protein and its truncated form in host cells. From 1-10 are S1190-Fc, respectively, about 185KD; S1148 Fc, about 180KD; S900 Fc, about 175 D; S318-1190 Fc, about 160KD; S511-1190 Fc, about 155KD; S685 Fc, Approximately 155 KD; S511 Fc, approximately 140 KD; S681-1190 Fc, approximately 120 KD; S317 Fc, approximately 85 KD; S318-510-Fc, approximately 67 KD. It can be confirmed by this figure that the optimized S fusion protein gene and its fragment are highly expressed in the host cell, and the original sequence before optimization is difficult to express in mammalian cells, which proves that the expression optimization method is effective and feasible.
  • Figure 25 is a photograph of cell-transfected S317 Fc and ACE2, gpl20 and ACE2 not fused (magnification 100 times).
  • Figure 26 is a photograph of cell fusion of S318-510-FC with ACE2, S1190-Fc and ACE2 (magnification 100-fold).
  • Figure 27 is a photograph of cell-transfected S511-1190 Fc and ACE2, S681-1190 Fc and ACE2 not fused (magnification 100-fold).
  • ACE2 is a specific receptor for SARS-CoV virus, and the part that interacts with ACE2 and causes cell fusion is S318-510, which is amino acid 318 to 510 of S protein.
  • the present invention provides a SARS-CoV virus S protein gene sequence which can be expressed in a mammalian cell line, such as the sequence of the present invention SEQ ID NO: 1.
  • the present invention provides a recombinant expression plasmid comprising SEQ ID NO: 1, and the recombinant expression plasmid preferably comprises a eukaryotic PEAK series.
  • the present invention also provides a fusion protein of a SARS-CoV viral structural protein expressed by a SARS-CoV virus S protein gene sequence expressed in a mammalian cell line, the structure of which is: XYZ wherein X includes a SARS-CoV virus structure Protein S or M or E or N, or any truncated form of the above structural protein;
  • Y is a linking moiety consisting of 0-20 amino acids
  • transfection can express SARS-CoV viral structural proteins and their truncated forms of fusion protein and endogenous dihydrofolate reductase, d fr Recombinant plasmid, a mammalian cell expression strain was constructed.
  • the recombinant plasmid uses a mammalian eukaryotic expression vector with strong expression ability. Using a more powerful promoter to initiate gene expression, the SARS-CoV viral structural protein and its truncated form are highly expressed in mammalian cell expression systems.
  • the mammalian eukaryotic expression vector can use the PEAK series: for example, pEAK10, pEAK12, pEAK13, etc., pCDNA series: pCDNA3.0, pCDNA4.0, etc., pCDM series: pCDM7, pCDM8, pCDM10, pCDM12; preferred eukaryotic cells
  • the expression vector is pEAK13; the promoter is selected from the group consisting of CMV, EF1 ⁇ , Co YMV MV enhancer + chicken albumin promoter ⁇ SV40 promoter + enhancer; the preferred promoter is CMV enhancer + chicken albumin promoter.
  • the present invention replaces the secretory sequence preceding the target protein sequence with the well-known strong leader sequence of CD5 protein (CD5L), and enhances the secretion of the secreted expression of the target protein.
  • CD5L CD5 protein
  • the present invention employs a secretory expression vector to remove the original secretory sequence before the wild-type SARS-CoV viral structural protein gene, and adds a strong signal with a shear signal.
  • the guide sequence CD5L after being translated into a guide protein transmembrane signal peptide, directs the viral structural protein across the cell membrane and secretes it into the extracellular medium to achieve efficient separation from the host protein and DNA, simplifying the steps of protein purification.
  • the signal peptide translated into the leader sequence can be cleaved by the action of protein cleavage enzyme without affecting the structure of the viral protein.
  • the secretory sequence of the wild-type SARS-CoV viral structural protein with the CD5L sequence, as shown below -
  • the present invention synthesizes a plasmid encoding a fusion protein of a structural protein of the SARS-CoV virus, and encodes a gene encoding the structural protein, and replaces the codon encoding the same amino acid in the viral gene with a commonly used (preferred) codon in the human cell.
  • Structural protein genes are optimized for human coding.
  • the present invention also employs genetic optimization means.
  • the genetic optimization includes codon humanization and optimization.
  • the codon humanization refers to the difference and preference of codon usage in various organisms, and the present invention mainly uses human cells as a host, and the purpose is to apply to the human body, so that there is very little in the human body.
  • the used codon is replaced with a preferred codon that is frequently used in the human body.
  • the codon-optimized gene optimization method replaces the frequently used codons in the gene encoding of the fusion protein into frequently used codons.
  • the amino acid sequence corresponding to the wild-type SARS-CoV virus S protein was found by GenBank, and the codon corresponding to each amino acid was replaced with a codon with a higher frequency of use, thereby obtaining several optimized DNA sequences to enhance the protein.
  • a high-efficiency codon corresponding to an amino acid in a human body is used in place of a rare codon.
  • a codon of glycine (Gly) is replaced by GGC instead of other GGA/GGT/GGG, and glutamic acid (Glu) is replaced by GAG.
  • GAA, aspartic acid (Asp) replaces GAT and the like with GAC.
  • the frequency of use of codons in highly expressed genes in humans is listed in the table below.
  • the present invention performs codon substitution by the ratio of the frequency of use in the list, and selects a codon having a higher frequency by using a codon corresponding to the amino acid, thereby improving the expression efficiency of the protein.
  • the frequency of codon usage in high-expression genes in humans is as follows:
  • the present invention uses a gene coding optimization method to obtain a plurality of optimized DNA sequences, wherein the synthetic SRS-CoV S protein gene sequence of the present invention is preferably selected as shown in SEQ ID NO: 1.
  • the eukaryotic cell expression strain of the transfected recombinant plasmid of the present invention is selected from the group consisting of CHO, 293 and Vera cell lines and derived cells thereof.
  • a suitable host cell strain capable of high expression is selected, and an expression cell line is established, and 293 cells, CHO cells or Vera cells, and derived cells of the above cells (derivati Ve ) are selected.
  • the constructed recombinant plasmid contains the anti-puromycin gene, and the cells transfected into the S protein or its truncated form of the gene are screened, and finally quantitatively characterized by ELISA or Western Blotting, and the optimal expression strain is selected. .
  • 293E, 293ET and CHO cells have higher expression levels, especially CHO cells.
  • the selected high-volume constant expression cell line is subjected to cell domestication culture, thereby further increasing the expression level of viral protein, and providing conditions for batch preparation and industrial production.
  • CGMCC General Microbiology Center
  • the screening drug used in the construction of the eukaryotic cell expression strain of the present invention is puromycin, and the drug used for acclimation of the cell to increase the protein expression amount is methotrexate.
  • the recombinant plasmid constructed by the present invention uses a gene resistant to puromycin, which is a drug capable of killing eukaryotic cells.
  • puromycin is a drug capable of killing eukaryotic cells.
  • the addition of puromycin to the cell culture medium can be used to screen for successful transfection with the difference in tolerance to puromycin from cells containing the anti- puromycin gene and cells without the gene.
  • the endogenous dihydrofolate reductase (dhfr) gene is also present in the recombinant plasmid. Therefore, methotrexate can be used for cell domestication and increase protein expression.
  • the full-length structural protein fusion protein of the SARS-CoV virus expressed in the present invention (E-Fc, M-Fc, N-Fc, S-Fc) is shown in Fig. 1.
  • the S protein of the SARS-CoV virus expressed by the present invention and its truncated form of any of the fusion proteins (317-Fc, 511-Fc, 685-Fc, 900-Fc, 1148-Fc, 1190-Fc, 318-510- Fc, 318-1190-Fc, 511-1190-Fc and 6811-190-Fc) are shown in Figures 23 and 24.
  • the construction of the cell expression strain avoids the instability of the transient transfection state of the cells, and can increase the yield by selecting the dominant strain for amplification, and further increase the expression amount by means of cell domestication, thereby realizing the batch of the protein. Preparation or industrial production.
  • the cell expression strain produces 10 ⁇ ⁇ of the above recombinant protein per million cells per unit of cells under normal growth conditions every 24 hours. After the cells were counted, the cells were cultured, and the medium for cell culture was collected three days later, and the expression amount of the cell strain was measured by an ELISA method, and a series of truncated forms of the S protein and the S protein were obtained by calculation. With the method of the present invention, a high expression amount of a fusion protein of a structural protein of various SARS-CoV viruses and a truncated form thereof can be finally obtained. In the medium of the cells, the expression amount was higher than 10 ⁇ g/10 6 cells/24 hours.
  • the yield of S1190-FC (full length S protein removed from the transmembrane region) was higher than 10 ug/10 6 cells/24 hours; the yield of S truncated form protein (S318-510-FC) was higher than 30 wg/10 6 Cells / 24 hours.
  • step (3) Purifying the protein expressed by the step (2). Due to its secretory expression, it simplifies the steps of protein purification, reduces the possibility of denaturation in protein purification, and is effectively separated from host proteins and DNA. After purifying the target protein with an affinity column and molecular sieve, the purity can reach 99% or more. As shown in Figure 4. It was verified by high performance liquid chromatography-mass spectrometry.
  • the purified protein expressed by the present invention has its corresponding biological activity in a living organism.
  • the purified S1190-FC expressed in the present invention can bind to the receptor ACE2 of the S protein, as shown in Fig. 5, Fig. 6, Fig. 7, and Figure 9 shows.
  • S1190-Fc can also be fused to cells with its receptor and into the cell, as shown in Figure 8, Figure 10, and Figure 11.
  • the purified fusion protein expressed by the present invention can be used to prepare a vaccine to prevent infection of the SARS-CoV virus.
  • the invention completes a series of truncated forms of S protein and S protein through a series of optimization of S protein, and studies the functions of each part, and obtains a large number of results and data, and the s protein and Its different truncated forms of application provide the basis.
  • the present invention further completes cell experiments and in vivo experiments in which S protein interacts with ACE2, and finds that S protein can cause down-regulation of ACE2 expression, and down-regulation of ACE2 can aggravate acute lung injury.
  • the present invention and further experiments demonstrate that the site which mainly binds to the ACE2 receptor and causes the ACE2 receptor to down-regulate is the amino acid 318 amino acid to the 510 amino acid of the S protein.
  • the purified fusion protein expressed by the present invention immunizes a mouse to produce a highly potent neutralizing antibody that neutralizes the SARS-CoV virus.
  • mice Five-week-old female bait c mice were divided into two groups, 5 in each group. One group was injected with 50 ⁇ g of S1190-FC plus adjuvant at 0, 2, and 4 weeks, and the other group was injected with the same dose of Fc. Add adjuvant as a control, and collect blood in the sixth week.
  • the heat inactivated serum was subjected to a microneutralization assay to detect the presence of neutralizing antibodies. After double dilution of the heat-inactivated serum, the titer of the neutralizing antibody was measured by a microneutralization assay.
  • Neutralizing antibodies were added to the gradient in 96-well plates, three wells per gradient, and then each well was spiked with TCID50 dose of SARS-CoV-infected Vero E6 monolayer adherent cells, detected on the third and fourth days.
  • Viral cytopathic effect (CPE) using the RM formula to calculate the gradient of CPE in 50% of the wells, and finally the titer of the neutralizing antibody.
  • CPE Viral cytopathic effect
  • the results of the present invention prove that the antibody titer of the S1190-FC group and the control group are significantly different, and the mice immunized by the S1190-FC can produce a large number of effective neutralizing antibodies, which can effectively prevent the infection of the SARS-CoV virus. See Example 11 for specific steps. The result is shown in Figure 22.
  • the present invention demonstrates that immunization of mice with any truncated form of the S protein can produce neutralizing antibodies to SARS-CoV viruses of varying potency, which in turn prevents SARS-CoV infection.
  • the fusion protein of the present invention can be used to prepare a virus detection kit. '
  • the invention verifies the strong immunogenicity of the S protein by animal experiments, and can be used as a detection antigen to detect corresponding antibodies in the blood.
  • the antigenic protein which is expressed in the mammalian host cell system and which can react with the corresponding antibody against the SARS-CoV virus in the human body is purified and then ligated to the microplate, and the principle of ELISA is used.
  • the corresponding antibody appearing in the blood can adsorb the S protein attached to the microplate and further react with the labeled antibody, so that a positive result is detected, thereby assisting diagnosis.
  • the fusion proteins of the invention can be used to prepare or screen for drugs that are resistant to SARS-CoV infection.
  • the S protein obtained by the present invention can also be used for screening therapeutic drugs.
  • the pathogenesis of the SARS-CoV virus is mainly achieved by the interaction of the S protein with the receptor ACE2. Therefore, in the screening of drugs against the SARS-CoV virus, a drug capable of inhibiting the binding of the S protein to the ACE2 receptor can be selected. Including small molecule compounds, peptides and genetically engineered drugs, thereby inhibiting the invasion of SARS-CoV viruses into cells.
  • the present inventors have demonstrated that mice can produce a large amount of neutralizing antibodies by injecting S1190 protein into mice, and the resulting antibodies can inhibit 100 times the TCID50 of SARS-CoV virus infected Vero E6 cells.
  • the fusion protein of the present invention can be used to prepare an antibody against SARS-CoV virus infection.
  • the S protein obtained by the invention can also be used for screening monoclonal antibodies, especially humanized monoclonal antibodies, and the selected monoclonal antibodies can specifically bind to the S protein, thereby preventing the binding of the S protein to the receptor ACE2. Therefore, it can be used as a therapeutic drug for SARS, or passive immunization of the population.
  • amino acid sequence of the SARS-CoV viral structural protein described in the present specification is derived from GenBank NC-004718.
  • the present invention also relates to a method for removing, modifying or mutating the amino acid fragment of amino acid 318 to amino acid 510 of the structural protein S of the SARS-CoV virus, or removing, modifying or mutating to express the 318th position of the structural protein S of the SARS-CoV virus.
  • the present invention also relates to the DNA sequence or the amino acid expressed by the DNA in the preparation prevention
  • the vaccine includes a DNA vector vaccine, a protein vaccine, and a viral vector vaccine.
  • the DNA sequence or amino acid thus obtained is intended to prevent the SARS-CoV viral structural protein S from binding to or reducing the ability to bind to ACE2.
  • Acute respiratory distress syndrome is the most severe form of acute lung injury.
  • Acute respiratory distress syndrome is characterized by pulmonary edema caused by increased vascular permeability, increased inflammatory cells, and severe hypoxia.
  • the predisposing factors for ARDS are diverse, including sepsis, inhalation, and pneumonia caused by SARS coronavirus or avian/human influenza virus.
  • Experimental data of the present invention show that acute lung injury includes mice Infection with SARS leads to a significant decrease in ACE2, an important enzyme in the renin angiotensin system. The damage to the renin-angiotensin system caused by the injury led to a shift in the direction of the increase in Angll.
  • the renin-angiotensin system plays a very important role in maintaining blood pressure stability and hydroelectric balance.
  • Angiotensin-converting enzyme 2 (ACE2) shares homology with ACE and is a negative-regulating component of the RAS system.
  • SARS virus infection in vivo resulted in a significant decrease in ACE2 expression in the lungs of mice.
  • both human and mouse lungs express ACE2, there is no knowledge of the function of ACE2 in the lungs.
  • ace2 deletion To understand the role of ACE2 in acute lung injury and lung failure, we examined the effects of ace2 deletion in an experimental model. This model reproduces the pathological manifestations of pulmonary failure common in many human diseases such as sepsis, acid inhalation, and acute lung injury caused by SARS and avian influenza virus type A infection.
  • the wild-type mice treated with the fusion protein S1190-FC showed a decrease in the expression of ACE2 protein in the lung tissue.
  • Wild type mice were treated with fusion protein S1190-FC and control Fc protein, respectively, and then detected by Western blotting using an antibody against ACE2.
  • the present inventors have found that S1190-Fc-treated mice result in decreased expression of ACE2 protein in lung tissue. As shown in Figure 20.
  • mice Infusion of the acid or salt in the lungs of wild-type mice and the addition of the fusion protein S1190-FC resulted in changes in lung rebound rate.
  • the mice were divided into 4 groups, 5 to 7 in each group. The two groups were subjected to acid perfusion, and the fusion protein S1190-FC and control Fc were added. The two groups were subjected to salt perfusion, and the fusion protein S1190-FC and control Fc were also added. , per mouse perfusion The amount is 5.5 nmol/kg fusion protein S1190-Fc or 5.5 nmol/kg control Fc.
  • Figure 13 is a pathological section of mouse lung tissue.
  • the lung tissue of the mouse treated in the same manner as in the description of Fig. 12 was made into a pathological section, and the results were in agreement with the results described in the description of Fig. 12.
  • lung tissue showed significant edema and acute lung injury.
  • the addition of the fusion protein S1190-FC significantly aggravated the degree of acute lung injury.
  • Figure 14 shows the results of lung injury scores. This result confirms the results of Fig. 12 and Fig. 13 and demonstrates that acute lung injury occurs in lung tissue under acid perfusion conditions. Compared with the control Fc, the addition of the fusion protein S1190-FC significantly aggravates the degree of acute lung injury. There was a significant difference (p ⁇ 0.01).
  • Figure 15 is the result of the wet-dry lung tissue weight ratio. This result confirms the results of Figures 12, 13, and 14. It was demonstrated that in the case of acid-induced acute lung injury, the degree of pulmonary edema caused by the addition of the fusion protein S1190-FC group was more serious than that of the control Fc group, and the wet-dry lung tissue weight ratio was larger, compared with the control Fc group. The ratio was significantly different (p ⁇ 0.05).
  • mice The wild type mouse lung acid or salt perfusion and the fusion protein S1190-FC or fusion protein S318-510-FC caused changes in lung rebound rate.
  • the mice were divided into 5 groups, 5 to 7 in each group, 3 of which were subjected to acid perfusion, and the fusion protein S1190-Fc, fusion protein S318-510-FC and control Fc were added respectively, and the two groups were subjected to salt perfusion.
  • the fusion protein S318-510-FC and control Fc were separately injected at a dose of 5.5 nmol/kg of fusion protein or control Fc per dose.
  • fusion protein S1190-FC After intraperitoneal injection of the fusion protein S1190-FC, S1190-Fc was detected in the lung tissue.
  • the fusion protein S1190-FC was detected by Western blotting and protein G agarose using an anti-Fc antibody, and the control Fc of the control was not detected. As shown in Figure 18.
  • fusion protein S1190-FC Accumulate in bronchial epithelial cells (left column, magnified 100 times), inflammatory secretory cells (middle column, magnified 200 times) and alveolar cells (right column, magnified 200 times), which is also the site of acute lung injury, and found S1190 -FC first accumulates in the site of acute lung injury. As shown in Figure 19.
  • S1190 binds to ACE2 receptor and down-regulates ACE2, causing or aggravating acute lung injury.
  • the site that mainly binds to ACE2 receptor and causes ACE2 receptor down-regulation is amino acid 318 to amino acid 510 of S protein.
  • the fragment of amino acid 318 to amino acid 510 of the S protein itself can cause or aggravate acute lung injury, so the amino acid sequence needs to be removed or modified in the preparation of the vaccine.
  • the truncated form of the SARS-CoV viral structural protein S includes any truncated form in which amino acid 318 to amino acid 510 are removed.
  • ACE2 is a receptor for the SARS-CoV viral structural protein S.
  • cell experiments and in vivo experiments in which S protein interacts with ACE2 have revealed that S protein can cause down-regulation of ACE2, and down-regulation of ACE2 can aggravate acute lung injury.
  • the present invention and further experiments have found that the site which mainly binds to the ACE2 receptor and causes the ACE2 receptor to down-regulate is the amino acid 318 amino acid to the 510 amino acid of the S protein.
  • the SARS-CoV viral structural protein S includes an S protein that is unable to bind to the ACE2 receptor or to reduce its ability to bind to the ACE2 receptor by mutation or modification, and any truncated form thereof.
  • the present invention demonstrates that down-regulation of ACE2 may lead to further deterioration of the body's acute lung injury and other pathological processes, and the site that mainly binds to the ACE2 receptor and causes the ACE2 receptor to down-regulate is amino acid 318 to amino acid 510 of the S protein.
  • Figure 25 is a photograph of cell-transfected S317 Fc and ACE2, gpl20 and ACE2 not fused (magnification 100 times).
  • Figure 26 is a photograph of cell fusion of S318-510-FC with ACE2, S1190-Fc and ACE2 (magnification 100-fold).
  • Figure 27 is a photograph of cell-transfected S511-1190 Fc and ACE2, S681-1190 Fc and ACE2 not fused (magnification 100-fold).
  • ACE2 is a specific receptor for SARS-CoV virus
  • S318-510 is amino acid 318 to 510 of S protein.
  • the invention discloses that the S protein is mutated or modified, in particular, the amino acid 318 amino acid to the 510 amino acid of the S protein is removed, mutated and modified, so that it can not bind to the ACE2 receptor or reduce the binding ability to the ACE2 receptor, in which The selection of a truncated form of S protein with high-efficiency humoral and cellular immunity to SARS-CoV virus is needed to prepare a highly effective and safe anti-SARS-CoV virus vaccine.
  • the vaccine includes a DNA vector vaccine, a protein vaccine, and a viral vector vaccine. detailed description:
  • the whole gene of SEQ ID No: 1 was synthesized by Shanghai (China) Boya Biotechnology Co., Ltd. using a gene synthesis method well known in the art.
  • the well-known whole-genome artificial synthesis method uses 100 base oligonucleotide primers corresponding to each other and corresponding PCR amplification primers, and the oligonucleotide primers overlap by 20 bases in two parts to form a complete gene, and are connected. Annealing, PCR amplification, synthesis of whole genes.
  • Example 2 Plasmid construction and identification
  • S317 forword: 5 'GGCGCTAGCCAGCGACCTGGACCGCTGC3 '
  • the PCR amplification kit (2 X pfu PCR Master Mix, Cat No: KP-201, Beijing Tianwei Times Technology Co., Ltd.) was used according to the kit instructions and was added to the 50 ⁇ l reaction system.
  • the denaturation temperature was 94V for 5 minutes, and then entered into the following 30-40 cycles, the denaturation temperature was 94 ° C for 1 minute, the annealing temperature was 55 ° C for 30 seconds, the extension reaction was 72 V for 1-2 minutes, and at the end of the cycle reaction, 72 ⁇ 10 minutes was given. Prolonged response.
  • the obtained PCR product was subjected to electrophoresis using 5 ⁇ l of 1% agarose gel (Agarose, TED & HY Bio Co: Ltd Cat NO: A9918).
  • the correct PCR product was purified using a PCR clean-up kit (VITAGENE, Cat No: 110310-05), and the resulting purified product was stored in 25 ⁇ l ⁇ (Molecular Cloning Protocol Guide, Second Edition).
  • the Fc fragment sequence was derived from the human IgG Fc fragment DNA sequence in GenBank and synthesized by Shanghai Boya Biotechnology Co., Ltd.
  • pEAK13 as a vector, digested with restriction enzymes EcoR I, Not I, and then ligated, transformed, and detected (the specific procedure is as follows) to obtain plasmid pEAK13 CD5L Fc.
  • Fragment source Other plasmids containing this gene in our laboratory.
  • the insert DR was first digested from its vector by restriction endonucleases Pst I, Bgl II. Then, pEAK13 CD5L Fc was used as a vector, digested with restriction endonucleases Pst l and Bgl II, and then ligated, transformed, and detected (the specific procedure is as follows) to obtain plasmid pEAK13 CD5L Fc DR.
  • the vector DNA or PCR product is added to the restriction enzyme buffer system for digestion for 1-3 hours.
  • Total volume of digestive system 20 ⁇ 1 Add 1 ⁇ g DNA, 2 ⁇ 1 10XBSA (0.1% BSA), 2 ⁇ 1 10XNEB Buffer, 0.5 ⁇ restriction endonucleases Nhe I and BamH I (all restriction enzymes) , 0.1% BSA, NEB Buffer was purchased from NEW ENGLAND BioLabs® Inc, USA).
  • the vector DNA digestion system was supplemented with 0.5 ⁇ l of alkaline phosphatase (Promega, USA, CatNo: M182A) to remove the phosphate groups from the digested ends of the vector.
  • the comb is removed, placed in an electrophoresis tank, and TAE buffer containing 500 ug/L Ethidium Bromide Promega, USA, Cat# H5041) (Molecular Cloning Experimental Guide, Second Edition), each in the well 15-20 ⁇ l of the digested vector DNA and the digested PCR product were added.
  • DNAMarker such as: ⁇ -Hind III, DL2000 (TaKaRa Biotechnology (Dalian) Co. Ltd) was added to determine the fragment size of the DNA.
  • Electrophoresis 60-80V electrophoresis instrument DYY-6C, Beijing Liuyi Instrument Factory), 20-60 minutes.
  • the electrophoresed gel was transferred to ultraviolet light (UV-IV UV analyzer, Beijing New Technology Application Research Institute), photographed, and the desired DNA bands were cut.
  • UV-IV UV analyzer Beijing New Technology Application Research Institute
  • the cut gel containing the desired DNA band was placed in a 1.5 ml centrifuge tube, and the gel was dropped to the bottom of the tube by a short high-speed centrifugation, and the gel was melted by heating at 65 Torr.
  • the ligation system Divide the ligation system into two parts (20 ⁇ l each), add 2-4 ⁇ l of the gel containing the DNA fragment to be inserted to one of the ligation systems, and add the corresponding volume of deionized water as a negative control.
  • the amount of carrier and insert DNA in the ligation system is maintained at about 1:2, and the total volume of the low melting gel at 1.5% of the 20 ⁇ l system is no more than 6 ⁇ l).
  • LB agar containing no ampicillin on the surface of agar containing LB agar culture containing 50 ⁇ g/ml ampicillin (Molecular Cloning Protocol Guide, Second Edition Reagent Preparation Method, Ampicillin (Huabei Pharmaceutical Factory, China)) -6ml, used after solidification.
  • the competent cells Immediately after the competent cells have melted, the ligation product and the negative control are added.
  • Each ⁇ ⁇ ⁇ chemically competent bacteria is added to a volume of 5-8 ⁇ M, gently mixed, and placed on ice for 15-30 minutes.
  • the cell suspension was aspirated and placed in a petri dish just added to the LB medium, and cultured at 37 ° C for 12-16 hours to grow monoclonal colonies.
  • the monoclonal colonies were picked with a toothpick into 4 ml of LB liquid medium containing ampicillin, placed in a 37 °C shaker (table thermostat THZ-D, Pei Ying), 250-280 rpm, shaken for 7-8 hours.
  • the liquid is long to saturated.
  • Plasmid DNA was extracted using the plasmid mini-kit (Beijing Tianwei Times Technology Co., Ltd., DP-103) according to the method provided.
  • the plasmid obtained by the small extraction was dissolved in 50-60 ⁇ 1TE, and 10 ⁇ l was digested with restriction endonucleases Nhe I and BamH I, and the corresponding strain corresponding to the plasmid was selected by restriction enzyme digestion.
  • the correct plasmid was digested and sequenced (Shanghai Boya Biotechnology Co., Ltd. and Shanghai Shenggong Bioengineering Technology Service Co., Ltd.) for further confirmation.
  • the recombinant plasmid was extracted in large quantities by CsCl density gradient centrifugation (for the experimental method, see Molecular Cloning Experiment Guide >> Second Edition).
  • E transfects the cells to determine the high amount of correct expression of the fusion protein.
  • CALBIOCHEM® CLONTECH kills cells that have not been transferred to DNA.
  • ElectroPac BasicTM Power Supply Available from BIO-RAD, Catalog
  • Electrophoresis Device (Mini-PROTEAN® 3 Cell): Available from BIO-RAD, Catalog Numbers: 165-3301, 165-3302
  • Sample source Medium taken from transfected cells, culture medium of the constructed cell strain, and purified protein.
  • SDS polyacrylamide gel was poured.
  • the separation gel concentration is 10%, and the concentration of the gel is 5% (for the preparation method, see Molecular Cloning Test Guide, Second Edition, pp. 883-884, Science and Technology Press).
  • the potting glass plate is taken out from the fixing frame, and installed in the electrophoresis apparatus according to the instructions, and the entire electrophoresis cassette is filled with 1 X electrophoresis buffer (for the preparation method, see Molecular Cloning Test Guide, Second Edition, page 884, Science and Technology Press).
  • the initial voltage is 80V.
  • the voltage is 120V and continue electrophoresis until the bromophenol blue dye reaches the bottom of the separation gel or all of the gel is removed. Turn off the power (about 120 minute).
  • Transfer conditions Constant current 300 mA, time 120 minutes.
  • the blocking solution was discarded, and the primary antibody was diluted with 2% chicken egg white blocking solution and allowed to stand at room temperature for 3 hours or 4 ° C overnight.
  • the membrane was washed 3 times with TBST for 10 minutes each time.
  • Discard TBST dilute the secondary antibody with 2% chicken egg white blocking solution, and combine for 1 to 2 hours at room temperature (for the protein label of the directly available secondary antibody such as Fc, after blocking, directly add the secondary antibody diluted in the blocking solution for binding) .
  • the membrane was washed 3 times with TBST for 10 minutes each time.
  • the film In the dark room, the film is exposed in a cassette, then developed for 4-5 minutes, and fixed for 4-5 minutes.
  • the film is Kodak X-Omat BT Film, manufactured by China Shantou Kodak Co., Ltd., manufactured by Eastman Kodak Company, USA. Specifications: 12.7 X 17.8cm, Emulsion No.: 031222104) (Development powder and fixing powder purchased from Tianjin Hebei Photosensitive material factory)
  • By qualitatively testing the cell monoclonal supernatant by Western Blotting we detected a band that matched the expected size.
  • Example 5 Detection of the expression level of fusion protein by ELISA
  • the concentration of the protein contained in the medium can be detected by an ELISA method.
  • the reagent used in the ELISA is
  • the ELISA test method is as follows:
  • sensitizers A and B substrate reagent ⁇ / ⁇
  • susceptor After mixing eight times to the sensitizer after mixing, 100 ⁇ l per well, and keep it at room temperature for 30 minutes.
  • the expression level of the S protein and its truncated form protein is above 10 g per 24 hours of expression per million cells.
  • the expression level of the fusion protein S1190-FC in the supernatant can reach above 10 w g, and the expression level of the fusion protein S1148 Fc can reach more than 20 in the supernatant, and the expression level of the fusion protein S900 Fc can reach more than 20 in the supernatant.
  • the fusion protein S685 Fc can express more than 20 g in the supernatant, the fusion protein S511 Fc can express more than 20 ⁇ g in the supernatant, and the fusion protein S317 Fc can express up to 20 g in the supernatant.
  • the fusion protein S318-1190 Fc can express more than 10 ⁇ g in the supernatant, and the fusion protein S318-510-FC can express more than 30 g in the supernatant, and the fusion protein S511-1190 Fc is above.
  • the expression level of the fusion protein can reach 10 ⁇ g or more, and the fusion protein S681-1190 Fc can express more than 10 in the supernatant.
  • Vero E6 cells have the receptor ACE2 of S protein, which mainly interacts with amino acids 318 to 510 of S protein. By utilizing the binding properties of the receptor and the ligand, it is possible to confirm the correct folding of the protein, that is, the conformation is correct.
  • Vero E6 cells or 293 cells transfected with ACE2 were digested with PBS/2 mM EDTA, and divided into several portions, which were placed in a centrifuge tube.
  • the cells were resuspended separately with a cell culture medium containing the S protein or its truncated form, and an IMDM (purchased from GIBCO) medium supplemented with serum (purchased from Hyclone) was used as a negative control.
  • IMDM purchased from GIBCO
  • serum purchased from Hyclone
  • Resuspension cells such as FITC/anti-human IgG (purchased from Jackson ImmunoResearch) or FITC/anti-His (purchased from Sigma) (if there is no fluorescently labeled secondary antibody, you need to use it first)
  • Vero E6 cells are known to have the receptor ACE2 of the S protein, and the S protein is down-regulated by the ACE2 receptor. Vero E6 cells can be used.
  • the medium (containing 10% fetal bovine serum (Hyclone)) grown to 50%-70% of Vero E6 cells in a 10 cm cell culture dish (Greiner bio-one) was removed, and washed three times with PBS.
  • the serum-free medium was added and cultured in a C02 incubator (SANYO, MCO-15AC) at 37 ° C for 1 hour.
  • control Fc and one of the cells to which the fusion protein S1190-FC was added were placed at 4 ° C and slowly rotated, and the other portion was placed at 37 ° C and slowly rotated, and the process was maintained for 3 hours.
  • the 293ET cells in the middle of the logarithmic growth phase were digested with trypsin, and after the cells were rounded, they were added to DMEM medium (purchased from GIBCO) and blown into individual cells.
  • the cells were digested with trypsin, counted, and the cells were mixed in two wells and placed in a well of a 12-well cell culture plate.
  • the number of cells per well was: 2X 10 4 , 4X 10 4 , 6X 10 4 , 8 X 10 4 , 1 X 10 5 .
  • the cells were harvested and lysates, 12000rpm, 2min, 4 ° C by centrifugation (BECKMAN COULTER TM, Microiuge® 22R Centrifoge ).
  • Proteins bearing the Fc tag were purified using a Protein A column, and proteins with a 6His tag were purified using a nickel column.
  • the Fc-tagged protein was purified using Amersham's ProteinA protein column. (Amersham Biosciences AB, Sweden; CAT NO: 17-04020-03)
  • Dialysis The collected supernatant is dialyzed.
  • the components of the dialysate were: 11.54 mM/L Na 2 HP04, 8.46 mM/L NaH 2 P04 (Beijing Chemical Plant, China), ImM EDTA (Promega USA), pH 7.0.
  • the dialysis time is generally not less than 8 hours, and the volume of the dialysate should be at least 20 times the volume of the supernatant. '
  • the purified protein samples were identified by Western Blotting and Coomassie Brilliant Blue staining with SDS polyacrylamide gel.
  • the protein concentration was determined by Lowry method (Lowry method kit was purchased from Tianxiang Bonding Co., Ltd., CAT NO: TB090- 1 ).
  • Example 11 Detection of Neutralizing Antibody in Serum by Vaccine After immunization with S1190-FC, the titer of neutralizing antibody was produced in vivo.
  • Neutralizing antibodies were added to the gradient in 96-well plates, three wells per gradient, and then each well was spiked with 100% of the TCID50 dose of SARS-CoV-infected Vero E6 monolayer adherent cells, the third and fourth days.
  • Viral cytopathic effect (CPE), using the RM formula to calculate the gradient of CPE in 50% of the wells, and finally the titer of the neutralizing antibody.
  • Example 12 Lung Elasticity Analysis Experiment 1) Mice aged 2.5 to 3 months were divided into 5 groups of 5 to 7 mice each.
  • the amount of ventilation is measured using a constant flow ventilator that controls the flow of gas.
  • VRM gas flow replenishment adjustment
  • VRM air flow replenishment adjustment
  • mice were again injected intraperitoneally with S1190-Fc, S318-510-Fc, or control Fc (each 5. 5 nmol/kg).
  • control Fc each 5. 5 nmol/kg.
  • Example 12 The right lung of the mouse as described in Example 12 was used as a sample, and lung tissue was fixed with 3.7% formalin and embedded in paraffin.
  • Example 14 1) In each of the four sections in Example 14, four fields of view were randomly selected, and each group of 16 fields of view was scored by blind method according to a prescribed scoring standard.
  • the scoring range includes: alveolar congestion, hemorrhage, neutrophil infiltration, alveolar wall thickness, and transparent membrane formation.

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Description

重组 SARS-CoV病毒结构蛋白的融合蛋白及其制备方法和应用 技术领域
本发明涉及 SARS-CoV病毒结构蛋白的融合蛋白及其在哺乳动物细胞中的高量 表达； 用所述融合蛋白在制备防治 SARS- CoV病毒感染的基因工程疫苗和药物的用 途； 以及用所述融合蛋白在制备检测 SARS- CoV病毒感染试剂盒的用途。 本发明还 涉及 SARS- CoV病毒结构蛋白 S的有毒片段的发现； 以及设计预防 SARS-CoV病毒 感染的各类疫苗。 技术背景
严重急性呼吸系统综合征 (Severe Acute Respiratory Syndrome, SARS)的病原体 是一种新型冠状病毒 (SARS-CoV), 其宿主范围广， 传播速度快， 且可通过飞沬甚 至空气传播， 危害极大。 因此， SARS-CoV病毒感染的预防、 治疗及检测的研究 迫在眉睫。
由于 SARS-CoV病毒疫苗的应用对象是人，疫苗的稳定和安全可靠是最基本、 最重要的要求。 而基因工程疫苗的研究已较为成熟， 与此要求最为相符。
SARS-CoV病毒存在 S、 M、 N和 E四种结构蛋白。实验生物信息学的研究表 明 S蛋白和 N蛋白具有强烈的免疫原性， 是疫苗研究的主要抗原。 M和 E也存在 一定的免疫原性， 都有可能成为有效疫苗。 其中 S蛋白成为有效疫苗的可能性最 大。
但由于 s蛋白本身的特点， 使其在成功表达全长的有活性的 s蛋白及 s蛋白 的纯化方面存在重重困难。
由于 s蛋白存在大量的翻译后修饰位点， 主要是糖基化位点， 致使通过原核 细胞表达或酵母表达的蛋白将不能正确地折叠， 影响其生物活性。 只有在哺乳动 物细胞中表达， S蛋白才能进行正确地修饰，折叠和加工处理，也更接近自然状态， 否则将会严重影响疫苗的免疫效果。但是， S病毒原编码的 S蛋白在哺乳动物细胞 表达系统中表达量很低， 难以在实际中应用。
因此， 为制备有效的 SARS-CoV 病毒疫苗， 必须解决三个问题： 一是使 SARS-CoV病毒 S蛋白在哺乳动物细胞中有效表达， 并在纯化过程中尽可能选择 合适的条件， 使所表达的蛋白能够与宿主蛋白和 DNA有效分离， 以保证病毒蛋白 的结构尤其立体结构不被破坏； 二是提高 S蛋白的表达产量， 使疫苗的生产更为 经济。 三是疫苗的安全性， SARS- CoV病毒致病机理的研究有限， SARS- CoV病毒 是如何导致急性肺损伤， 心脏功能衰竭， 以及免疫系统崩溃的途径并不清楚， 现 有技术 SARS疫苗的制备中存在安全隐患。 而且现有技术未能解决这个问题， 因 此， 制备安全有效的 SARS-CoV病毒疫苗需要进一歩的发明研究。 发明内容
为了克服现有技术的不足， 本发明的首要目的是提供一种能够在哺乳动物细 胞株中表达的 SARS-CoV病毒 S蛋白基因片段。
本发明的第二个目的是将 SARS病毒的结构蛋白及其截短形式的融合蛋白在 哺乳动物细胞表达系统中进行高量表达和纯化。
本发明的第三个目的是用所得到的 SARS病毒的结构蛋白的融合蛋白，包括 S 蛋白的融合蛋白， 制备预防 SARS-CoV病毒感染的基因工程疫苗； 我们的研究发 现， S蛋白与其受体 ACE2的结合可导致或加剧机体的急性呼吸窘迫综合症，研发 安全有效的疫苗需要删除或修饰 S蛋白中与其受体 ACE2结合的片段。
本发明的第四个目的是用所得到的 SARS-CoV病毒的结构蛋白的融合蛋白， 制备检测 SARS-CoV病毒感染的试剂盒。
本发明的第五个目的是用所得到的 S 蛋白融合蛋白制备和筛选治疗 SARS-CoV病毒感染的药物。
本发明的第六个目的是用 SARS-CoV病毒 S蛋白的已去除， 突变， 或修饰其 氨基酸 318— 510 片段并使 S 蛋白不能与其受体 ACE2 结合的片段制备预防 SARS-CoV病毒感染的疫苗， 包 DNA疫苗， 蛋白疫苗， 和病毒载体疫苗等。
在本文中， S蛋白截短形式的描述以如下方式表示： "Sa-b"
表示从野生型 SARS-CoV病毒的全长的 S蛋白的第 a位氨基酸到第 b位氨基 酸。
当 a是起始第一个氨基酸时， 以 "Sb"方式表示。
例如， S318-510是指该基因片段表达出的蛋白是整个 SARS-CoV病毒 S蛋白 的 318到 510位氨基酸， S511是指该基因片段表达出的蛋白是整个 SARS-CoV病 毒 S 蛋白的 1 到 511 位氨基酸， S685 是指该基因片段表达出的蛋白是整个 SARS-CoV病毒 S蛋白的 1到 685位氨基酸， 依此类推。
本发明的技术解决方案包括：
一种 SARS-CoV病毒结构蛋白的融合蛋白， 其结构为： X-Y-Z,
其中 X包括 SARS-CoV病毒结构蛋白 S或 M或 E或 N， 或以上结构蛋白的 任意截短形式， 所述 SARS-CoV病毒结构蛋白 S包括去除、 修饰或者突变第 318 位氨基酸到 510位氨基酸任意氨基酸片段， 或者去除、 修饰或突变第 318位氨基 酸到 510位氨基酸片段；
Y是连接部分， 由 0-20个任何氨基酸组成； Z是包含铰链区、 CH2、 CH3结构域的人 的？0及其变体或蛋白标签。 所述蛋白标签包括且不限于六组氨酸 (6XHis)标签、聚乙二醇 (PEG)标签和人血 清白蛋白 (Human serum albumin, HS A)标签。
所述 SARS-CoV病毒结构蛋白 S包括蛋白 S的全长或其任意截短形式。
所述 SARS-CoV病毒结构蛋白 S是不能与受体 ACE2结合或者降低与 ACE2 结合能力的蛋白。
所述的 Y优选是两个氨基酸， 所述的氨基酸为赖氨酸和精氨酸。
本发明还涉及一种能够在哺乳动物细胞株中表达的 SARS-CoV病毒 S蛋白基 因， 其特征在于所述的基因为 SEQ ID NO: 1。
本发明进一步涉及一种含有 SEQ ID NO: 1的重组表达质粒， 所述的质粒包括 真核 PEAK系列。
本发明还进一步涉及哺乳动物细胞株， 其含有所述的能够表达 SARS-CoV病 毒 S蛋白基因的所述的 SARS-CoV病毒结构蛋白的融合蛋白。该哺乳动物细胞株， 包括 CHO、 293和 Vero细胞株及其衍生细胞株。
本发明还涉及所述的 SARS-CoV病毒结构蛋白的融合蛋白的制备方法，包括：
( 1 ) 转染能够表达权利要求 1 所述融合蛋白及内源性二氢叶酸还原酶 (endogenous dihydrofolate reductase) (dhfr)的重组表达质粒，构建哺乳动物细胞表达 株；
(2)该哺乳动物细胞表达株在正常生长状态下每 24小时每百万细胞在其培 养基中产生 10 μ§以上重组蛋白；
(3 )纯化通过步骤（2)表达的蛋白。
在该方法中，所述的重组表达质粒含有的融合蛋白的引导序列，该序列是 CD5 蛋白的引导序列。 所述的重组表达质粒， 其结构蛋白的编码基因进行人工合成， 用人体细胞内常用或者偏好密码子替换相同氨基酸的病毒基因中的密码子序列， 对病毒的结构蛋白进行人类密码子优化。 所述的表达 SARS-CoV病毒的结构蛋白 的融合蛋白， 其用人体细胞常用或者偏好密码子合成的 SARS-CoV的 S蛋白基因 如 SEQ ID NO: 1所示。
构建哺乳动物细胞表达株所用的筛选药物优选包括嘌呤霉素和氨甲蝶呤。 在该方法步骤（2)是每 24小时每百万细胞在其培养基中可产生 30 μ§或者以 上的重组蛋白。 本发明还涉及所述的融合蛋白在制备预防 SARS-CoV病毒的疫苗中的用途； 以及在制备 SARS-CoV病毒检测试剂盒中的用途； 在制备或筛选抗 SARS-CoV病 毒感染的药物中的用途； 在制备防止 SA S-CoV病毒感染的抗体中的用途。
本发明还特别涉及一种去除、 修饰或者突变表达 SARS-CoV病毒结构蛋白 S 第 318位氨基酸到 510位氨基酸任意氨基酸片段， 或者去除、 修饰或突变表达
SARS-CoV病毒结构蛋白 S第 318位氨基酸到 510位氨基酸片段序列的 DNA序列 以及由这种 DNADNA表达的氨基酸。
本发明进一步涉及所述的 DNA序列或者所述的 DNA表达的氨基酸在制备预 防 SARS-CoV病毒的疫苗中的用途， 所述的疫苗包括 DNA载体疫苗， 蛋白疫苗， 病毒载体疫苗。 附图说明- 图 1是用 Western Blotting的方法确定优化后的 E、 M、 N和 S的融和蛋白宿 主细胞中表达的检测结果。从左到右分别为 E-Fc、 M-Fc、 N-Fc和 S- Fc。 结果证明 SARS-CoV病毒的四种结构蛋白均可在宿主细胞中得到较好的表达。
图 2是 S1190基因片段插入表达载体后酶切鉴定的琼脂糖凝胶电泳结果。 从 左向右三个泳道依次为 λ -Hind III Marker, S1190, DL2000 Marker。 λ -Hind ΙΠ Marker从小到大（从底端到上端 )依次为 564bp (从图中较难分辨)、 2027bp、 2322bp、 4361bp、 6557bp、 9416bp、 23130bp; DL2000 Marker从小到大 （从底端到上端） 依次为 100bp、 250bp、 500bp、 750bp、 1000bp、 2000bp, 证明 SI 190的基因片段 已经插入到表达载体中。
图 3是用 Western Blotting的方法确定融合蛋白 S1190-Fc在宿主细胞中表达的 检测结果， 证明融合蛋白 S1190-FC可以在宿主细胞中得到较好的表达， 表达出的 蛋白大小约为 185KD。
图 4是纯化后的融合蛋白 S1190-FC聚丙烯酰胺凝胶的考马斯亮蓝染色的结果， 证明可以得到较为纯净的融合蛋白 S1190-Fc。
图 5是融合蛋白 S1190-FC在 4°C结合 Vero E6细胞的细胞流式结果。由结果可 知，融合蛋白 S1190-FC与 Vero E6细胞结合。通过国际上已发表的文献可知， Vera E6细胞表面存在 S蛋白的受体 ACE2， ACE2的结合 S蛋白的区域为 318位到 510 位氨基酸。 所以， 用此细胞粘附融合蛋白 S1190-Fc， 可检测表达出的融合蛋白 S1190-FC的活性。 图中空白的峰形区域是阴性对照（PBS缓冲液）， 阴影部分是实 验结果， 证明融合蛋白 S1190-Fc粘附 Vero E6细胞。
图 6是融合蛋白 S1190-FC与转染了人源 ACE2 (hACE2)的 293E细胞结合的 细胞流式结果。 转染了 hACE2的 293E细胞与融合蛋白 S1190-Fc在 4°C进行结合 后， 再与 FITC标记的抗 Fc的抗体结合， 未转染的 293E细胞结合 Fc抗体作为阴 性对照 （空白峰形区域）， 然后用流式进行检测。 结果证明转染了 hACE2的 293E 细胞与融合蛋白 S1190-FC结合， 发生明显位移 （阴影部分)。
图 7是融合蛋白 S1190-FC与转染了鼠源 ACE2 (mACE2) 的 293E细胞结合 的细胞流式结果。 转染了 mACE2的 293E细胞与融合蛋白 S1190-Fc在 4°C进行结 合后， 再与 FITC标记的抗 Fc的抗体结合， 未转染的 293E细胞结合 Fc抗体作为 阴性对照（空白峰形区域），然后用流式进行检测。结果证明转染了 mACE2的 293E 细胞与融合蛋白 S1190-FC结合， 发生明显位移 （阴影部分）。
图 8的左图是 293ET细胞分别转染了 ACE2及 S1190基因后发生了细胞融合 的照片 （放大 100倍）； 右图是 293ET细胞分别转染了 CD4及 S1190基因后未发 生细胞融合的照片（放大 100倍）。 证明蛋白 S1190与 ACE2结合， 并能够导致细 胞融合。
图 9是 S1190-FC与 ACE2受体的免疫共沉淀（IP) 的结果。 将分别转染了 S1190-FC和 ACE2, 对照 Fc和 ACE2的细胞裂解， 然后用 Western Blotting的方法 进行检测。 左侧的第一条条带是转染了对照 Fc和 ACE2的细胞裂解液作为对照， 第二条条带是转染了对照 Fc和 ACE2的细胞裂解液 IP结果，第三条条带是转染了 S1190-FC和 ACE2的细胞裂解液作为对照，第四条条带是转染了 S1190-Fc和 ACE2 的 IP结果。 结果证明了蛋白 S1190与 ACE2受体结合， Fc对 S1190与受体 ACE2 的结合没有影响。
图 10是用培养细胞做 ACE2受体下调实验的结果。 将 Vero E6细胞分别在 4
°C (篮线）和 37°C (红线） 与融合蛋白 S1190-FC充分作用， 同时用 Fc作为对照 (黑线）， 然后用抗 Fc的抗体进行检测。 结果证明在 37Ό时， 融合蛋白 S1190-FC 与受体 ACE2相互作用， 并导致 ACE2受体下调。
图 11是用培养细胞做 ACE2受体下调实验的结果。 将 Vero E6细胞分别在 4 。C (篮线） 和 37Ό (红线）与融合蛋白 S1190-FC充分作用， 同时用 Fc作为对照 (黑线），然后用抗 ACE2的抗体进行检测。结果证明在 37°C时，融合蛋白 S1190-FC 与受体 ACE2相互作用， 并导致 ACE2受体下调。
图 12是野生型小鼠肺部酸或盐灌注并加注融合蛋白 S1190-FC的肺弹回率变化 的结果。将小鼠分为 4组，每组 5到 7只，两组进行酸灌注,并加注融合蛋白 S1190-FC 和对照 Fc， 两组进行盐灌注， 同样加注融合蛋白 S1190-FC和对照 Fc，每只小鼠灌 注剂量为 5.5 nmol/kg融合蛋白 S1190-Fc或 5.5 nmol/kg对照 Fc。 结果证明野生型 小鼠酸灌注加注融合蛋白 S1190-FC组与酸灌注加注对照 Fc组相比，在全部的测量 时间内弹回率的变化具有显著性差异 （p < 0.05)， 酸灌注加注融合蛋白 S1190-FC 组肺弹回率变化幅度明显高于酸灌注加注对照 Fc组。 证明在酸灌注条件下， 融合 蛋白 S1190-FC加重急性肺损伤。
图 13是小鼠肺组织病理切片。 将图 11附图说明中同样处理的小鼠肺组织制 成病理切片， 结果与图 11附图说明中所述结果吻合。 在酸灌注条件下， 肺组织出 现明显水肿， 发生急性肺损伤， 与对照 Fc相比，加入了融合蛋白 S1190-FC后明显 加剧了急性肺损伤的程度。
图 14是肺损伤评分结果。 此结果印证了图 11和图 12的结果， 证明在酸灌注 条件下，肺组织发生急性肺损伤， 与对照 Fc相比，加入了融合蛋白 S1190-FC后明 显加剧了急性肺损伤的程度， 两者具有显著性差异 （ρ < ο.οι )。
图 15是湿干肺组织重量比的结果。 此结果印证了图 11、 12、 13的结果。 证 明在酸诱导的急性肺损伤的情况下， 相较于对照 Fc组， 加入融合蛋白 S1190-FC 组所造成的肺水肿程度更为严重， 湿干肺组织重量比更大， 与对照 Fc组相比具有 显著性差异 （p < 0.05)。
图 16 野生型小鼠肺部酸或盐灌注并加注融合蛋白 S1190-FC 或融合蛋白
S318-510-FC的肺弹回率变化的结果。 将小鼠分为 5组， 每组 5到 7只， 其中 3组 进行酸灌注， 并分别加注融合蛋白 S1190-Fc、 融合蛋白 S318-510-FC和对照 Fc， 2 组进行盐灌注， 并分别加注融合蛋白 S318-510-FC和对照 Fc, 每只灌注剂量为 5.5 nmol/kg融合蛋白或对照 Fc。 结果证明野生型小鼠酸灌注加注融合蛋白 S1190-Fc 组或融合蛋白 S318-510-FC组与酸灌注加注对照 Fc组相比， 在全部的测量时间内 弹回率的变化具有显著性差异 （p < 0.05)。 证明在酸灌注条件下， S1190-FC和 S318-510-FC都可以加重急性肺损伤。
图 17是 ACE2敲除小鼠肺部酸或盐灌注并加注融合蛋白 S1190-FC的肺弹回率 变化的结果。 过程与分组情况可参照图 11的说明。 结果证明在 ACE2敲除的小鼠 中， 在酸灌注条件下， S1190-FC和对照 Fc对肺组织的弹回率的变化没有显著性差 异。
图 18 腹膜内局部注射融合蛋白 S1190-FC 后， 可在肺组织匀浆中检测到 S1190-Fc。用抗 Fc的抗体通过 Western blotting和蛋白 G琼脂糖的方法可检测到融 合蛋白 S1190-Fc， 对照的鼠的 Fc则没有检测到。
图 19是检测融合蛋白 S1190-FC的肺免疫组化的结果。 结果表明， 融合蛋白
S1190-FC聚积在支气管上皮细胞（左列， 放大 100倍）， 炎性分泌细胞（中列， 放 大 200倍）和肺泡细胞（右列，放大 200倍），这也是急性肺损伤容易发生的部位， 证明 S1190-Fc首先聚积在急性肺损伤部位。
图 20用融合蛋白 S1190-FC处理过的野生型小鼠，肺组织 ACE2蛋白表达量下 降。 分别用融合蛋白 S1190-FC和对照 Fc蛋白处理野生型小鼠， 然后用抗 ACE2 的抗体通过 Western blotting的方法进行检测。 结果证明 S1190-Fc处理过的小鼠导 致肺组织 ACE2蛋白表达量下降。
图 21是检测野生型小鼠肺组织的 Angll水平的结果。 将野生型小鼠用酸或盐 灌注肺组织， 并加注融合蛋白 S1190-FC或对照 Fc蛋白， 3小时后， 用 EIA测定肺 组织 Angll的水平。 结果证明在酸处理组， 肺组织注入融合蛋白 S1190-FC与对照 Fc蛋白的 Angll的水平有明显差异（p < 0.05)，酸处理组并加注融合蛋白 S1190-Fc 的野生型小鼠肺组织 Ang ll的水平明显增加， 远髙于酸处理过并加注对照 Fc蛋白 的小鼠的肺组织 Angll的水平。
图 22是小鼠用 S1190-FC免疫后， 体内产生中和抗体的滴度 （橙色）。 将五周 龄的雌性 balb/e小鼠分为两组，每组 5只，一组在 0周、 2周、 4周注射 50 μ g S1190-Fc 加佐剂进行免疫， 另一组注射同样剂量的 Fc加佐剂作为对照 （蓝色）， 第六周采 血收集血清。 将热灭活的血清进行微量中和分析检测中和抗体的存在。 结果证明， S1190-FC组与对照组抗体滴度有明显差异， 经 S1190-FC免疫后的小鼠能够产生大 量有效的中和抗体， 可有效阻止 SARS-CoV的感染。
图 23是 S基因及其片段插入表达载体后酶切鉴定结果的电泳图。从左向右依 次为 λ -Hind III Marker, S317, S318-510, S318-1190, S511-1190, S685, S900, S1148, S1190, DL2000 Marker。 λ -Hind III Marker从小到大（从底端到上端） 依次为 564bp (此条带从图中较难分辨）， 2027bp, 2322bp, 4361bp， 6557bp, 9416bp, 23130bp; DL2000 Marker从小到大（从底端到上端）依次为 100bp， 250bp, 500bp, 750bp, lOOObp, 2000bp。 通过本图可以确定， S蛋白基因及其片段正确插入了表 达载体。
图 24是用 Western Blotting的方法确定优化后的 S融合蛋白及其截短形式在宿 主细胞中表达的检测结果。 从 1-10分别为 S1190-Fc， 约为 185KD; S1148 Fc, 约 为 180KD; S900 Fc, 约为 175 D; S318-1190 Fc, 约为 160KD; S511-1190 Fc， 约为 155KD; S685 Fc, 约为 155KD; S511 Fc， 约为 140KD; S681-1190 Fc, 约为 120KD; S317 Fc,约为 85KD; S318-510-Fc, 约为 67KD。 通过本图可以确定， 优 化后的 S融合蛋白基因及其片段在宿主细胞中得到了较高量的表达， 而优化前的 原始序列在哺乳动物细胞中难以表达， 证明了表达优化方法有效可行。
图 25是细胞转染 S317 Fc与 ACE2， gpl20与 ACE2未见融合的照片 （放大 100倍）。
图 26是细胞转染 S318-510-FC与 ACE2， S1190-Fc与 ACE2发生融合的照片 (放大 100倍）。
图 27是细胞转染 S511-1190 Fc与 ACE2， S681-1190 Fc与 ACE2未见融合的 照片 （放大 100倍）。
用 293细胞分别转染上述 S蛋白的几种截短形式与 ACE2受体，或 gpl20 (HIV 表面蛋白)与 ACE2受体， 然后将转染 24小时的两种细胞混合在一起， 48小时拍 照。 以上六张照片说明， ACE2是 SARS-CoV病毒的特异性受体， 与 ACE2发生 作用并导致细胞融合的部分是 S318-510, 即 S蛋白的 318位到 510位氨基酸。
以下将结合附图详细说明本发明。 由此可见， 本发明提供一种能够在哺乳动 物细胞株中表达的 SARS-CoV病毒 S蛋白基因序列， 例如本发明所述的序列 SEQ ID NO: 1。
另外， 本发明还提供一种含有 SEQ ID NO: 1的重组表达质粒， 所述的重组表 达质粒优选包括真核 PEAK系列。
本发明还提供经过能够在哺乳动物细胞株中表达的 SARS-CoV病毒 S蛋白基 因序列表达出来的一种 SARS-CoV病毒结构蛋白的融合蛋白， 其结构为： X-Y-Z 其中 X包括 SARS-CoV病毒结构蛋白 S或 M或 E或 N， 或以上结构蛋白的 任意截短形式；
Y是连接部分， 由 0-20个任何氨基酸组成；
Z是包含铰链区、 CH2、 CH3结构域的人 的？(：及其变体或蛋白标签。 由此可见， 通过转染能够表达 SARS-CoV病毒结构蛋白及其任意截短形式的 融合蛋白及内源性二氢叶酸还原酶 (endogenous dihydrofolate reductase, d fr)的重组 质粒， 构建哺乳动物细胞表达株。
该重组质粒使用了强表达能力的哺乳动物真核细胞表达载体。 利用更为强大 的启动子， 来启动基因的表达， 使得 SARS-CoV病毒结构蛋白及其截短形式在哺 乳动物细胞表达系统中得到了高量表达。 所述哺乳动物真核细胞表达载体可使用 PEAK系列： 例如 pEAK10、 pEAK12、 pEAK13等， pCDNA系列： pCDNA3.0、 pCDNA4.0等， pCDM系列： pCDM7、 pCDM8、 pCDM10、 pCDM12; 优选的真 核细胞表达载体是 pEAK13;所述启动子选自 CMV、EF1 α、 Co YMV MV enhancer + chicken albumin promoter ^ SV40 promoter + enhancer； 优选的启动子是 CMV enhancer + chicken albumin promoter。
本发明将目的蛋白序列前的分泌序列更换为公知的 CD5 蛋白的强引导序列 (CD5L), 强化引导目的蛋白的分泌性表达。 为了使所表达的病毒蛋白能够与宿主蛋白和 DNA有效分离，本发明采用分泌 表达的载体,去除野生型 SARS-CoV病毒结构蛋白基因前的原始的分泌序列， 添加 一段带有剪切信号的强大的引导序列 CD5L，引导序列翻译后成为引导蛋白跨膜的 信号肽， 引导病毒结构蛋白穿过细胞膜， 分泌到细胞外的培养基中， 达到与宿主 蛋白及 DNA有效分离， 简化蛋白提纯的步骤， 降低蛋白提纯的难度， 减小蛋白在 提纯中变性的可能性的目的。 并且引导序列翻译成的信号肽能够通过蛋白剪切酶 的作用将其剪切下来， 从而不影响病毒蛋白的结构。 用 CD5L序列替换野生型 SARS-CoV病毒结构蛋白的分泌序列， 如以下所示的序列-
5'ATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGG ATGCTGGTCGCTTCCTGCCTCGGAGCG 3，。
本发明将表达 SARS-CoV病毒的结构蛋白的融合蛋白的质粒， 其结构蛋白的 编码基因进行人工合成， 用人体细胞内常用 （偏好） 密码子替换病毒基因中编码 相同氨基酸的密码子， 对病毒的结构蛋白基因进行人类编码优化。
为使 SARS-CoV病毒结构蛋白及其截短形式在哺乳动物细胞表达系统中的表 达量更高， 本发明还采用基因优化的手段。 所述基因优化包括密码子人源化和最 佳化。
所述密码子人源化是指多种生物都不同程度的存在密码子利用的差异及偏 爱， 由于本发明主要使用人源的细胞作为宿主， 并且目的是应用于人体， 因此将 人体内极少被利用的密码子替换为人体内频繁被使用的偏爱密码子。
采用密码子最佳化的基因优化手段， 是将融合蛋白的基因编码中表达宿主体 内极少被利用的密码子替换为被频繁使用的密码子。 通过 GenBank査找出野生型 SARS-CoV病毒 S蛋白所对应的氨基酸序列， 将每个氨基酸所对应的密码子更换 为使用频率比例较高的密码子， 从而得到若干优化 DNA序列， 以提高蛋白的表达 在本发明中， 使用人体内氨基酸相对应的高效密码子替代稀有密码子， 例如， 甘氨酸（Gly)的密码子用 GGC代替其它的 GGA/GGT/GGG,谷氨酸 (Glu)用 GAG 代替 GAA,天冬氨酸 (Asp)用 GAC代替 GAT等。
为清楚说明， 下表中列出了密码子在人体内高表达基因中的使用频率。 本发 明按下列表中使用频率的比例进行密码子置换， 将氨基酸所对应的密码子选择使 用频率较高的密码子， 提高了蛋白的表达效率。 人体高表达基因中密码子使用频 率比例如下：
氨基酸 密码子 数量 /1000 比例
Gly GGG 905.00 18.70 0.24
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C6ZlOO/900ZM3/X3d 1"809εΐ/900Ζ OAV Leu TTA 78.00 1.61 0.02
Phe TTT 337.00 6.96 0.20
Phe TTC 1378.00 28.48 0.80
Ser TCG 325.00 6.72 0.09
Ser TCA 167.00 3.45 0.05
Ser TCT 453.00 9.36 0.13
Ser TCC 958.00 19.80 0.28
Arg CGG 611.00 12.63 0.21
Arg CGA 184.00 3.80 0.06
Arg CGT 211.00 4.36 0.07
Arg CGC 1086.00 0.37
Gin CAG 2023.00 C 41.81 0.88
Gin CAA 289.00 5.97 0.13
His CAT 237.00 4.90 0.21
His CAC 871.00 18.00 0.79
Leu CTG 2885.00 59.62 0.58
Leu CTA 167.00 3.45 0.03
Leu CTT 242.00 5.00 0.05
Leu CTC 1278.00 26.41 0.26
Pro CCG 482.00 9.96 0.17
Pro CCA 457.00 9.44 0.16
Pro CCT 569.00 11.76 0.19
Pro CCC 1410.00 29.14 0.48 本发明用基因编码优化的方法， 得到了若干优化的 DNA序列， 其中本发明优 先选用的合成的 SARS-CoV的 S蛋白基因序列， 如 SEQ ID NO: 1所示。
本发明的转染重组质粒的真核细胞表达株选自 CHO、 293和 Vera细胞株及其 衍生细胞。
选择合适的能高量表达的宿主细胞株，建立表达细胞株，选用 293细胞、 CHO 细胞或 Vera细胞， 以及以上细胞的衍生细胞（derivatiVe)。利用构建的重组质粒中 含有抗嘌呤霉素 (puromycin)基因， 筛选转染进 S蛋白或其截短形式的基因的细胞， 最后用 ELISA或 Western Blotting的方法进行定量定性，挑选出最优表达株。其中， 293E, 293ET及 CHO细胞表达量较高， 尤其以 CHO细胞为最佳。 将挑选出的高 量恒定表达细胞株进行细胞驯化培养， 进一步提高病毒蛋白的表达量， 为批量制 备及产业化生产提供条件。
本发明中所涉及的相关哺乳动物细胞表达株已经于 2005年 7月 6日在中国微 生物菌种保藏管理委员会普通微生物中心 （CGMCC)保藏， 其保藏号分别为 1408、 1409、 1410。
本发明构建真核细胞表达株所用的筛选药物是嘌呤霉素（puromycin), 细胞驯 化提高蛋白表达量所用的药物为氨甲蝶呤 （ methotrexate )。
本发明构建的重组质粒选用了抗嘌昤霉素 (puromycin)的基因， 嘌呤霉素 (puromycin)是一种可以杀伤真核细胞的药物。 将细胞转染进带有抗嘌呤霉素 (puromycin)的基因的重组质粒后， 可以提高细胞对嘌呤霉素的耐受性。 在细胞培 养基中梯度加入嘌呤霉素 (puromycin), 利用含有抗嘌呤霉素基因的细胞与不含该 基因的细胞对嘌呤霉素的耐受性的差异， 可以用来筛选转染成功的哺乳动物细胞。 该重组质粒中还存在二氢叶酸还原酶 (endogenous dihydrofolate reductase; dhfr)基 因， 因此， 氨甲蝶呤（methotrexate)可用来进行细胞驯化， 提高蛋白表达量高。
本发明所表达的 SARS-CoV病毒的全长的结构蛋白的融合蛋白 （E-Fc, M-Fc, N-Fc, S-Fc)如图 1所示。本发明所表达的 SARS-CoV病毒的 S蛋白及其任意截短 形式的融合蛋白 (317-Fc, 511-Fc, 685-Fc, 900-Fc, 1148-Fc, 1190-Fc, 318-510-Fc, 318-1190-Fc, 511-1190-Fc和 681-1190-Fc)如图 23和图 24所示。
而细胞表达株的构建则避免了细胞瞬时转染状态的不稳定， 并可以通过选择 表达优势株进行扩增来提高产量， 还可以通过细胞驯化等手段来进一步提高表达 量， 从而实现蛋白的批量制备或产业化生产。
此质粒的具体构建过程见实施例 1和 2， 细胞表达株的构建过程见实施例 3。
(2)细胞表达株在正常生长状态下每 24小时每百万细胞在其培养基中产生 10 μ§以上重组蛋白。将细胞计数后，进行培养，三天后收集细胞培养用的培养基， 通过 ELISA的方法检测细胞株的表达量， 通过计算， 得到 S蛋白及 S蛋白的一系 列截短形式的表达量。用本发明的方法，最终可以得到高表达量的多种 SARS-CoV 病毒的结构蛋白及其截短形式蛋白的融合蛋白。 在细胞的培养基中， 表达量高于 10 y g/106细胞 /24小时。 其中 S1190-FC (去掉跨膜区的全长的 S蛋白）产量高于 10 u g/106细胞 /24小时； S截短形式蛋白 （S318-510-FC)产量可高于 30 w g/106细 胞 /24小时。
(3 ) 纯化通过步骤 （2)表达的蛋白。 由于是分泌性表达， 简化了蛋白纯化 的步骤， 减小蛋白提纯中变性的可能性， 并能够与宿主蛋白及 DNA有效分离。将 目的蛋白用亲和柱和分子筛提纯后， 其纯度可达 99%以上。 如图 4所示。 并为高 效液相色谱一质谱实验所验证。
具体步骤见实施例 10。
本发明所表达提纯的蛋白具有其在生物机体内相应的生物活性。 例如， 本发 明所表达提纯的 S1190-FC可与 S蛋白的受体 ACE2结合， 如图 5， 图 6， 图 7， 和 图 9所示。 S1190-Fc亦可与具有其受体的细胞融合并进入细胞， 如图 8， 图 10， 和图 11所示。
本发明所表达纯化的融合蛋白可以用来制备疫苗， 防止 SARS-CoV病毒的感 染。
本发明通过对 S蛋白的一系列优化，完成了 S蛋白及 S蛋白的一系列截短形式 的表达， 并对其各部分的功能进行了研究， 得到了大量的结果与数据， 对 s蛋白 及其不同截短形式的应用提供了依据。 本发明进一步完成了 S蛋白与 ACE2相互 作用的细胞实验及体内实验，发现了 S蛋白可导致 ACE2表达的下调，并且 ACE2 的下调可加剧急性肺损伤。 本发明并通过进一步的实验证明， 其主要结合 ACE2 受体并引起 ACE2受体下调的位点为 S蛋白第 318位氨基酸到 510位氨基酸。 因 此在制备疫苗时， 需要去掉此段氨基酸序列或者对 S蛋白进行突变或修饰使其不 能结合 ACE2受体或降低与 ACE2受体结合能力， 尤其是对 S蛋白第 318位氨基 酸到 510位氨基酸的突变与修饰， 以防止引发一系列的病理过程； 可挑选具有合 适免疫原性， 能引发机体适当的细胞免疫反应及体液免疫反应， 产生有效地中和 抗体的 S蛋白的截短形式或 S蛋白的不同突变体或修饰后产物来制备疫苗， 从而 预防 SARS-CoV病毒的感染。
本发明所表达纯化的融合蛋白免疫小鼠， 可产生中和 SARS-CoV病毒的高效 中和抗体。
五周龄的雌性 bait c小鼠分为两组， 每组 5只， 一组在 0周、 2周、 4周注射 50 μ g S1190-FC加佐剂进行免疫， 另一组注射同样剂量的 Fc加佐剂作为对照， 第六 周采血收集血清。 将热灭活的血清进行微量中和分析检测中和抗体的存在。 将热 灭活的血清进行双重稀释后， 用微量中和分析检测中和抗体的滴度。 在 96孔板中 梯度加入中和抗体，每个梯度加三个孔，然后每个孔加入 SARS-CoV感染 Vero E6 单层贴壁细胞的 TCID50剂量的 100倍，第三天和第四天检测病毒致细胞病变影响 (CPE), 用 RM公式计算在 50%的孔中能够完全抑制 CPE的梯度， 最后得到中和 抗体的滴度。 本发明结果证明， S1190-FC 组与对照组抗体滴度有明显差异， 经 S1190-FC免疫后的小鼠能够产生大量有效的中和抗体， 可有效阻止 SARS-CoV病 毒的感染。 具体步骤见实施例 11。 结果如图 22所示。 本发明说明， S蛋白的任意 截短形式的融合蛋白免疫小鼠可产生不同效价的 SARS-CoV病毒的中和抗体， 不 同程度阻止 SARS-CoV病毒的感染。 本发明的融合蛋白可以用来制备病毒检测试剂盒。 '
用本发明所得到的病毒结构蛋白及其不同截短形式作为一种检测 SARS-CoV 病毒的新试剂， 来检测血液中相应抗体的存在， 以确定是否存在 SARS-CoV病毒 感染的可能性。 本发明通过动物实验验证了 S蛋白的强免疫原性， 可作为检测性 抗原检测血液中的相应抗体。 将在哺乳动物宿主细胞系统中表达出来的具有抗原 性的， 能与人体内抗 SARS-CoV病毒的相应抗体发生反应的蛋白进行提纯后， 连 接到酶标板上，利用 ELISA的相关原理，制成检测试剂盒。当人体感染 SARS-CoV 病毒后， 血液中出现的相应抗体可吸附连接到酶标板上的 S蛋白， 经过标记性抗 体的进一步反应， 从而呈现阳性结果被检测出来， 从而协助诊断。
本发明的融合蛋白可以用来制备或筛选抗 SARS-CoV感染的药物。
本发明所得到的 S蛋白还可用于筛选治疗性药物。 SARS-CoV病毒的致病机 理主要是通过 S蛋白与受体 ACE2的相互作用实现， 因此， 在筛选抗 SARS-CoV 病毒的药物时， 可选择能够抑制 S蛋白与 ACE2受体的结合的药物， 包括小分子 化合物、 多肽和基因工程药物， 从而抑制 SARS-CoV病毒侵入细胞。 本发明已经 证明通过小鼠体内注射 S1190蛋白， 小鼠可产生大量的中和抗体， 产生的抗体可 抑制 100倍的 SARS-CoV病毒感染 Vero E6细胞的 TCID50。
本发明的融合蛋白可以用来制备防止 SARS-CoV病毒感染的抗体。
本发明所得到的 S蛋白还可用于筛选单克隆抗体， 尤其是人源化单克隆抗体， 筛选出的单克隆抗体可以特异性的与 S蛋白结合， 从而阻止 S蛋白与受体 ACE2的 结合， 因此， 可用作 SARS的治疗药物， 或对人群进行被动免疫。
本说明书中所述 SARS- CoV 病毒结构蛋白的氨基酸序列来源于 GenBank NC— 004718。
此外， 本发明还涉及一种去除、 修饰或者突变表达 SARS-CoV病毒结构蛋白 S第 318位氨基酸到 510位氨基酸任意氨基酸片段， 或者去除、 修饰或突变表达 SARS-CoV病毒结构蛋白 S第 318位氨基酸到 510位氨基酸片段序列的 DNA序列， 以及所述的 DNA序列表达的氨基酸。
本发明还涉及所述的 DNA序列或者所述的 DNA表达的氨基酸在制备预防
SARS-CoV病毒的疫苗中的用途。所述的疫苗包括 DNA载体疫苗， 蛋白疫苗， 病 毒载体疫苗。
由此获得的 DNA序列或者氨基酸其目的都是为使所述 SARS-CoV病毒结构蛋 白 S不能与受体 ACE2结合或降低与 ACE2结合能力。
急性呼吸窘迫综合症（ARDS)是各种急性肺损伤中最为严重的一种。 急性呼吸 窘迫综合症是以血管通透性增强导致的肺水肿， 炎症细胞的增加和严重缺氧为特 征的。 ARDS的诱病因素是多种多样的，包括败血症,吸入性的，以及由 SARS冠状病 毒或禽 /人流感病毒引发的肺炎。本发明的实验数据显示急性肺损伤包括小鼠体内 感染 SARS会导致一个肾素血管紧张素系统中一个重要的酶 --ACE2的明显下降。损 伤导致的肾素血管紧张素体系失调， 导致了平衡向 Angll增加的方向移动。 ACE2 和肾素血管紧张素体系的这个全新角色似乎与其血管紧缩作用无关， 而与血管渗 透性的调节相联联。 尽管 ACE2的其他代谢产物， 例如缓激肽， 可能在体内发挥着 重要的作用， 本发明的实验的数据提供了 ACE2是通过 Angll来发挥它调节作用的 第一个证据。
肾素-血管紧张素系统（RAS)对于维持血压稳定， 水电介质平衡起着非常重要 的作用。 血管紧张素转换酶 2 (ACE2)与 ACE有同源性， 是 RAS系统的负调性成分。 有趣的是， 体内实验性 SARS病毒感染可导致小鼠肺脏 ACE2表达明显下降。 尽管 人和小鼠肺脏都表达 ACE2, 但有关肺中 ACE2的功能还一无所知。 为了弄清 ACE2 在急性肺损伤和肺脏功能衰竭中的作用，我们在实验模型中检测 ace2缺失的影响。 这个模型再现了在许多人疾病如败血症、 酸吸入、 及 SARS和禽流感病毒 A型感染 导致的急性肺损伤中常见的肺功能衰竭的病理表现。
发明人发现 S蛋白与 ACE2的结合可导致 ACE2在机体内表达的下调， 如图 10， 图 11， 和图 20所示， 并且 ACE2的下调通过肺部的 RAS系统的信号传导途 径可引起或加剧急性肺损伤， 如图 12， 图 13， 图 14， 图 15， 图 16， 图 17， 图 18， 图 19， 和图 21所示。
抗 Fc的抗体和培养细胞株的实验发现 ACE2受体下调。 将 Vera E6细胞分别 在 4°C (篮线）和 37Ό (红线）与融合蛋白 S1190-FC充分作用， 同时用 Fc作为对 照（黑线）， 然后用抗 Fc的抗体进行检测。 本发明人发现， 在 37°C时， 融合蛋白 S1190-FC与受体 ACE2相互作用， 并导致 ACE2受体下调。 如图 10所示。
抗 ACE2的抗体和培养细胞株的实验发现 ACE2受体下调。将 Vero E6细胞分 别在 4Ό (篮线）和 37°C (红线）与融合蛋白 S1190-FC充分作用， 同时用 Fc作为 对照（黑线）， 然后用抗 ACE2的抗体进行检测。本发明人发现， 在 37Ό时， 融合 蛋白 S1190-Fc与受体 ACE2相互作用， 并导致 ACE2受体下调。 如图 11所示。
融合蛋白 S1190-FC处理过的野生型小鼠， 其肺组织 ACE2蛋白表达量下降。 分别用融合蛋白 S1190-FC和对照 Fc蛋白处理野生型小鼠，然后用抗 ACE2的抗体 通过 Western blotting的方法进行检测。 本发明人发现， S1190-Fc处理过的小鼠导 致肺组织 ACE2蛋白表达量下降。 如图 20所示。
野生型小鼠肺部酸或盐灌注并加注融合蛋白 S1190-FC导致肺弹回率变化。 将 小鼠分为 4组， 每组 5到 7只， 两组进行酸灌注， 并加注融合蛋白 S1190-FC和对 照 Fc，两组进行盐灌注， 同样加注融合蛋白 S1190-FC和对照 Fc， 每只小鼠灌注剂 量为 5.5 nmol/kg融合蛋白 S1190-Fc或 5.5 nmol/kg对照 Fc。 结果证明野生型小鼠 酸灌注加注融合蛋白 S1190-FC组与酸灌注加注对照 Fc组相比，在全部的测量时间 内弹回率的变化具有显著性差异 （p < 0.05)， 酸灌注加注融合蛋白 S1190-FC组肺 弹回率变化幅度明显高于酸灌注加注对照 Fc组。 如图 12所示。 本发明人发现， 在酸灌注条件下， 融合蛋白 S1190-FC加重急性肺损伤。
图 13是小鼠肺组织病理切片。 将图 12附图说明中同样处理的小鼠肺组织制 成病理切片， 结果与图 12附图说明中所述结果吻合。 在酸灌注条件下， 肺组织出 现明显水肿， 发生急性肺损伤， 与对照 Fc相比，加入了融合蛋白 S1190-FC后明显 加剧了急性肺损伤的程度。
图 14是肺损伤评分结果。 此结果印证了图 12和图 13的结果， 证明在酸灌注 条件下，肺组织发生急性肺损伤， 与对照 Fc相比，加入了融合蛋白 S1190-FC后明 显加剧了急性肺损伤的程度， 两者具有显著性差异 （p < 0.01 )。
图 15是湿干肺组织重量比的结果。 此结果印证了图 12、 13、 14的结果。 证 明在酸诱导的急性肺损伤的情况下， 相较于对照 Fc组， 加入融合蛋白 S1190-FC 组所造成的肺水肿程度更为严重， 湿干肺组织重量比更大， 与对照 Fc组相比具有 显著性差异 （p < 0.05)。
野生型小鼠肺部酸或盐灌注并加注融合蛋白 S1190-FC 或融合蛋白 S318-510-FC导致肺弹回率变化。 将小鼠分为 5组， 每组 5到 7只， 其中 3组进行 酸灌注， 并分别加注融合蛋白 S1190-Fc、融合蛋白 S318-510-FC和对照 Fc， 2组进 行盐灌注， 并分别加注融合蛋白 S318-510-FC和对照 Fc， 每只灌注剂量为 5.5 nmol/kg融合蛋白或对照 Fc。 结果证明野生型小鼠酸灌注加注融合蛋白 S1190-Fc 组或融合蛋白 S318-510-FC组与酸灌注加注对照 Fc组相比， 在全部的测量时间内 弹回率的变化具有显著性差异 （p < 0.05)。 如图 16所示。 本发明人发现， 在酸灌 注条件下， S1190-FC和 S318-510-FC都可以加重急性肺损伤。
ACE2敲除小鼠肺部酸或盐灌注并加注融合蛋白 S1190-FC导致肺弹回率变化。 过程与分组情况可参照图 12的说明。 结果显示， 在 ACE2敲除的小鼠中， 在酸灌 注条件下， S1190-FC和对照 Fc对肺组织的弹回率的变化没有显著性差异，如图 17 所示。 本实验说明； S1190-FC是通过与 ACE2的结合引起或导致急性肺损伤。
腹膜内局部注射融合蛋白 S1190-FC后， 可在肺组织勾浆中检测到 S1190-Fc。 用抗 Fc的抗体通过 Western blotting和蛋白 G琼脂糖的方法可检测到融合蛋白 S1190-FC, 对照的鼠的 Fc则没有检测到。 如图 18所示。
融合蛋白 S1190-FC在小鼠肺部的定位。免疫组化结果表明，融合蛋白 S1190-FC 聚积在支气管上皮细胞（左列， 放大 100倍）， 炎性分泌细胞（中列， 放大 200倍） 和肺泡细胞 (右列，放大 200倍），这也是急性肺损伤容易发生的部位，发现 S1190-FC 首先聚积在急性肺损伤部位。 如图 19所示。
S1190-FC对野生型小鼠肺组织的 Angll水平的影响。 将野生型小鼠用酸或盐 灌注肺组织， 并加注融合蛋白 S1190-FC或对照 Fc蛋白， 3小时后， 用 EIA测定肺 组织 Angll的水平。 结果证明在酸处理组， 肺组织注入融合蛋白 S1190-FC与对照 Fc蛋白的 Angll的水平有明显差异（p < 0.05)，酸处理组并加注融合蛋白 S1190-Fc 的野生型小鼠肺组织 Angll的水平明显增加， 远高于酸处理过并加注对照 Fc蛋白 的小鼠的肺组织 Angll的水平， 如图 21所示。
上述实验结果说明： S1190通过与 ACE2受体结合并下调 ACE2, 引起或加剧 急性肺损伤，其主要结合 ACE2受体并引起 ACE2受体下调的位点为 S蛋白第 318 位氨基酸到 510位氨基酸。 S蛋白第 318位氨基酸到 510位氨基酸的片段本身即可 引起或加剧急性肺损伤， 因此在制备疫苗时， 需要去掉或修饰此段氨基酸序列。
所述 SARS-CoV病毒结构蛋白 S的截短形式包括去除了第 318位氨基酸到 510 位氨基酸的任意截短形式。根据国际上已经发表的文献可知， ACE2是 SARS-CoV 病毒结构蛋白 S的受体。 本发明通过 S蛋白与 ACE2相互作用的细胞实验及体内 实验， 发现了 S蛋白可导致 ACE2的下调， 并且 ACE2的下调可加剧急性肺损伤。 本发明并通过进一步的实验发现， 其主要结合 ACE2受体并引起 ACE2受体下调 的位点为 S蛋白第 318位氨基酸到 510位氨基酸。 因此在制备疫苗时， 需要去掉 或修饰此段氨基酸序列， 防止引发一系列的病理过程； 可挑选具有合适免疫原性， 能引发机体适当的细胞免疫反应及体液免疫反应， 产生有效地中和抗体的 s蛋白 的截短形式 （已去掉或修饰此段氨基酸序列）来制备疫苗。
SARS-CoV病毒结构蛋白 S包括通过突变或修饰使其不能结合 ACE2受体或降 低与 ACE2受体结合能力的 S蛋白及其任意截短形式。本发明证实， ACE2下调可 导致机体急性肺损伤进一步恶化等病理过程， 并且主要结合 ACE2受体并引起 ACE2受体下调的位点为 S蛋白第 318位氨基酸到 510位氨基酸。所以在制备疫苗 时， 需要对 S蛋白进行突变或修饰使其不能结合 ACE2受体或降低与 ACE2受体 结合能力， 尤其是对 S蛋白第 318位氨基酸到 510位氨基酸的突变与修饰。 通过 突变与修饰， 可以即保持较好的免疫原性， 能够引起机体的细胞免疫与体液免疫， 又不至于引发机体的病理损伤。 因此， 通过对 S蛋白进行突变或修饰， 使其不能 结合 ACE2受体或降低与 ACE2受体结合能力， 也是制备基因工程疫苗的一条重 要的途径。
图 25是细胞转染 S317 Fc与 ACE2， gpl20与 ACE2未见融合的照片 （放大 100倍）。
图 26是细胞转染 S318-510-FC与 ACE2， S1190-Fc与 ACE2发生融合的照片 (放大 100倍）。
图 27是细胞转染 S511-1190 Fc与 ACE2， S681-1190 Fc与 ACE2未见融合的 照片 （放大 100倍）。
用 293细胞分别转染上述 S蛋白的几种截短形式与 ACE2受体，或 gpl20 (HIV 表面蛋白)与 ACE2受体， 然后将转染 24小时的两种细胞混合在一起， 48小时拍 照。 以上六张照片说明， ACE2是 SARS-CoV病毒的特异性受体， 与 ACE2发生 作用并导致细胞融合的部分是 S318-510, 即 S蛋白的 318位到 510位氨基酸。 S 蛋白的其他截短形式， 去掉或修饰 S318-510片段， 不会与 ACE2发生作用并导致 细胞融合， 也不会引起或加剧急性肺损伤， 是比较安全的候选疫苗。
本发明说明， 对 S蛋白进行突变或修饰， 尤其是对 S蛋白第 318位氨基酸到 510位氨基酸进行去除，突变与修饰，使其不能结合 ACE2受体或降低与 ACE2受 体结合能力， 在其中挑选具有对 SARS-CoV病毒高效体液免疫和细胞免疫的 S蛋 白截短形式， 是制备高效安全的抗 SARS-CoV病毒疫苗的需要。
本发明涉及的所述的 DNA序列或者所述的 DNA表达的氨基酸在制备预防 SARS-CoV病毒的疫苗中的用途。所述的疫苗包括 DNA载体疫苗， 蛋白疫苗， 和 病毒载体疫苗。 具体实施方式：
实施例 1 SEO ID No: 1 的全基因人工合成
SEQ ID No: 1 的全基因采用现有技术公知的基因合成方法， 委托上海 (中国) 博亚生物技术有限公司人工合成。
公知的全基因人工合成方法采用相互对应的 100个碱基的寡核苷酸引物及相 应 PCR扩增引物，寡核苷酸引物两两部分有 20个碱基重叠， 构成完整基因，经过 连接， 退火， PCR扩增， 合成全基因。 实施例 2质粒构建及鉴定
(l) PCR产物的获得：
A引物设计及合成
S317: forword:5 'GGCGCTAGCCAGCGACCTGGACCGCTGC3 '
reverse: 5， CGCGGATCCGTCGGGGAAGCGCACGACGTC3 '
S510 :forword:5， GGCGCTAGCCAGCGACCTGGACCGCTGC3 ' reverse: 5' CGCGGATCCGTCACGGTGGCGGGGGCGTTC3' S685： forword:5'GGCGCTAGCCAGCGACCTGGACCGCTGC3'
reverse: 5 'CGCGGATCCGTGGCGCCCAGGCTCATGGTG3 '
S900: forword:5'GGCGCTAGCCAGCGACCTGGACCGCTGC3'
reversed ' CGCGGATCCGTCTCGTACAGCACGTTCTG3 '
S I 148: forword:5' GGCGCTAGCCAGCGACCTGGACCGCTGC3 '
reverse: 5， CGCGGATCCGTCAGGTCCACGTCGGGGCTG3'
S1190: forword:5， GGCGCTAGCCAGCGACCTGGACCGCTGC3 '
Reverse： 5 ' CTCACATGTATGGATCCTTCTGCTCGTACTTGCCCAG3 ' S318-510: forword:5 ' GGCGCTAGCCATCACCAACCTGTGCCCC3 '
reversed' CGCGGATCCGTCACGGTGGCGGGGGCGTTC3 '
S318-1190: forword:5' GGCGCTAGCCATCACCAACCTGTGCCCC3' reverse: 5 ' CTCACATGTATGGATCCTTCTGCTCGTACTTGCCCAG3 '
S511-1190: forword:5'GGCGCTAGCCTGCGGGCCCAAGCTGAGC3' reverse: 5'CTCACATGTATGGATCCTTCTGCTCGTACTTGCCCAG3'
S681-1190: forword: 5 ' GGCGCTAGCCCTQGGCGCCGACAGCAGC3 '
reverse: 5'CTCACATGTATGGATCCTTCTGCTCGTACTTGCCCAG3 '
以上引物均由上海 (中国)博亚生物技术有限公司合成
B PCR扩增产物
使用 PCR仪 (eppendorf Mastercycler, Germany)进行扩增反应
引物（ g/ w l) 0.5 l
模板 PUC18 S 1 μ g (用限制性内切酶 EcoR137°C酶切孵育一小时后）
PCR扩增试剂盒 (2 X pfu PCR Master Mix, Cat No:KP-201，北京天为时代科技有 限公司：)按试剂盒说明使用,加至 50 μ 1反应体系。首先变性温度 94V 5分钟，然后 进入如下 30-40个循环， 变性温度 94°C 1分钟， 退火温度 55 °C 30秒， 延伸反应 72V 1-2分钟， 循环反应结束时， 再给予 72Ό 10分钟的延长反应。
所得 PCR产物取 5 μ 1， 用 1%琼脂糖凝胶 (Agarose, TED&HY Bio Co:Ltd Cat NO:A9918)电泳检测。
检测正确的 PCR 产物用 PCR clean-up kit 试剂盒（VITAGENE, Cat No : 110310-05)纯化,所得纯化产物用 25 μ 1 ΤΕ (分子克隆实验指南第二版)溶解保 存。
(2)插入片段的合成 CD5L-top:
5'AATTCGCCGCCACCATGCCCATGGGGTCTCTGCAACCGCTGGCCACCT ACCTGCTGG(
CD5L-bottom:
5'CATGGATGCTAGCGCTCCGAGGCAGGAAGCGACCAGCATCCCCAGCA 由上海博亚生物技术有限公司合成上述序列。
Fc段序列来源于 GenBank中的人源 IgG Fc段 DNA序列， 由上海博亚生物技 术有限公司进行合成。
(3)质粒构建及初步检测
A首先将合成的单链复性成双链， 作为插入片段。
B构建重组载体 pEAK13 CD5L Fc
载体： pEAK13
插入片段： CD5L、 Fc段
我们以 pEAK13作为载体， 用限制性内切 EcoR I、 Not I消化， 然后连接、 转 化、 检测 （具体过程如下述）， 得到质粒 pEAK13 CD5L Fc
C构建重组载体 pEAK13 CD5L Fc DR
载体： pEAK13 CD5L Fc
插入片段： DR (内源性二氢叶酸还原酶基因）
片段来源： 本实验室保存的其它含有该基因的质粒。
先用限制性内切酶 Pst I、Bgl II将插入片段 DR从其载体上消化下来。然后以 pEAK13 CD5L Fc作为载体，用限制性内切酶 Pst l、 Bgl II消化，然后连接、转化、 检测 （具体过程如下述）， 得到质粒 pEAK13 CD5L Fc DR
D构建含有优化后的 S蛋白基因及其片段的重组载体 载体： pEAK13 CD5L Fc DR
插入片断： PCR产物 S317， S511， S685, S900, S1148, S1190, S318-510, S318-1190, S511-1190, S681-1190。
a酶切消化
在限制性内切酶缓冲体系中加入载体 DNA或 PCR产物消化 1-3小时。消化体 系总体积 20 μ 1，加入 1 μ g DNA, 2 μ 1 10XBSA(0.1%BSA), 2 μ 1 10XNEB Buffer, 0.5 μ ΐ限制性内切酶 Nhe I 和 BamH I (所有限制性内切酶， 0.1% BSA， NEB Buffer均购自 NEW ENGLAND BioLabs® Inc,USA), 载体 DNA的消化体系需另外 加入 0.5 μ 1 的碱性磷酸酶 (Promega,USA,CatNo:M182A)使载体消化末端去掉磷酸 基团。
倒 1.5%的低熔点胶（Promega，USA, CatNo:v2111 ) 8.5ml在放有梳子的电泳玻 璃片 (75 X 50mmPre-Cleaned Micro Slides Plain, coming, USA ,No2974)上，等其冷却 凝固。
胶固化后拔去梳子， 置于电泳槽， 加入含有 500 u g/L Ethidium Bromide Promega,USA,Cat#H5041)的 TAE缓冲液 (《分子克隆实验指南》第二版)，在加样 孔中各加 15-20 μ 1的消化后的载体 DNA和消化后的 PCR产物。同时加 DNAMarker 如： λ -Hind III， DL2000 (TaKaRa Biotechnology(Dalian)Co.Ltd)以确定 DNA的片 段大小。
电泳 60-80V(电泳仪 DYY-6C型， 北京六一仪器厂)， 20-60分钟。
将电泳后的胶移到紫外线 (UV-IV紫外分析仪， 北京市新技术应用研究所)下， 照相， 并切下需要的 DNA条带。
将切下的含有所需 DNA条带的胶置于 1.5ml离心管中， 短暂高速离心使胶落 到管底， 65Ό加热使胶融化。
b连接
准备连接缓冲体系 40 μ 1:力 Π 5 μ 1 10 XNEB Buffer 4， 2 μ 1 100XBSA, 5 μΐ 10 X Ligation Additions, 0.5 μ 1 T4 DNA连接酶， 2-4 μ 1载体 DNA， 去离子水补足总 体积至 40 μ 1(Τ4 DNA连接酶,100 88 NEB Buffer均购自 NEW ENGLAND BioLabs® Inc,US A)。
将连接体系均分两份（各 20 μ 1)， 在其中一份连接体系中加 2-4 μ 1含有待插 入 DNA片段的胶， 另一份加入相应体积的去离子水作阴性对照（在连接体系中载 体和插入 DNA量保持在 1:2左右， 20 μ 1体系 1.5%的低熔点胶的总体积不超过 6 μ 1)。
混匀， 室温 1—3小时。
转化感受态细胞 MC1061(由本实验室制备， 制备方法见 <<分子克隆实验指南 »第二版化学感受态细菌制备的实验方法)。
c转化
从 -70°C冰箱中取出化学感受态细胞置于冰上融化。
在含有 50 μ g/ml氨苄青霉素的 LB琼脂培养皿 (《分子克隆实验指南》第二版 试剂配制方法， 氨苄青霉素 (华北制药厂， 中国)的琼脂表面倒上不含氨苄青霉素的 LB琼脂 5-6ml， 凝固后使用。 在感受态细胞刚刚融化后， 马上加入连接产物和阴性对照， 每 ΙΟΟ μ Ι化学感 受态细菌加入 5-8 μ ΐ的体积， 轻轻混匀， 置于冰上 15— 30分钟。
37°C水浴 5分钟。
将细胞悬液吸出， 均勾铺在刚刚加入 LB培养基的培养皿中， 37°C培养 12— 16小时， 可长出单克隆菌落。
d鉴定
将单克隆菌落用牙签挑到 4ml含有氨苄青霉素的 LB液体培养基中， 置于 37 °C摇床 (台式恒温振荡器 THZ-D,培英)， 250-280rpm,摇 7—8小时使菌液长至饱和。
使用质粒小提试剂盒（北京天为时代科技有限公司， DP-103), 根据其提供的 方法提取质粒 DNA。
小提所得的质粒溶于 50-60 μ 1TE,取 10 μ 1用限制性内切酶 Nhe I 和 BamH I酶切消化检测， 挑出酶切检测正确的质粒所对应的菌株。
e测序
将酶切检测正确的质粒进行测序 （上海博亚生物技术有限公司及上海生工生 物工程技术服务有限公司）， 进行进一步的确认。
f 质粒的大量提取
用 CsCl密度梯度离心法大量提取重组后的质粒（实验方法见《分子克隆实验 指南 >>第二版）。
E转染细胞， 确定融合蛋白的高量正确表达。
计数 2 X 105个细胞分到 6孔细胞培养板的每个孔， 24小时后， 分别用脂质体
(lipofectamine™ 2000购自 Invitrogen™)转染构建好的含有 S蛋白及其不同截短形 式的重组质粒。 收集培养 3天和 6天的培养基， 用 Western Blotting方法和细胞流 式技术进行检测。 用 Western Blotting方法检测融合蛋白分子量的大小， 用细胞流 式技术检测融合蛋白的活性 （具体操作方法如下述）。 实施例 3 建立恒定表达细胞株
(1)用限制性内切酶 Avrll (购自 NEW ENGLAND BioLabs® Inc,US A)酶切大 约 10 u g的重组质粒， 取少量酶切产物电泳确定酶切完全后， 将剩余的酶切产物 用纯化试剂盒（购自 V-Gene公司）进行纯化， 回收 DNA, 去除其中的酶及蛋白 等。
(2)用胰酶消化细胞，加入培养基， 吹打成单个细胞。计数 2X 105个细胞分到 6孔细胞培养板的每个孔。
(3) 24小时后，分别用脂质体 (lipofectamine™ 2000购自 Invitrogen™)转染水（阴 性对照）及 0.5 g酶切纯化后的 DNA (根据试剂盒提供的操作方法进行操作）。 (4) 48小时后分至 12孔细胞培养板 (每个 6孔细胞培养板的孔的细胞均分到 4 个 12 孔细胞培养板的孔）， 梯度加入不同浓度的筛选药物 puromysin (购自
CALBIOCHEM® CLONTECH )， 杀死未转入 DNA的细胞。
(5) 72小时后， 选择阴性对照全部死亡， 转染 DNA的细胞有适量存活的药物 浓度所对应的细胞。 将孔内细胞用有限稀释法分单个细胞到细胞培养用 96孔板。
(6)等待 10天左右，挑选细胞单克隆，用 ELISA及 Western Blotting方法进行 检测。
用 Western Blotting方法进行检测， 确定蛋白分子量的大小， 从而确定基因的 正确表达。 用 ELISA试剂盒检测蛋白的浓度， 选取能进行高量表达的细胞系。 用 细胞流式技术检测蛋白的活性（具体操作方法如下述）。 实施例 4用 Western Blotting的方法检测融合蛋白的分子量
所用电泳、 转膜装置及试剂：
电泳仪（PowerPac Basic™ Power Supply)： 购自 BIO-RAD， Catalog
Number: 164-5050电泳装置 (Mini-PROTEAN® 3 Cell):购自 BIO-RAD, Catalog Numbers: 165-3301， 165-3302
电转膜装置 (Mini Trans-Blot® Electrophoretic Transfer Cell): 购自 BIO-RAD, Catalog Numbers: 170-3930, 170-3935
试剂：
均购自 SIGMA-ALDRICH
CORPORATION · BOX14508 · ST. LOUIS · MISSOURI 63178 · USA
样品来源： 取自转染细胞的培养基、 构建好的细胞株的培养基以及纯化的蛋 白。
操作方法：
电泳上样前将样品加入到等体积的 2X电泳加样缓冲液 (配制方法参见分子克 隆试验指南第二版第 890页科学技术出版社）中， 97。C， 加热 5分钟。
灌制 SDS聚丙烯酰胺凝胶。 分离胶浓度 10%, 浓缩胶浓度 5% (配制方法参见 分子克隆试验指南第二版第 883-884页科学技术出版社)。
将灌胶玻璃板从固定框架上取出，按说明书安装到电泳装置中，用 1 X电泳缓 冲液（配制方法参见分子克隆试验指南第二版第 884页科学技术出版社 ）注满 整个电泳盒。
用枪头小心的将 15 μ 1预先经过加热变性的样品 (含 7.5微升上清， 7.5微升加 样缓冲液)依次注入到凝胶加样孔内。
按说明书正确连接电泳装置， 接通电源。
开始电泳， 起始电压为 80V， 当溴酚兰染料前端进到分离胶后， 提高电压至 120V继续电泳，直至溴酚兰染料达到分离胶底部或全部泳出凝胶，关闭电源 (大约 需要 120分钟)。
配制 1升转膜缓冲液 (配制方法参见分子克隆试验指南第二版第 892页 科学技 术出版社)， 并置于 4°C预冷。
电泳结束后， 切断电源打开电泳盒， 将内盒中的玻璃板取出， 切去浓缩胶， 转移分离胶于预先注入转膜缓冲液的容器中。
戴上手套切一块略大于分离胶的硝酸纤维素膜（Amersham, Catalog
No:RPN303C), 再切两块同样大小的滤纸， 将硝酸纤维素膜， 滤纸和两块海绵分 别浸于三个盛有转膜缓冲液的容器中。
按照转膜仪说明书， 安装转膜装置。转膜条件： 恒定电流 300毫安， 时间 120 分钟。
转膜结束后将膜小心取出， 置于盛有 2%鸡卵清封闭液（SIGMA® ALBUMIN,
CHICKEN EGG, Catalog number: A-5253 )的容器里，在平缓摇动的摇床上室温封闭 1小时 （或 4°C， 过夜）。
封闭结束后， 弃去封闭液， 用 2%鸡卵清封闭液稀释一抗加入， 室温结合 3小 时或 4°C过夜。
一抗结合结束后， 用 TBST洗膜 3次， 每次 10分钟。
弃去 TBST, 用 2%鸡卵清封闭液稀释二抗加入， 室温结合 1到 2小时 （对于 Fc等直接可用二抗的蛋白标签，封闭完后，直接加入封闭液稀释的二抗进行结合）。
抗体结合结束后， 用 TBST洗膜 3次， 每次 10分钟。
按照说明书配制 Western检测显色液（Santa Cruz Biotechnology, Inc. Catalog Number:sc-2048), 并将配制好的 Western检测显色液均匀滴加于膜结合蛋白的一 吸尽过多的 Western检测显色液， 用保鲜膜小心的将膜包好， 置于 X射线摄 影暗匣 (中国 汕头市粤华医疗器械厂有限公司 型号: ΑΧ- Π ,规格: 127 X 178mm) 中。
暗室中， 将胶片置于暗盒中曝光， 然后显影 4-5分钟， 定影 4-5分钟。 （胶片 为 Kodak X-Omat BT Film, 由中国汕头柯达股份有限公司分装， 美国伊士曼柯达 公司制造。 规格： 12.7 X 17.8cm， 乳剂编号： 031222104) (显影粉与定影粉购自天 津河北感光材料厂） 通过对细胞单克隆上清进行 Western Blotting定性检测， 我们检测到了跟预期 大小相符的条带。 实施例 5 用 ELISA的方法检测融合蛋白的表达量
培养基中含有的蛋白的浓度可用 ELISA的方法予以检测。 ELISA所用试剂为
BD Biosciences公司的 BD Pharmingen™ ELISA试剂盒。
ELISA检测方法如下：
( 1 )将细胞上清液、阴性对照（加入牛血清的培养基）、阳性对照及标准（呈 梯度稀释的已知浓度的人的 IgG抗体， 用于蛋白定量）加入 96孔酶标板， 每孔 ΙΟΟμΙ, 过夜。每个样品加三个孔， 以鉴定区别阳性结果与污染所致的假阳性结果。
(2) 次日， 将 96孔板中的培养液吸出， 用洗液（wash solution)洗一次， 每 孔 200μ1。
(3 ) 加入分析液（assay diluent) ， 每孔 200μ1， 在摇床上室温摇 1小时。
(4)加入分析液稀释的一抗， 每孔 ΙΟΟμΙ, 在摇床上室温摇 3小时。
(5 )一抗结合结束后， 用洗液洗 3次， 每孔 200μ1
(6)加入用分析液稀释的 HRP标记的二抗 （对于 Fc等直接可用二抗的蛋白 标签， 用分析液结合完后， 直接加入分析液稀释的二抗进行结合）， 每孔 100μ1， 室温下摇一小时。
(7)用洗液洗八遍， 200μ1每孔。
(8)将等体积的感光剂 A、 B (substrate reagent Α/Β)混匀， 避光。 将洗完八 遍的酶标板加入混匀后的感光剂， 每孔 100μ1， 常温避光 30分钟。
(9) 30分钟后加入终止液 （stop solution) ， 每孔 50μ1， 终止反应。
( 10)酶标仪 450nm读数。
( 11 )根据读数， 计算蛋白含量。
经检测， 以每百万细胞在 24小时的表达量计， S蛋白及其截短形式蛋白的表 达量都在 lO g以上。 融合蛋白 S1190-FC在上清中的表达量可达 lOw g以上， 融 合蛋白 S1148 Fc在上清中的表达量可达 20 以上， 融合蛋白 S900 Fc在上清中 的表达量可达 20 以上， 融合蛋白 S685 Fc在上清中的表达量可达 20 g以上， 融合蛋白 S511 Fc在上清中的表达量可达 20 μ g以上， 融合蛋白 S317 Fc在上清中 的表达量可达 20 g以上， 融合蛋白 S318-1190 Fc在上清中的表达量可达 10 μ g 以上， 融合蛋白 S318-510-FC 在上清中的表达量可达 30 g 以上， 融合蛋白 S511-1190 Fc在上清中的表达量可达 10 μ g以上，融合蛋白 S681-1190 Fc在上清中 的表达量可达 10 以上。 实施例 6用流式细胞技术检测表达蛋白的活性
已知 Vero E6细胞存在 S蛋白的受体 ACE2,该受体主要与 S蛋白第 318位到 510位氨基酸作用。 利用受体与配体相互结合的特性， 可以确认蛋白的正确折叠， 即构象正确。
流式检测方法-
(1)将 Vero E6细胞或转染了 ACE2的 293细胞用 PBS/2mMEDTA消化下来， 均分为若干份， 置于离心管中。
(2)离心 （ Eppendorf CentrifUge 5415D)， lOOOrpm, 10min。
(3)用含有 S蛋白或其截短形式的细胞培养基分别重悬细胞，用加入血清（购 自 Hyclone) 后的 IMDM (购自 GIBCO)培养基作为阴性对照。
(4) 4°C旋转混匀 l-2h。
(5)离心 （同上）， lOOOrpm, 10min。 弃上清。
(6) 力口入二抗 FITC/anti-human IgG (购自 Jackson ImmunoResearch ) 或 FITC/anti-His (购自 Sigma)等重悬细胞（若没有荧光标记的相关二抗， 则需先用
—抗结合）。
(7) 4°C旋转混匀 30min-lh。
(8) 4 °C离心（ BECKMAN COULTERTM Microfuge® 22R Centrifuge )， 1 OOOrpm, lOmino
(9) PBS重悬， 用流式细胞仪（BECKMAN COULTERTM EPICS ELITE EST) 进行检测。 实施例 7 用流式细胞技术检测 ACE2受体下调
已知 Vero E6细胞存在 S蛋白的受体 ACE2,检测 S蛋白下调 ACE2受体的作 用， 可用 Vero E6细胞。
检测方法：
( 1 ) 将 10cm细胞培养皿（Greiner bio-one)中生长至 50%-70%满的 Vero E6 细胞的培养基（含有 10%胎牛血清 （Hyclone))去掉， 用 PBS洗三遍。
(2) 加入无血清培养基在 37°C的 C02培养箱（SANYO, MCO-15AC) 中 培养 1小时。
(3) 用 PBS洗一遍，加入 2mM EDTA/PBS, 37°C的 C02培养箱中 20-30min。
(4) 将变圆的细胞吹打下来， 均分为三份。
(5 ) lOOOrpm , lOmin 离心（BECKMAN COULTERTM， Microfoge® 22R Centrifuge), 用 800 μ 1无血清培养基重悬， 并加入适量 EDTA。
(6) 三份细胞中的一份加入对照用 Fc， 另外两份分别加入 50 μ g的融合蛋 白 S1190-Fc。
(7) 将对照 Fc与其中一份加入了融合蛋白 S1190-FC的细胞置于 4°C并缓慢 旋转， 将另一份置于 37°C并缓慢旋转， 此过程维持 3小时。
(8) lOOOrpm, lOmin, 4。C离心。
(9) PBS重悬后， 1000rpm， lOmin, 4°C离心。
( 10)将 FITC标记的抗 Fc的抗体稀释到 PBS中，并重悬细胞（在检测 ACE2 时， 需先加抗 ACE2的一抗， 再加 FITC标记的二抗）。
( 11 ) 4°C， 缓慢旋转 30min。
( 12) lOOOrpm, lOmin, 4。C离心。
PBS重悬后，用流式细胞仪（BECKMAN COULTER™ EPICS ELITE EST)进 行检测。 实施例 8 细胞融合实验
( 1 ) 用胰酶消化处于对数生长中期状态良好的的 293ET细胞， 待细胞变圆 后， 加入 DMEM培养基（购自 GIBCO), 吹打成单个细胞。
(2) 计数 2X 105个细胞分到 6孔细胞培养板的每个孔。
(3 ) 24小时后， 分别用脂质体 (lipofectamine™ 2000购自 InvitrogenTM)转染 所需质粒。
(4) 24小时后， 用胰酶将细胞消化下来， 计数， 将细胞两两混和， 放入 12 孔细胞培养板一个孔。 每孔每种细胞数分别为： 2X 104 , 4X 104 , 6X 104 , 8 X 104, 1 X 105。
(5) 48-72小时后拍照 （Nikon Eclipse TE2000-U), 可见明显的细胞融合， 阴性对照未见融合。 实施例 9证明蛋白 S1190与 ACE2受体相互作用的 IP实验
( 1 ) 用 S1190-Fc和 ACE2, 对照 Fc和 ACE2两组质粒分别转染细胞。
(2) 转染后 36小时， 将细胞置于冰上预冷， 并用预冷的 PBS洗 3遍。 (3 ) 加入含有蛋白酶抑制剂的细胞裂解液， 裂解 20-30min。
(4) 收集细胞及裂解液， 12000rpm , 2min， 4 °C离心（BECKMAN COULTERTM, Microiuge® 22R Centrifoge)。
(5) 将上清移入新管， 加入适量 Protein G-Agarose， 4°C缓慢旋转过夜。 (6) 将上清与磁珠 12000rpm， 5min, 4。C离心。
(7) 去上清， 加入适量裂解液重悬， 并缓慢旋转 20min。
(8) 12000rpm, 5min， 4°C离心。
(9) 去上清， 加入与沉淀等体积的 2 X Western Blotting上样缓冲液， 97°C， 5min。
( 10)快速离心， 取上清， 做 Western Blotting检测。 实施例 10蛋白提纯
带有 Fc标签的蛋白用 Protein A的柱子提纯， 带有 6His标签的蛋白用镍柱提 纯。
用 Fc标记的蛋白采用 Amersham公司的 ProteinA蛋白柱进行纯化。（ Amersham Biosciences AB, Sweden; CAT NO: 17— 04020— 03 )
( 1 ) 收集恒定表达细胞株培养的 3天上清。
(2) 透析：收集的上清进行透析。透析液的成分为： 11.54mM/L的 Na2HP04， 8.46mM/L的 NaH2P04(北京化工厂，中国）， ImM的 EDTA(Promega U.S.A), pH7.0。 透析的时间一般不少于 8小时， 透析液的体积最少应为上清体积 20 倍。 '
(3 ) 过滤：透析完的液体进行过滤。采用的滤膜是 Millipore公司生产的 0.45 μ m的 Durapore membrane filters (Millipore, Ireland; CATNO:HVLP04700)。 (4) 纯化： 纯化的步骤按照 Amersham公司提供的产品说明书上的 protocol 进行， 采用的仪器是 Bio— Rad公司生产的 Econo Gradient Pump Kits (Bio— Rad U.S.A)。
(5 ) 纯化完的蛋白样品经过 Western Blotting和考马斯亮蓝对 SDS聚丙稀酰 胺凝胶染色来鉴定， 蛋白浓度的测定用 Lowry法（Lowry法试剂盒购自天象 邦定公司， CAT NO: TB090— 1 )。
带有 6His标签的蛋白提纯方法同上。 实施例 11 : 疫苗在血清中中和抗体的检测 小鼠用 S1190-FC免疫后， 体内产生中和抗体的滴度。
( 1 ) 将五周龄的雌性 ba ^、鼠分为两组， 每组 5只。
(2) 一组在 0周、 2周、 4周注射 50 μ g S1190-Fc加等量佛氏佐剂进行免 疫， 另一组注射同样剂量的 Fc加同等剂量佛氏佐剂作为对照。
(3) 第二， 四， 六周采血收集血清。 (4) 将热灭活的血清进行双重稀释。
(5) 用微量中和分析检测中和抗体的滴度。
在 96孔板中梯度加入中和抗体，每个梯度加三个孔，然后每个孔加入 SARS-CoV 感染 Vero E6单层贴壁细胞的 TCID50剂量的 100倍， 第三天和第四天检测病毒致 细胞病变影响 (CPE)， 用 RM公式计算在 50%的孔中能够完全抑制 CPE的梯度， 最 后得到中和抗体的滴度。 实施例 12肺弹性分析实验 1 ) 将 2.5到 3月龄的小鼠分为 5组， 每组 5到 7只。
2) 用 (ketamin) (75 mg/kg)和甲苯噻嗪（xylazine) (20 mg/kg)腹膜内注射， 进 行麻醉。
3 ) 气管切开后用用可控制气流量的恒定流量通风机测定换气量。
4) 利用气流量补充调整（VRM) (25 cmH20, 3 sec)标准化流量记录并作为基
5 ) 进行酸或盐溶液处理前 30 分钟， 分别在小鼠腹膜内注射 S1190-Fc， S318-510-FC, 或对照 Fc (每只 5. 5 nmol/kg)。
6) 用盐酸 (pH=1.5; 2 ml/kg)或盐水溶液进行气管内灌输后，然后测量气流量补 充调整（VRM)(35 cm¾0, 3 seconds),将所有的实验动物通气 3小时 ( 02 1.0)， 并记录肺弹性分析。
7) 全部 PEEP (PEEPt)并且压力稳定 (Pplat)后在呼气末和吸气梗塞时测量， as Pplat minus PEEPt) VT, 在通气期间， 每 30分钟分别计算一次肺弹性。
8) 酸或盐溶液处理后 1小时和 2小时，将小鼠再次分别腹膜内注射 S1190-Fc， S318-510-Fc, 或对照 Fc (每只 5. 5 nmol/kg)。 实施例 13 小鼠的免疫组化实验
1 ) 将如实施例 12所述的小鼠右肺作为样本， 用 3.7%的福尔马林固定肺组织 并用石 蜡包埋。
2) 切成 5 μπι的组织切片。
3) 用 72°C的 EDTA预处理。
用羊抗人 Fc 的夺克隆抗体 (Jackson Immunological Research, Inc.)染色。 用 Vectastatin ABC试剂盒检测特异性着色部分。 实施例 14 H&E染色
1 )如实施例 12中进行酸或盐处理并腹腔内注射 S1190-FC或对照 Fc 2)将小鼠右肺作为样本， 用 3.7%的福尔马林固定肺组织并用石蜡包埋。
3)切成 5 μιη厚的组织切片。
4)用 haematoxylin禾卩 eosin染色
5)在显微镜下照像 实施例 15 肺损伤评分：
在酸吸入后， 用 S1190-FC和对照 Fc处理的小鼠的肺损伤的半定量评估。
1 ) 实施例 14中每张切片随机选取 4个视野， 每组 16个视野， 根据规定的评分标准， 用盲法评分。
2)评分范围包括： 肺泡充血， 出血， 中性粒细胞渗透， 肺泡壁厚度， 及透明膜形成等。
3)评分标准： 极低的损伤为 0分， 轻微损伤为 1分， 中度损伤为 2分， 严重损伤为 3 分， 最高程度的损伤为 4分。

Claims

权 利 要 求 书
1. —种 SARS-CoV病毒结构蛋白的融合蛋白， 其结构为： X-Y-Z,
其中 X包括 SARS-CoV病毒结构蛋白 S或 M或 E或 N， 或以上结构蛋白的 任意截短形式， 所述 SARS-CoV病毒结构蛋白 S包括去除、 修饰或者突变第 318 位氨基酸到 510位氨基酸任意氨基酸片段， 或者去除、 修饰或突变第 318位氨基 酸到 510位氨基酸片段；
Y是连接部分， 由 0-20个任何氨基酸组成；
Z是包含铰链区、 CH2、 CH3结构域的人 01的？0及其变体或蛋白标签。
2.根据权利要求 1所述的 SARS-CoV病毒结构蛋白的融合蛋白，其特征在于 所述蛋白标签包括且不限于六组氨酸 (6XHis)标签、聚乙二醇 (PEG)标签和人血清白 蛋白 (Human serum albumin， HS A)标签。
3.根据权利要求 1所述的 SARS-CoV病毒结构蛋白的融合蛋白，其特征在于 所述 SARS-CoV病毒结构蛋白 S包括蛋白 S的全长或其任意截短形式。
4.根据权利要求 1所述的 SARS-CoV病毒结构蛋白的融合蛋白，其特征在于 所述 SARS-CoV病毒结构蛋白 S是不能与受体 ACE2结合或者降低与 ACE2结合 能力的蛋白。
5. 根据权利要求 1所述的 SARS-CoV病毒结构蛋白的融合蛋白，其特征在于 所述的 Y是两个氨基酸， 所述的氨基酸为赖氨酸和精氨酸。
6.—种能够在哺乳动物细胞株中表达的 SARS-CoV病毒 S蛋白基因， 其特征 在于所述的基因为 SEQ ID NO: 1。
7.含有 SEQ ID NO: 1的重组表达质粒。
8. 根据权利要求 7所述的重组表达质粒， 其特征在于所述的质粒包括真核
PEAK系列。
9. 哺乳动物细胞株， 其含有如权利要求 6所述的能够表达 SARS-CoV病毒 S 蛋白基因的如权利要求 1所述的 SARS-CoV病毒结构蛋白的融合蛋白。
10. 根据权利要求 9所述的哺乳动物细胞株， 其特征在于包括 CHO、 293和 Vero细胞株及其衍生细胞株。
11.根据权利要求 10所述的哺乳动物细胞株， 其特征在于所述的细胞株是中 国微生物菌种保藏管理委员会普通微生物中心保藏的， 保藏号分别为 1408、 1409、 1410的细胞株。
12. 如权利要求 1所述的 SARS-CoV病毒结构蛋白的融合蛋白的制备方法， 包括-
( 1 ) 转染能够表达权利要求 1 所述融合蛋白及内源性二氢叶酸还原酶 (endogenous dihydrofolate reductase) (dhfr)的重组表达质粒，构建哺乳动物细胞表达 株；
(2)该哺乳动物细胞表达株在正常生长状态下每 24小时每百万细胞在其培 养基中产生 10 μ§以上重组蛋白；
(3 ) 纯化通过步骤（2)表达的蛋白。
13. 根据权利要求 12所述的融合蛋白的制备方法， 其特征在于所述的重组表 达质粒含有的融合蛋白的引导序列， 该序列是 CD5蛋白的引导序列。
14. 根据权利要求 12所述的融合蛋白的制备方法， 其特征在于所述的重组表 达质粒， 其结构蛋白的编码基因进行人工合成， 用人体细胞内常用或者偏好密码 子替换相同氨基酸的病毒基因中的密码子序列， 对病毒的结构蛋白进行人类密码 子优化。
15. 根据权利要求 12所述的融合蛋白的制备方法， 其特征在于所述的表达 SARS-CoV病毒的结构蛋白的融合蛋白， 其用人体细胞常用或者偏好密码子合成 的 SARS-CoV的 S蛋白基因如 SEQ ID NO: 1所示。
16. 根据权利要求 12所述的融合蛋白的制备方法， 其特征在于所述的哺乳动 物细胞表达株包括 CHO、 293和 Vero细胞株及其衍生细胞株。
17.根据权利要求 16所述的融合蛋白的制备方法，其特征在于所述的哺乳动物 细胞表达株是中国微生物菌种保藏管理委员会普通微生物中心保藏的， 保藏号分 别为 1408、 1409、 1410的细胞株。
18. 根据权利要求 12所述的融合蛋白的制备方法， 其特征在于构建哺乳动物 细胞表达株所用的筛选药物包括嘌昤霉素和氨甲蝶呤。
19. 根据权利要求 12所述的融合蛋白的制备方法， 其中所述的步骤 （2)是 每 24小时每百万细胞在其培养基中可产生 30 μΕ或者以上的重组蛋白。
20. 根据权利要求 1〜5 中任一项权利要求所述的融合蛋白在制备预防 SARS-CoV病毒的疫苗中的用途。
21. 根据权利要求 1〜5中任一项权利要求所述的融合蛋白在制备 SARS-CoV 病毒检测试剂盒中的用途。
22. 根据权利要求 1〜5 中任一项权利要求所述的融合蛋白在制备或筛选抗 SARS-CoV病毒感染的药物中的用途。
23 . 根据权利要求 1〜5 中任一项权利要求所述的融合蛋白在制备防止
SARS-CoV病毒感染的抗体中的用途。
24. 一种去除、 修饰或者突变表达 SARS-CoV病毒结构蛋白 S第 318位氨基 酸到 510位氨基酸任意氨基酸片段， 或者去除、 修饰或突变表达 SARS-CoV病毒 结构蛋白 S第 318位氨基酸到 510位氨基酸片段序列的 DNA序列。
25. 由权利要求 22所述的 DNA表达的氨基酸。
26.根据权利要求 22或 23所述的 DNA序列或者所述的 DNA表达的氨基酸 在制备预防 SARS-CoV病毒的疫苗中的用途。
27.根据权利要求 24所述的用途，所述的疫苗包括 DNA载体疫苗，蛋白疫苗， 病毒载体疫苗。
33
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US20210284716A1 (en) 2020-03-11 2021-09-16 Immunitybio, Inc. ACE2-Fc Trap
US11857620B2 (en) 2020-03-11 2024-01-02 Immunitybio, Inc. Method of inducing immunity against SARS-CoV-2 using spike (s) and nucleocapsid (N)-ETSD immunogens delivered by a replication-defective adenovirus
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WO2022191742A1 (en) * 2021-03-11 2022-09-15 Dukhovlinov Ilya Vladimirovich A method for assessment of the cellular immune
CN113444745A (zh) * 2021-05-25 2021-09-28 安徽省立医院(中国科学技术大学附属第一医院) 病毒s蛋白突变体假病毒盘的构建及其疫苗评价应用
CN113897346A (zh) * 2021-09-16 2022-01-07 四川大学 能够提高与SARS-CoV-2 S蛋白亲和力的ACE2突变组合及应用
CN114230643A (zh) * 2021-12-23 2022-03-25 东莞市松山湖中心医院(东莞市石龙人民医院、东莞市第三人民医院、东莞市心血管病研究所) 一种SASR-Cov-2病毒S重组蛋白、细胞株及构建方法与应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1468865A (zh) * 2003-06-04 2004-01-21 杭州华大基因研发中心 重组的sars病毒n蛋白羧基端肽段与gst的融合蛋白及方法
CN1570115A (zh) * 2003-07-16 2005-01-26 陈克勤 优化的sars冠状病毒刺突蛋白基因
WO2005028497A2 (en) * 2003-09-15 2005-03-31 The Brigham And Women's Hospital Inc. Receptor binding peptides derived from the sars s protein

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1468865A (zh) * 2003-06-04 2004-01-21 杭州华大基因研发中心 重组的sars病毒n蛋白羧基端肽段与gst的融合蛋白及方法
CN1570115A (zh) * 2003-07-16 2005-01-26 陈克勤 优化的sars冠状病毒刺突蛋白基因
WO2005028497A2 (en) * 2003-09-15 2005-03-31 The Brigham And Women's Hospital Inc. Receptor binding peptides derived from the sars s protein

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LI W.H. ET AL.: "Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus", NATURE, vol. 426, 27 November 2003 (2003-11-27), pages 450 - 454, XP002342002 *
MARRA M.A. ET AL.: "The Genome Sequence of the SARS-Associated Coronavirus", SCIENCE, vol. 30, 30 May 2003 (2003-05-30), pages 1399 - 1404, XP002269483 *
WONG S.K. ET AL.: "A 193-Amino Acid Fragment of the SARS Coronavirus S Protein Efficiently Binds Angiotensin-converting Enzyme 2", J. BIOL. CHEM., vol. 279, no. 5, 30 January 2004 (2004-01-30), pages 3197 - 3201, XP002332535 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111333704A (zh) * 2020-02-24 2020-06-26 军事科学院军事医学研究院微生物流行病研究所 新型冠状病毒covid-19疫苗、制备方法及其应用
WO2021170113A1 (zh) * 2020-02-29 2021-09-02 南京金斯瑞生物科技有限公司 ACE-2-Fc融合蛋白治疗冠状病毒的方法
CN113388027A (zh) * 2020-03-12 2021-09-14 中国科学院武汉病毒研究所 抗新型冠状病毒的抗体及其制备方法和应用
CN113388027B (zh) * 2020-03-12 2022-07-22 中国科学院武汉病毒研究所 抗新型冠状病毒的抗体及其制备方法和应用
CN113881642A (zh) * 2021-07-21 2022-01-04 上海市食品药品检验研究院 一种新型冠状病毒假病毒及其构建方法和应用
CN115894713A (zh) * 2022-09-22 2023-04-04 武汉滨会生物科技股份有限公司 异源三聚体化融合蛋白、组合物及其应用
CN115894713B (zh) * 2022-09-22 2023-08-01 武汉滨会生物科技股份有限公司 异源三聚体化融合蛋白、组合物及其应用

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