CN115353552B - Method for reducing protein denaturation temperature, mutant and application thereof - Google Patents
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Abstract
The invention discloses a method for reducing protein denaturation temperature, and a mutant with reduced denaturation temperature is obtained, which can be used for preparing an attenuated live vaccine. According to the invention, through analyzing the existing protein structure, large side chain amino acids such as tryptophan of a structural core are selected, the codon base composition is analyzed, and mutation is carried out on the amino acids with 2-3 different base composition codons, so that the probability of reversion mutation is low. The structural core of the mutant protein is hollow, and the thermal stability of the mutant protein is changed. The invention performs reverse genetic operation on the mutant with reduced protein denaturation temperature or the combination thereof, and selects the mutant which can survive in nasal cavity or upper respiratory tract but can not survive in vivo so as to achieve the aim of constructing attenuated live vaccine. Compared with the traditional attenuated live vaccine, the method provided by the invention has the advantages that passage is not needed, the development time is short, the prepared attenuated live vaccine is not needed to be injected and inhaled through nasal cavity, and the method has a great application prospect in the field of preparation of pathogenic attenuated live vaccines.
Description
Technical Field
The invention relates to the technical fields of bioengineering and molecular biology, in particular to a method for reducing protein denaturation temperature, a mutant with reduced protein denaturation temperature obtained by the method, and application of the mutant in preparation of attenuated live vaccines.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Proteins have primary, secondary, tertiary and quaternary structures, which are maintained by bonds and interactions between amino acids. The folding of a protein depends on the amino acid sequence, and the protein is encoded by the gene that determines its amino acid sequence. The amino acids are assembled sequentially in primary structure, the proper interactions of the amino acids form a secondary structure, the energy within the protein is finally minimized, the protein is folded into a stable conformation, a stable 3D structure is generated, and the 3D structure provides stability for the protein, so that the protein has functional activity.
Protein stability includes dynamic stability and thermal stability, where the denaturation temperature of the protein is a visual indicator of protein thermal stability. Protein engineering aims to overcome the natural limitations of proteins under harsh conditions by increasing their stability and activity, and in most cases the higher the denaturation temperature, the better the stability of the protein and the longer the time to remain active in the environment.
In the field of enzyme engineering, optimizing various industrial enzymes, diagnostic enzymes, antibodies, antigens and other products has important significance: in most cases, it is desirable that the higher the thermal stability of these products, the more the amount of related protein products can be reduced, which has a key effect on cost reduction. To meet this demand, various methods for improving the thermal stability of proteins have been developed. For example: introducing disulfide bond, increasing ion interaction, multimerization, chemical crosslinking immobilization, site-directed mutagenesis and the like. These methods are beneficial to increasing the thermal denaturation temperature of the protein and increasing its working life.
The above-mentioned improvement of protein denaturation temperature and enhancement of stability have been studied and applied, but in special cases, the reduction of protein thermal denaturation temperature will also have important uses. For example: allowing one of the enzymatic reactions to occur only at a desired low temperature; another important potential use is the design of vaccines, whereby by lowering the temperature of thermal denaturation of the naturally encoded proteins of the virus, the virus is in an unstable state in the human environment, and strains with greatly reduced toxicity can be obtained, thus making it possible to obtain a specific attenuated vaccine. However, few methods for reducing the thermal denaturation temperature of proteins have been studied. In the studies of the applicant, it was found that only in the case of site-directed mutagenesis, the decrease in the denaturation temperature of the protein was likely to occur randomly, and if only one base was mutated, the back mutation was likely to occur. If the mutation amino acid has 2-3 codon bases which are different from the original amino acid codons, the probability of back mutation is reduced, and the low probability of back mutation can be ensured. Through searching, a method for reducing the protein denaturation temperature by modifying a hydrophobic amino acid selected from a larger side chain of a protein structural core, a mutant with reduced protein denaturation temperature obtained by the method, and application of the mutant in preparation of attenuated live vaccines have not been reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for reducing the protein denaturation temperature, a mutant with reduced protein denaturation temperature obtained by the method and application of the mutant in preparation of attenuated live vaccines.
The method for reducing the protein denaturation temperature comprises the following steps:
(1) Analyzing and selecting mutant amino acid sites of a single domain of the protein, analyzing related protein structures disclosed by a PDB database (the website is https:// www1.Rcsb. Org), selecting hydrophobic amino acids on a larger side chain of a structural core for modification, and trying to prepare mutant amino acids with codons not easy to return mutation;
(2) Designing an amino acid mutation primer, mutating an escherichia coli expression plasmid containing a single structural domain gene for encoding the protein to obtain an expression plasmid of mutant amino acids with at least 2-3 codon bases different from original amino acid codons, and then transforming escherichia coli to obtain a designed expression strain of the mutant amino acids;
(3) Culturing the obtained Escherichia coli expression strain to OD 600 Performing IPTG induction expression at 0.6-0.8, and collecting thallus to obtain target protein-producing thallus;
(4) Crushing and centrifuging the collected thalli, adding supernatant containing target protein to a Ni-NTA affinity chromatographic column, then carrying out enzyme digestion by using PPase or ULP1, eluting the target protein by using an elution buffer solution, and obtaining the high-purity mutant protein which does not need further purification;
(5) After the mutant protein is obtained, a protein denaturation temperature measurement experiment is carried out by using a fluorescent quantitative PCR reagent Protein Thermal Shift Dye Kit, a melting curve of ROX fluorescent signals along with the temperature rise is obtained, the denaturation temperature of the mutant protein is calculated according to the melting curve, and whether the mutant with the protein denaturation temperature reduced is obtained is determined.
In the method for reducing the denaturation temperature of protein, the following steps are adopted: the single domain of the protein in the step (1) is preferably novel coronavirus structural protein N-terminal domain NTD, novel coronavirus structural protein N-protein C-terminal domain CTD or novel coronavirus nonstructural protein Nsp7; the selected mutant amino acid site is preferably tryptophan at position 108 or tryptophan at position 132 of the N-terminal domain of the N-protein of the novel coronavirus structural protein, tryptophan at position 301, tryptophan at position 330 or leucine at position 331 of the C-terminal domain of the N-protein of the novel coronavirus structural protein, glutamine at position 29 and 31 of the Nsp7 structural core of the novel coronavirus non-structural protein; the hydrophobic amino acid of the larger side chain of the structural core is preferably tryptophan, phenylalanine, histidine or tyrosine.
In the above method for reducing protein denaturation temperature, a further preferred embodiment is: the single domain of the protein in the step (1) is novel coronavirus structural protein N-end domain NTD, the selected mutation amino acid site is 132 th tryptophan of N-end domain NTD of the novel coronavirus structural protein N-end domain, and the hydrophobic amino acid of the larger side chain of the structural core is tryptophan. Because tryptophan (W) is encoded by only one codon TGG, while other amino acids have codon degeneracy, encoded by 2-6 codons, tryptophan is mutated and all three bases are the most preferred, unlike TGG, with the smallest probability of back mutation.
In the method for reducing the denaturation temperature of protein, the following steps are adopted: the mutant amino acid of step (2) in which the 2-3 codon bases are different from the codons of the original amino acid is preferably leucine, proline, histidine, glutamine, isoleucine, threonine, asparagine, lysine, valine, alanine, aspartic acid or glutamic acid; the escherichia coli expression vector is preferably a pET-15b, pET-32a or pET-28b-MBP vector; the E.coli is preferably E.coli BL21 (DE 3).
The invention provides a method for obtaining novel mutant with reduced denaturation temperature of N-terminal domain NTD protein of coronavirus structural protein, which comprises the following steps:
(1) Downloading an NTD structure in a PDB database (the website is https:// ww1.Rcsb. Org/structure/7 CDZ), opening the structure in Coot software, analyzing the amino acid position of the structure, wherein the tryptophan at 132 is positioned in a structural core, and selecting the tryptophan at 132 for mutation;
(2) Synthesizing an NTD gene by using a gene synthesis technology, amplifying by polymerase chain reaction to obtain a nucleotide sequence of the coding NTD gene, and connecting the nucleotide sequence into a pET-15b expression vector by seamless cloning to obtain an expression plasmid; mutation of tryptophan at position 132 into histidine which is different from the three bases of the codon TGG, wherein the amino acid codon is CAT; designing an amino acid mutation primer NTD W132H upstream primer: GGTATTATTcatGTGGCAACCGAAGGTGCA, NTD W132H downstream primer: GGTTGCCACatgAATAATACCATCCTTATTTGCACCATA by quick change method, obtaining NTD W132H mutant expression plasmid, transforming the expression plasmid into E.coli BL21 (DE 3) to obtain expression strain of mutant amino acid of tryptophan (W) mutated into histidine (H);
(3) Inoculating the expression strain into LB culture medium, culturing the strain to OD 600 Adding IPTG to perform induction expression for 0.6-0.8, collecting thalli, resuspending thalli, crushing and centrifuging at high pressure, adding supernatant containing target protein to a Ni-NTA affinity chromatographic column, then performing PPase enzyme digestion overnight, eluting the target protein by using an elution buffer solution to obtain high-purity mutant protein which does not need further purification, and naming the high-purity mutant protein as N-NTD W132H mutant;
(4) The concentration and purity of the mutant protein N-NTD W132H are identified by polyacrylamide gel electrophoresis;
(5) After obtaining mutant protein N-NTD W132H, using a fluorescent kit Thermal Shift Dye Kit, detecting ROX fluorescence change by using a fluorescent quantitative PCR instrument Quantum studio 3Real-Time PCR System to obtain a melting curve of fluorescence along with temperature rise, and using software Protein Thermal Shift TM The melting curve was analyzed by Software v1.0, and the denaturation temperature of the N-NTD W132H mutant protein was calculated, which revealed that the denaturation temperature was reduced from 48.9℃to 37℃in the wild-type, and it was confirmed that the mutant having reduced protein denaturation temperature was obtained.
The mutant with reduced protein denaturation temperature, which is prepared by the method or a combination of more than one of the mutants, is characterized in that: the mutants are respectively N-NTD W132T or N-NTD W108L at the temperature of 32-34 ℃; N-CTD W330NL331W at 35-36 ℃; N-NTD W132H at 37 ℃; N-NTD W108H, nsp W29E Q F or Nsp 7W 29EQ31W at 38deg.C; N-CTD W301F at 39-40 ℃; nsp 7W 29Q or Nsp 7W 29T at 41-42 ℃; nsp 7W 29E at 43-45 ℃; N-CTD W330N L W at 35-36 ℃, N-NTD W132H at 37 ℃ and Nsp 7W 29E Q F at 38 ℃ are combined; N-CTD W330N L W at 35-36 ℃, N-NTD W132H at 37 ℃ and Nsp 7W 29E Q W at 38 ℃ are combined; combining N-NTD W132T at 32-34 ℃, N-CTD W330N L W at 35-36 ℃ and Nsp 7W 29E Q F at 38 ℃; combining N-NTD W132T at 32-34 ℃, N-CTD W330N L W at 35-36 ℃ and Nsp 7W 29E Q W at 38 ℃; combining N-NTD W108L at 32-34 ℃, N-CTD W330N L W at 35-36 ℃ and Nsp 7W 29E Q F at 38 ℃; combining N-NTD W108L at 32-34 ℃, N-CTD W330N L W at 35-36 ℃ and Nsp 7W 29E Q W at 38 ℃; combining N-CTD W330N L W at 35-36 ℃, N-NTD W132H at 37 ℃ and Nsp 7W 29Q at 41-42 ℃; combining N-CTD W330N L W at 35-36 ℃, N-NTD W132H at 37 ℃ and Nsp 7W 29T at 41-42 ℃; combining N-NTD W132H at 37 ℃, nsp 7W 29E Q F at 38 ℃ and N-CTD W301F at 39-40 ℃; combining N-NTD W132H at 37 ℃, nsp 7W 29E Q W at 38 ℃ and N-CTD W301F at 39-40 ℃; combining N-NTD W108H at 38 ℃ and N-CTD W301F at 39-40 ℃; combining N-CTD W330N L W at 35-36 ℃ with N-NTD W108H at 38 ℃; N-NTD W132H at 37℃and Nsp 7W 29E Q F at 38℃are combined; N-NTD W132H at 37℃and Nsp 7W 29E Q W at 38℃are combined; combining N-CTD W330N L W at 35-36 ℃ with N-NTD W132H at 37 ℃; combining N-NTD W132H at 37 ℃ and N-CTD W301F at 39-40 ℃; combining Nsp 7W 29E Q F at 38 ℃ with N-CTD W301F at 39-40 ℃; nsp 7W 29W E Q W at 38 ℃ and N-CTD W301F at 39-40 ℃ were combined.
Among the above mutants, further preferred mutants are respectively: N-CTD W330N L W at 35-36 ℃; N-NTD W132H at 37 ℃; N-NTD W108H, nsp W29E Q F or Nsp 7W 29E Q31W at 38deg.C; N-CTD W330N L W at 35-36 ℃, N-NTD W132H at 37 ℃ and Nsp 7W 29E Q F at 38 ℃ are combined; N-CTD W330N L W at 35-36 ℃, N-NTD W132H at 37 ℃ and Nsp 7W 29E Q W at 38 ℃ are combined; combining N-CTD W330N L W at 35-36 ℃ with N-NTD W108H at 38 ℃; N-NTD W132H at 37℃and Nsp 7W 29E Q F at 38℃are combined; N-NTD W132H at 37℃and Nsp 7W 29E Q W at 38℃are combined; N-CTD W330N L W at 35-36℃and N-NTD W132H at 37℃are combined.
The mutant with reduced protein denaturation temperature is applied to preparation of attenuated live vaccines.
Wherein: the application method comprises the steps of amplifying cDNA of a virus to be prepared through RT-PCR, splicing the cDNA into full length, carrying out gene mutation by utilizing mutants or combinations thereof with reduced protein denaturation temperature to obtain mutant genes, carrying out in vitro transcription to obtain cRNA, then introducing competent cells through transfection or electroporation by a conventional method, carrying out virus rescue to generate infectious and high-titer progeny viruses, and further preparing the attenuated live vaccine.
Further, the attenuated live vaccine strain, a freeze-drying protective agent (the volume percentage of skim milk is 0.5%, the mass percentage of trehalose is 6.0%, the mass percentage of inositol is 0.5%, the mass percentage of dextran-40 is 0.5%, the volume percentage of glycerin is 0.5%, the mass percentage of urea is 0.1%), a storage protective agent (the mass percentage of sodium glutamate is 0.5% and the mass percentage of thiourea group is 0.5%), a dispersing agent (the mass percentage of leucine is 0.5% and the mass percentage of poloxamer is 0.05%) and deionized water are mixed to obtain a mixed solution containing viruses, and the mixed solution is subjected to vacuum freeze-drying to prepare a freeze-drying vaccine, so that the vaccine is convenient to store and transport, and the powder vaccine is preferably inhaled into nasal cavities for use.
The technical scheme of the invention has the beneficial technical effects that:
(1) The mutation of large side chain amino acid in the protein structure nucleus is selected, so that the protein structure nucleus is easy to generate a cavity, the denaturation temperature is further influenced, and a proper mutant is obtained.
(2) The mutated amino acids are all caused by mutation of 2-3 bases, and through combination of mutation of several amino acids, the mutant sites are more, the probability of occurrence of high-toxicity back mutant is low, and the safety is high.
(3) Besides respiratory viruses, the invention can also be used for producing other pathogen vaccines.
The invention provides a method for reducing protein denaturation temperature, one or more combined mutants with reduced protein denaturation temperature are obtained by the method, and the application of the mutants in preparation of attenuated live vaccines is provided. The establishment of the method of the invention provides a new idea for vaccine development and other bioengineering products, and the achievement of the method has certain theoretical significance and application value and wide application prospect.
According to the invention, through analyzing the protein structure, large side chain amino acids such as tryptophan of the structural core are selected, and through mutation, the structural core of the protein is made to generate a cavity, so that the thermal stability of the protein is changed, and through the combination of several mutant proteins, mutants at the survival temperature of nasal cavity or upper respiratory tract can be selected, so that the purpose of constructing attenuated live vaccine is achieved. Compared with the traditional attenuated live vaccine, the method has the advantages that passage is not needed, development time is shortened, injection is not needed, and nasal inhalation is needed. Compared with the traditional inactivated vaccine, the effective is enhanced under the condition of ensuring the safety, and the more comprehensive immunogenicity is caused. Therefore, the method has great application prospect and economic value in the field of pathogen attenuated live vaccines.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a diagram of the amino acid structure of the interaction of tryptophan at position 108 (left) and tryptophan at position 132 (right) of the novel N protein NTD structural core of coronavirus structural protein.
FIG. 2 is a diagram showing the structure of the amino acid interactions between tryptophan at position 301 (left) and tryptophan at position 330 (right) of the CTD structural core of the novel coronavirus structural protein N.
FIG. 3 is a diagram of the amino acid structure of tryptophan at position 29 of the novel coronavirus nonstructural protein Nsp7 structural core.
FIG. 4 is a graph of NTD wild-type and mutant protein denaturation temperature measurement.
FIG. 5 is a graph showing the denaturation temperature of CTD wild-type and mutant proteins.
FIG. 6 is a graph showing the denaturation temperature of Nsp7 wild-type and mutant proteins.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
The present invention uses techniques and methods conventional in the fields of genetic engineering and molecular biology. Those skilled in the art may utilize other conventional techniques, methods and reagents in the art based on the embodiments provided herein and are not limited to the specific examples of the invention. In the examples described below, the experimental methods used, not specifically described, are conventional methods, and reference may be made, for example, to the "molecular cloning Experimental guidelines" (Sambrook and Russell, 2001).
In the following examples:
the expression vectors pET-15b, pET-32a and pET-28b are purchased from vast biological technology Co., ltd
The E.coli BL21 (DE 3) was purchased from Beijing all gold biotechnology Co.
The PPase was purchased from the division of bioengineering (Shanghai).
The mutant primers were purchased from the division of biological engineering (Shanghai).
The 2 XPrimer star Mix was purchased from Bao organism.
The Ni column was purchased from GE healthcare.
In the examples described below, materials, reagents, strains, vectors and the like used, unless otherwise specified, are all commercially available.
Embodiments of the present invention will be described in detail below with reference to the attached drawings, and it should be noted that, without conflict, the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other.
Example 1: modification of N-terminal domain NTD of novel coronavirus structural protein N-protein and mutant screening thereof
1. Downloading NTD structure in PDB database (website is https:// ww1.Rcsb. Org/structure/7 CDZ), opening in Coot software, analyzing amino acid position, and selecting 108 th tryptophan or 132 th tryptophan of structural core for mutation. The analysis result is shown in figure 1.
2. The tryptophan (W) at the two sites was mutated to amino acids different from all three bases of the tryptophan codon TGG, respectively, namely: CTT (leucine L), CCT (proline P), CAT (histidine H), CAA (glutamine Q), ATT (isoleucine I), ACT (threonine T), AAT (asparagine N), AAA (lysine K), GTT (valine V), GCT (alanine a), GAT (aspartic acid D), GAA (glutamic acid E).
3. The NTD gene (Huada gene) is synthesized by utilizing a gene synthesis technology, the nucleotide sequence of the coding NTD gene is obtained through Polymerase Chain Reaction (PCR) amplification, and is connected into a pET-15b expression vector to obtain an expression plasmid NTD-pET-15b.
4. Design of amino acid mutation primer
NTD W108X upstream primer: AGCCCGCGTGxxxTATTTTTATTTATTCTGGGCACCGGCC
NTD W108X downstream primer: AATAAATAyyACGCGGGCTCAGATCTTTTCA
NTD W132X upstream primer: GGTATATTXXxGTGGCAACCGAAGGTGCA
NTD W132X downstream primer: GGTTGCCACyyyAATATATATATATTTTGCACCATA
Wherein X corresponds to leucine L, proline P, histidine H, glutamine Q, isoleucine I, threonine T, asparagine N, lysine K, valine V, alanine A, aspartic acid D, glutamic acid E; xxx corresponds to CTT, CCT, CAT, CAA, ATT, ACT, AAT, AAA, GTT, GCT, GAT, GAA; yyy corresponds to AAG, AGG, GTA, TTG, AAT, AGT, TTA, TTT, AAC, AGC, ATC, TTC.
5. The plasmid and the above primers were added to a system using quick change method, which was formulated as follows:
| reagent name | Dosage of |
| 2×Primer star Mix | 12.5μL |
| Upstream primer (10. Mu.M) | 1μL |
| Downstream primer (10. Mu.M) | 1μL |
| Above plasmid (20 ng/. Mu.L) | 1μL |
| Water and its preparation method | 9.5μL |
| Total (S) | 25μL |
After the PCR of the system is finished, obtaining mutant expression plasmids NTD W132L-pET-15b, NTD W132P-pET-15b, NTD W132H-pET-15b, NTD W132Q-pET-15b, NTD W132I-pET-15b, NTD W132T-pET-15b, NTD W132N-pET-15b, NTD W132K-pET-15b, NTD W132V-pET-15b, NTD W132A-pET-15b, NTD W132D-pET-15b, NTD W132E-pET-15b, NTD W108L-pET-15b, NTD 108P-pET-15b, NTD 108H-pET-15b, NTD 108Q-pET-15b, NTD 108T-pET-15b, NTD 108N-pET-15b, NTD 108K-108T-108B, NTD-pET-108T-15 b, NTD 108T-pET-108B, NTD-108T-pET-15 b, E.coli BL21 (DE 3) was transformed separately to obtain the designed mutant amino acid expression strain.
6. The expression strains were inoculated into LB medium containing ampicillin (containing pancreas per 1000 mL)Peptone 10g, yeast extract 5g, sodium chloride 10g, ampicillin 100 mg), and culturing the cells at 37℃and 200rpm until OD 600 About 0.8, cooled to 16℃and after 1 hour IPTG was added to the medium at a final concentration of 0.3mM for induction of expression, and cultured overnight at 200 rpm.
The following day the cells were collected (4 ℃,5000×g, centrifuged for 18 min, the supernatant discarded), and 30mL lysis buffer (25mM Tris pH 8.0,200mM sodium chloride) was added per liter to suspend the cells. High-pressure crushing the resuspended bacterial cells, centrifuging the crushed bacterial liquid (25200 Xg, 4 ℃ for 50 minutes), adding the supernatant containing the target protein to a Ni-NTA affinity chromatographic column, combining Ni with a histidine tag introduced into the protein, hanging the protein on the chromatographic column, flushing about 10 times of column volume by using the lysis buffer solution, removing nonspecifically adsorbed impurity proteins, adding 1 time of column volume of the lysis buffer solution, blocking the Ni-NTA affinity chromatographic column, adding His-carrying PPase, and carrying out enzyme digestion overnight to obtain the mutant protein with higher purity by flow-through: NTD W132L, NTD W132P, NTD W132H, NTD W132Q, NTD W132I, NTD W132T, NTD W132N, NTD W132K, NTD W132V, NTD W132A, NTD W132D, NTD W132E, NTD W108L, NTD W108P, NTD W108H, NTD W108Q, NTD W108I, NTD W108T, NTD W108N, NTD W108K, NTD W108V, NTD W108A, NTD W108D, NTD W108E.
Finally, the concentration and purity of the protein samples were identified by polyacrylamide gel electrophoresis.
7. Thermal Shift Dye Kit was purchased from Siemens, inc., where Dye was 1000X, diluted to 8X for use, diluted to 1mg/mL for protein concentration, and 25mM Tris pH 8.0,200mM sodium chloride was used as buffer. Is configured according to the following system:
| composition of the ingredients | Dosage of |
| 4 Xbuffer | 5μL |
| Protein (1 mg/mL) | 10μL |
| Dye(8×) | 2.5μL |
| Water and its preparation method | 2.5μL |
| Total (S) | 20μL |
The change in ROX fluorescence was detected using a fluorescent quantitative PCR instrument (Quantum 3 Real-Time PCR System) and the following procedure was followed:
| Step | Ramp rate | Temp(℃) | Time(mm:ss) |
| 1 | 100％ | 25.0 | 02:00 |
| 2 | 1％ | 99.0 | 02:00 |
using software Protein Thermal Shift TM The melting curve was analyzed by Software v1.0 to calculate the denaturation temperature of each mutant protein.
The results were as follows:
wherein "-" indicates that the mutein is insoluble and no useful protein is obtained.
The results indicate that mutation of tryptophan in the structural nucleus of the protein can indeed cause a large variation in protein stability, with a major decrease in denaturation temperature and an increase in denaturation temperature for a few mutants.
Screening mutant proteins with denaturation temperature reduced to 32-45 ℃: NTD W132T (32-34 ℃), NTD W108L (35 ℃), NTD W132H (37 ℃), NTD W108H (38 ℃).
Example 2: modification of novel coronavirus structural protein N-protein C-terminal domain CTD and mutant screening thereof
1. The CTD structure is downloaded in a PDB database (the website is https:// ww1.Rcsb. Org/structure/7C 22), opened in Coot software, the amino acid position is analyzed, and the 301 th tryptophan or the 330 th tryptophan of the structural core is selected to be mutated respectively. The analysis results are shown in figure 2.
2. The tryptophan (W) at the two sites was mutated to amino acids that differ from all three bases of the tryptophan codon TGG: CTT (leucine L), CCT (proline P), CAT (histidine H), CAA (glutamine Q), ATT (isoleucine I), ACT (threonine T), AAT (asparagine N), AAA (lysine K), GTT (valine V), GCT (alanine a), GAT (aspartic acid D), GAA (glutamic acid E); tryptophan at position 301 is mutated to TTT (phenylalanine F) with two different codon bases.
3. The CTD gene (Huada gene) is synthesized by utilizing a gene synthesis technology, the nucleotide sequence of the coding CTD gene is obtained through Polymerase Chain Reaction (PCR) amplification, and is connected into a pET-15b expression vector to obtain an expression plasmid CTD-pET-15b.
4. Design of amino acid mutation primer
CTD W301X upstream primer: TATAAAACATxxxCCGCAGATTGCCCAGTTTT
CTD W301X downstream primer: AATCTGCGGyyyATGTTTATAATCGGTGCCCTGG
CTD W330X upstream primer: AGCGGTACCxxxCTGACCTATAACCGGTGCAATTAAGC
CTD W330X downstream primer: ATAGGTCAGyyGGTACCGCTCGGGGTAACT
Wherein X corresponds to leucine, proline, histidine, glutamine, isoleucine, threonine, asparagine, lysine, valine, alanine, aspartic acid, glutamic acid, phenylalanine; xxx corresponds to CTT, CCT, CAT, CAA, ATT, ACT, AAT, AAA, GTT, GCT, GAT, GAA, TTT; yyy corresponds to AAG, AGG, GTA, TTG, AAT, AGT, TTA, TTT, AAC, AGC, ATC, TTC, AAA.
5. The plasmid and the above primers were added to a system using quick change method, which was formulated as follows:
| reagent name | Dosage of |
| 2×Primer star Mix | 12.5μL |
| Upstream primer (10. Mu.M) | 1μL |
| Downstream primer (10. Mu.M) | 1μL |
| Above plasmid (20 ng/. Mu.L) | 1μL |
| Water and its preparation method | 9.5μL |
| Total (S) | 25μL |
After the PCR of the above system was completed, mutant expression plasmids CTD W301L-pET-15b, CTD W301P-pET-15b, CTD W301H-pET-15b, CTD W301Q-pET-15b, CTD W301I-pET-15b, CTD W301T-pET-15b, CTD W301N-pET-15b, CTD W301K-pET-15b, CTD W301V-pET-15b, CTD W301A-pET-15b, CTD W301D-pET-15b, CTD W301E-pET-15b, CTD W301F-pET-15b, CTD 330L-pET-15b, CTD 330P-pET-15b, CTD 330H-pET-15b, CTD 330Q-pET-15b, CTD 330I-pET-15b, CTD 330T-pET-15b, CTD 330T-pET-330B, CTD-330T-330B, CTD-330B, and CTD-330D-pET-330B were obtained, E.coli BL21 (DE 3) was transformed separately to obtain the designed mutant amino acid expression strain.
6. The expression strains were inoculated into LB medium containing ampicillin (10 g per 1000mL of tryptone, 5g of yeast extract, 10g of sodium chloride, 100mg of ampicillin), and the cells were cultured at 37℃and 200rpm until the OD600 was about 0.8, cooled to 16℃and, after 1 hour, IPTG was added to the medium at a final concentration of 0.3mM for induction of expression, and cultured at 200rpm overnight.
The following day the cells were collected (4 ℃,5000×g, centrifuged for 18 min, the supernatant discarded), and 30mL lysis buffer (25mM Tris pH 8.0,200mM sodium chloride) was added per liter to suspend the cells. High-pressure crushing the resuspended bacterial cells, centrifuging the crushed bacterial liquid (25200 Xg, 4 ℃ for 50 minutes), adding the supernatant containing the target protein to a Ni-NTA affinity chromatographic column, combining Ni with a histidine tag introduced into the protein, hanging the protein on the chromatographic column, flushing about 10 times of column volume by using the lysis buffer solution, removing nonspecifically adsorbed impurity proteins, adding 1 time of column volume of the lysis buffer solution, blocking the Ni-NTA affinity chromatographic column, adding His-carrying PPase, and carrying out enzyme digestion overnight to obtain the mutant protein with higher purity by flow-through: CTD W301L, CTD W301P, CTD W301H, CTD W301Q, CTD W301I, CTD W301T, CTD W301N, CTD W301K, CTD W301V, CTD W301A, CTD W301D, CTD W301E, CTD W301F, CTD W330L, CTD W330P, CTD W330H, CTD W330Q, CTD W330I, CTD W330T, CTD W330N, CTD W330K, CTD W330V, CTD W330A, CTD W330D, CTD W330E.
Finally, the concentration and purity of the protein samples were identified by polyacrylamide gel electrophoresis.
7. Thermal Shift Dye Kit was purchased from Siemens, inc., where Dye was 1000X, diluted to 8X for use, diluted to 1mg/mL for protein concentration, and 25mM Tris pH 8.0,200mM sodium chloride was used as buffer. Is configured according to the following system:
| composition of the ingredients | Dosage of |
| 4 Xbuffer | 5μL |
| Protein (1 mg/mL) | 10μL |
| Dye(8×) | 2.5μL |
| Water and its preparation method | 2.5μL |
| Total (S) | 20μL |
The change in ROX fluorescence was detected using a fluorescent quantitative PCR instrument (Quantum 3 Real-Time PCR System) and the following procedure was followed:
| Step | Ramp rate | Temp(℃) | Time(mm:ss) |
| 1 | 100％ | 25.0 | 02:00 |
| 2 | 1％ | 99.0 | 02:00 |
using software Protein Thermal Shift TM The melting curve was analyzed by Software v1.0 to calculate the denaturation temperature of each mutant protein.
The results were as follows:
wherein "-" indicates that the mutein is insoluble and no useful protein is obtained.
After the above results, no mutant proteins with denaturation temperatures around 37℃were selected, and the present invention suggests that in mutants with better solubility and denaturation temperatures closest to 37℃the possibility of continuing to mutate the amino acids around tryptophan to achieve a further small range of changes in the denaturation temperature.
In this example, we selected CTD W330N mutants with denaturation temperatures of 48.46 ℃ for further engineering. Preliminary structural analysis, a mutation can be made to leucine (L) at position 331 of the hydrophobic amino acid near W330. The mutation of L331 into tyrosine (Y), lysine (K) and tryptophan (W) was chosen by the principle of changing as much as possible more codon bases.
The results were as follows:
the results indicate that mutation of tryptophan in the structural nucleus of the protein can indeed cause a large variation in protein stability, with a major decrease in denaturation temperature and an increase in denaturation temperature for a few mutants.
Screening mutant proteins with denaturation temperature reduced to 32-45 ℃: CTD W330N L W (35-36 ℃), CTD W330N L Y (35-36 ℃), CTD W301F (40 ℃).
Example 3: modification of novel coronavirus nonstructural protein Nsp7 and mutant screening thereof
1. The Nsp7 structure is downloaded in a PDB database (website is https:// ww1.Rcsb. Org/structure/7 DCD), opened in Coot software, the amino acid position is analyzed, and the 29 th tryptophan of the structural core is selected for mutation. The analysis results are shown in figure 3.
2. Tryptophan (W) is mutated to an amino acid which differs from the tryptophan codon TGG by three bases: CTT (leucine L), CCT (proline P), CAT (histidine H), CAA (glutamine Q), ATT (isoleucine I), ACT (threonine T), AAT (asparagine N), AAA (lysine K), GTT (valine V), GCT (alanine a), GAT (aspartic acid D), GAA (glutamic acid E).
3. The Nsp7 gene (Huada gene) is synthesized by utilizing a gene synthesis technology, the nucleotide sequence of the coding Nsp7 gene is obtained by Polymerase Chain Reaction (PCR) amplification, and is connected into a pET-21b expression vector to obtain an expression plasmid Nsp7-pET-21b.
4. Design of amino acid mutation primer
Nsp7W 29X upstream primer: AGCAAACTGGxxGCCCAGTGCGTTCAGCTG
Nsp7W 29X downstream primer: CTGGGCyyCAGTTTGCTGCTACTTTTCAAC
Wherein X corresponds to leucine, proline, histidine, glutamine, isoleucine, threonine, asparagine, lysine, valine, alanine, aspartic acid, glutamic acid; xxx corresponds to CTT, CCT, CAT, CAA, ATT, ACT, AAT, AAA, GTT, GCT, GAT, GAA; yyy corresponds to AAG, AGG, GTA, TTG, AAT, AGT, TTA, TTT, AAC, AGC, ATC, TTC.
5. The plasmid and the above primers were added to a system using quick change method, which was formulated as follows:
| reagent name | Dosage of |
| 2×Primer star Mix | 12.5μL |
| Upstream primer (10. Mu.M) | 1μL |
| Downstream primer (10. Mu.M) | 1μL |
| Above plasmid (20 ng/. Mu.L) | 1μL |
| Water and its preparation method | 9.5μL |
| Total (S) | 25μL |
After the PCR of the system is finished, mutant expression plasmids NSp7W 29L-pET-21b, NSp7W 29P-pET-21b, NSp7W 29H-pET-21b, NSp7W 29Q-pET-21b, NSp7W 29I-pET-21b, NSp7W 29T-pET-21b, NSp7W29N-pET-21b, NSp7W 29K-pET-21b, NSp7W 29V-pET-21b, NSp7W 29A-pET-21b, NSp7W29D-pET-21b and NSp7W 29E-pET-21b are obtained, and E.coli BL21 (DE 3) is transformed respectively to obtain the designed mutant amino acid expression strains.
6. The expression strains were inoculated into LB medium containing ampicillin (10 g per 1000mL of tryptone, 5g of yeast extract, 10g of sodium chloride, 100mg of ampicillin), and the cells were cultured at 37℃and 200rpm until the OD600 was about 0.8, cooled to 16℃and, after 1 hour, IPTG was added to the medium at a final concentration of 0.3mM for induction of expression, and cultured at 200rpm overnight.
The following day the cells were collected (4 ℃,5000×g, centrifuged for 18 min, the supernatant discarded), and 30mL lysis buffer (25mM Tris pH 8.0,200mM sodium chloride) was added per liter to suspend the cells. High-pressure crushing the resuspended bacterial cells, centrifuging the crushed bacterial solution (25200 Xg, 4 ℃ for 50 minutes), adding the supernatant containing the target protein to a Ni-NTA affinity chromatographic column, combining Ni with a histidine tag introduced into the protein, hanging the protein on the chromatographic column, flushing the column with the lysis buffer for about 10 times of the column volume to remove non-specifically adsorbed impurity proteins, eluting the protein with an elution buffer (25mM Tris pH 8.0,100mM sodium chloride, 250mM imidazole), wherein imidazole in the buffer has stronger affinity with Ni, and performing competitive elution on the target protein to obtain mutant proteins with higher purity: nsp7W 29L, nsp7W 29P, nsp W29H, nsp W29Q, nsp W29I, nsp W29T, nsp W29N, nsp W29K, nsp W29V, nsp7W 29A, nsp7W 29D, nsp W29E.
Finally, the concentration and purity of the protein samples were identified by polyacrylamide gel electrophoresis.
7. Thermal Shift Dye Kit was purchased from Siemens, inc., where Dye was 1000X, diluted to 8X for use, diluted to 1mg/mL for protein concentration, and 25mM Tris pH 8.0,200mM sodium chloride was used as buffer. Is configured according to the following system:
| composition of the ingredients | Dosage of |
| 4 Xbuffer | 5μL |
| Protein (1 mg/mL) | 10μL |
| Dye(8×) | 2.5μL |
| Water and its preparation method | 2.5μL |
| Total (S) | 20μL |
The change in ROX fluorescence was detected using a fluorescent quantitative PCR instrument (Quantum 3 Real-Time PCR System) and the following procedure was followed:
| Step | Ramp rate | Temp(℃) | Time(mm:ss) |
| 1 | 100％ | 25.0 | 02:00 |
| 2 | 1％ | 99.0 | 02:00 |
using software Protein Thermal Shift TM The melting curve was analyzed by Software v1.0 to calculate the denaturation temperature of each mutant protein.
The results were as follows:
wherein "-" indicates that the mutein is insoluble and no useful protein is obtained.
After the above results, no mutant protein having a denaturation temperature around 37℃was obtained, and the present invention suggests that in the mutant having a good solubility and a denaturation temperature closest to 37℃the possibility of further mutating the amino acid around tryptophan was selected to achieve a further small change in the denaturation temperature.
In this example we selected the Nsp 7W 29E mutant with a denaturation temperature of 44.59 ℃ for further engineering. Preliminary structural analysis, glutamine (E) at position 31 of the hydrophobic amino acid near W29 may be mutated. The mutation of Q31 into phenylalanine (F) and tryptophan (W) was chosen on the principle of changing as much more codon bases as possible.
The results were as follows:
the results indicate that mutation of tryptophan in the structural nucleus of the protein can indeed cause a large variation in protein stability, with a major decrease in denaturation temperature and an increase in denaturation temperature for a few mutants.
Screening mutant proteins with denaturation temperature reduced to 32-45 ℃: NSP 7W 29E Q F (38-39 ℃), NSP 7W 29E Q W (38-39 ℃), W29Q (41-42 ℃), W29T (41-42 ℃), W29E (43-45 ℃).
The protein denaturation temperature measurement in the above examples is shown in FIG. 4 (example 1), FIG. 5 (example 2) and FIG. 6 (example 3).
Example 4: construction of novel coronavirus nonstructural protein NTD W132H mutant and denaturation temperature measurement
1. The NTD structure is downloaded in PDB database (website is https:// ww1.Rcsb. Org/structure/7 CDZ), opened in Coot software, the amino acid position is analyzed, tryptophan at 132 is found in the structure core, and the tryptophan at 132 has tight interaction with surrounding amino acids, so that mutation is carried out. The analysis results are shown in the right graph of the attached figure 1.
2. The tryptophan (W) codon is TGG, and 12 amino acids are different from the TGG in three codon bases, and in this example, tryptophan at position 132 is preferably mutated to histidine (H) with CAT codon.
3. The NTD gene (Huada gene) is synthesized by utilizing a gene synthesis technology, the nucleotide sequence of the coding NTD gene is obtained through Polymerase Chain Reaction (PCR) amplification, and is connected into a pET-15b expression vector through seamless cloning, so that the expression plasmid NTD-pET-15b is obtained.
4. Design of amino acid mutation primer
NTD W132H upstream primer: GGTATTATTcatGTGGCAACCGAAGGTGCA
NTD W132H downstream primer: GGTTGCCACatgAATAATACCATCCTTATTTGCACCATA
5. The plasmid and the above primers were added to a system using quick change method, which was formulated as follows:
| reagent name | Dosage of |
| 2×Primer star Mix | 12.5μL |
| NTD W132H upstream primer (10. Mu.M) | 1μL |
| NTD W132H downstream primer (10. Mu.M) | 1μL |
| Above plasmid (20 ng/. Mu.L) | 1μL |
| Water and its preparation method | 9.5μL |
| Total (S) | 25μL |
After the PCR of the system is finished, mutant expression plasmids are obtained: NTD W132H was transformed into E.coli BL21 (DE 3) to obtain an expression strain of a mutant amino acid in which tryptophan (W) was mutated to histidine (H).
6. The expression strain was inoculated into LB medium containing ampicillin (10 g per 1000mL of tryptone, 5g of yeast extract, 10g of sodium chloride, 100mg of ampicillin), and the cells were cultured at 37℃and 200rpm until OD 600 About 0.8, cooled to 16℃and after 1 hour IPTG was added to the medium at a final concentration of 0.3mM for induction of expression, and cultured overnight at 200 rpm.
The following day the cells were collected (4 ℃,5000×g, centrifuged for 18 min, the supernatant discarded), and 30mL lysis buffer (25mM Tris pH 8.0,200mM sodium chloride) was added per liter to suspend the cells. High-pressure crushing the resuspended bacterial cells, centrifuging the crushed bacterial liquid (25200 Xg, 4 ℃ for 50 minutes), adding the supernatant containing the target protein to a Ni-NTA affinity chromatographic column, combining Ni with a histidine tag introduced into the protein, hanging the protein on the chromatographic column, flushing about 10 times of column volume by using the lysis buffer solution, removing nonspecifically adsorbed impurity proteins, adding 1 time of column volume of the lysis buffer solution, blocking the Ni-NTA affinity chromatographic column, adding His-carrying PPase, and carrying out enzyme digestion overnight to obtain the mutant protein NTD W132H with higher purity by flow-through.
Finally, the concentration and purity of the protein samples were identified by polyacrylamide gel electrophoresis.
7. Thermal Shift Dye Kit was purchased from Siemens, inc., where Dye was 1000X, diluted to 8X for use, diluted to 1mg/mL for protein concentration, and 25mM Tris pH 8.0,200mM sodium chloride was used as buffer. Is configured according to the following system:
| composition of the ingredients | Dosage of |
| 4 Xbuffer | 5μL |
| NTD W132H protein (1 mg/mL) | 10μL |
| Dye(8×) | 2.5μL |
| Water and its preparation method | 2.5μL |
| Total (S) | 20μL |
The change in ROX fluorescence was detected using a fluorescent quantitative PCR instrument (Quantum 3 Real-Time PCR System) and the following procedure was followed:
| Step | Ramp rate | Temp(℃) | Time(mm:ss) |
| 1 | 100％ | 25.0 | 02:00 |
| 2 | 1％ | 99.0 | 02:00 |
using software Protein Thermal Shift TM The melting curve was analyzed by Software v1.0 to calculate the denaturation temperature of the NTD W132H mutant protein.
The results were as follows:
the result shows that mutation of tryptophan at position 132 in the protein structure nucleus into histidine can indeed reduce the thermal stability of the protein, and the denaturation temperature is reduced from 48.9 ℃ to 37 ℃.
Example 5: mutant protein combinations with reduced denaturation temperature
To increase the chance of being rescued after virus engineering and to provide a variety of potential live attenuated vaccines, we combined the protein mutants selected in examples 1-4 with reduced denaturation temperatures, preferably by listing one or more combinations of mutant proteins with denaturation temperatures of 32-45 ℃.
Mutant proteins with denaturation temperatures of 32-34 ℃: N-NTD W132T, N-NTD W108L.
Mutant proteins with denaturation temperatures of 35-36 ℃): N-CTD W330N L W331.
Mutant protein with denaturation temperature of 37 ℃): N-NTD W132H.
Mutant protein with denaturation temperature of 38 ℃): N-NTD W108H, nsp W29E Q F, nsp W29E Q W.
Mutant proteins with denaturation temperatures of 39-40 ℃: N-CTD W301F.
Mutant proteins with denaturation temperatures of 41-42 ℃): nsp 7W 29Q, nsp 7W 29T.
Mutant proteins with denaturation temperature of 43-45 ℃: nsp 7W 29E.
Each protein domain, different denaturation temperatures, in combination, including, but not limited to, the following combinations:
(1)N-CTD W330N L331W(35-36℃)、N-NTD W132H(37℃)、Nsp7 W29E Q31F(38℃)；
(2)N-CTD W330N L331W(35-36℃)、N-NTD W132H(37℃)、Nsp7 W29E Q31W(38℃)；
(3)N-NTD W132T(32-34℃)、N-CTD W330N L331W(35-36℃)、Nsp7 W29E Q31F(38℃)；
(4)N-NTD W132T(32-34℃)、N-CTD W330N L331W(35-36℃)、Nsp7 W29E Q31W(38℃)；
(5)N-NTD W108L(32-34℃)、N-CTD W330N L331W(35-36℃)、Nsp7 W29E Q31F(38℃)；
(6)N-NTD W108L(32-34℃)、N-CTD W330N L331W(35-36℃)、Nsp7 W29E Q31W(38℃)；
(7)N-CTD W330N L331W(35-36℃)、N-NTD W132H(37℃)、Nsp7 W29Q(41-42℃)；
(8)N-CTD W330N L331W(35-36℃)、N-NTD W132H(37℃)、Nsp7 W29T(41-42℃)；
(9)N-NTD W132H(37℃)、Nsp7 W29E Q31F(38℃)、N-CTD W301F(39-40℃)；
(10)N-NTD W132H(37℃)、Nsp7 W29E Q31W(38℃)、N-CTD W301F(39-40℃)；
(11)N-NTD W108H(38℃)、N-CTD W301F(39-40℃)；
(12)N-CTD W330N L331W(35-36℃)、N-NTD W108H(38℃)；
(13)N-NTD W132H(37℃)、Nsp7 W29E Q31F(38℃)；
(14)N-NTD W132H(37℃)、Nsp7 W29E Q31W(38℃)；
(15)N-CTD W330N L331W(35-36℃)、N-NTD W132H(37℃)；
(16)N-NTD W132H(37℃)、N-CTD W301F(39-40℃)；
(17)Nsp7 W29E Q31F(38℃)、N-CTD W301F(39-40℃)；
(18)Nsp7 W29E Q31W(38℃)、N-CTD W301F(39-40℃)。
the above combinations of 18 different denaturation temperature mutants around 37℃were combined, but are not limited to the above combinations.
Example 6: rational targeted mutagenesis of viruses (reverse genetics)
Reverse genetics was performed on the mutant with reduced protein denaturation temperature by using one or several combinations of the mutants described in example 5 according to the properties of respiratory viruses (specific methods can be referred to patent a new crown vaccine of measles live vector, application publication No. CN 113293145A), and the genome mutation was performed to obtain a specific attenuated live vaccine.
The novel coronavirus cDNA was amplified by RT-PCR and spliced to full length using the protein mutants or combinations thereof with reduced denaturation temperature provided in example 5: N-NTD W132T or N-NTD W108L at 32-34 ℃; N-CTD W330N L W at 35-36 ℃; N-NTD W132H at 37 ℃; N-NTD W108H, nsp W29E Q F or Nsp 7W 29E Q31W at 38deg.C; N-CTD W301F at 39-40 ℃; nsp 7W 29Q or Nsp 7W 29T at 41-42 ℃; nsp 7W 29E at 43-45 ℃; N-CTD W330N L W at 35-36 ℃, N-NTD W132H at 37 ℃ and Nsp 7W 29E Q F at 38 ℃ are combined; N-CTD W330N L W at 35-36 ℃, N-NTD W132H at 37 ℃ and Nsp 7W 29E Q W at 38 ℃ are combined; combining N-NTD W132T at 32-34 ℃, N-CTD W330N L W at 35-36 ℃ and Nsp 7W 29E Q F at 38 ℃; combining N-NTD W132T at 32-34 ℃, N-CTD W330N L W at 35-36 ℃ and Nsp 7W 29E Q W at 38 ℃; combining N-NTD W108L at 32-34 ℃, N-CTD W330NL331W at 35-36 ℃ and Nsp 7W 29E Q F at 38 ℃; combining N-NTD W108L at 32-34 ℃, N-CTD W330N L W at 35-36 ℃ and Nsp 7W 29E Q W at 38 ℃; combining N-CTD W330N L W at 35-36 ℃, N-NTD W132H at 37 ℃ and Nsp 7W 29Q at 41-42 ℃; combining N-CTD W330N L W at 35-36 ℃, N-NTD W132H at 37 ℃ and Nsp 7W 29T at 41-42 ℃; combining N-NTD W132H at 37 ℃, nsp 7W 29EQ31F at 38 ℃ and N-CTD W301F at 39-40 ℃; combining N-NTD W132H at 37 ℃, nsp 7W 29E Q W at 38 ℃ and N-CTD W301F at 39-40 ℃; combining N-NTD W108H at 38 ℃ and N-CTD W301F at 39-40 ℃; combining N-CTD W330N L W at 35-36 ℃ with N-NTD W108H at 38 ℃; N-NTD W132H at 37℃and Nsp 7W 29E Q F at 38℃are combined; N-NTD W132H at 37℃and Nsp 7W 29E Q W at 38℃are combined; combining N-CTD W330N L W at 35-36 ℃ with N-NTD W132H at 37 ℃; combining N-NTD W132H at 37 ℃ and N-CTD W301F at 39-40 ℃; combining Nsp 7W 29E Q F at 38 ℃ with N-CTD W301F at 39-40 ℃; corresponding gene mutation is carried out by combining Nsp 7W 29E Q W at 38 ℃ and N-CTD W301F at 39-40 ℃ to obtain mutant genes, cRNA is obtained by in vitro transcription in a conventional manner, competent cells are introduced by transfection or electroporation, and infection and high-titer progeny viruses are generated by virus rescue, so that attenuated live vaccines are prepared.
The protein mutant with reduced denaturation temperature or the combination (virus) thereof is used for carrying out reverse genetic modification and genome mutation, so that the stability of the strain is reduced at the normal temperature of 37 ℃ of a human body, the activity of the strain is reduced, the strain cannot survive in the human body and can only survive in the nasal cavity and the upper respiratory tract with lower temperature, and the strain becomes an attenuated live vaccine.
Example 7: attenuated live vaccine freeze-dried product
The attenuated vaccine strain provided in example 6, a lyoprotectant (0.5% by volume of skim milk, 6.0% by mass of trehalose, 0.5% by mass of inositol, 0.5% by mass of dextran-40%, 0.5% by volume of glycerol, 0.1% by mass of urea), a storage protectant (0.5% by mass of sodium glutamate and 0.5% by mass of thiourea group), a dispersant (0.5% by mass of leucine and 0.05% by volume of poloxamer) and deionized water were mixed to obtain a virus-containing mixed solution; vacuum freeze drying to obtain vaccine freeze-dried product.
Conclusion: according to the invention, through protein structure analysis, mutation is carried out on large side chain amino acids such as tryptophan of a structural core, and a measurement experiment of mutant protein extraction and denaturation temperature, several mutants capable of reducing the denaturation temperature are discovered. The virus is genetically modified by combining mutant proteins with different denaturation temperatures through reverse genetics means, so that the virus can only survive below 37 ℃, thereby preparing the virus attenuated live vaccine, and the vaccine is prepared into a freeze-dried product which is convenient to store and transport.
It should be noted that the above examples are only for illustrating the technical solution of the present invention and are not limiting thereof. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can make modifications and equivalents to the technical solutions of the present invention as required, without departing from the spirit and scope of the technical solutions of the present invention.
Claims (2)
1. A method for obtaining novel mutants of coronavirus structural protein N-terminal domain NTD protein having reduced denaturation temperature, comprising the steps of:
(1) Downloading an NTD structure in a PDB database with a website of https of/(www 1.Rcsb. Org/structure/7CDZ, opening in Coot software, analyzing the amino acid position of the NTD structure, wherein tryptophan at 132 is positioned in a structural core, and selecting the 132 tryptophan for mutation;
(2) Synthesizing an NTD gene by using a gene synthesis technology, amplifying by polymerase chain reaction to obtain a nucleotide sequence of the coding NTD gene, and connecting the nucleotide sequence into a pET-15b expression vector by seamless cloning to obtain an expression plasmid; mutation of tryptophan at position 132 into histidine which is different from the three bases of the codon TGG, wherein the amino acid codon is CAT; designing an amino acid mutation primer NTD W132H upstream primer: GGTATTATTcatGTGGCAACCGAAGGTGCA, NTD W132H downstream primer: GGTTGCCACatgAATAATACCATCCTTATTTGCACCATA by quick change method, obtaining NTD W132H mutant expression plasmid, transforming the expression plasmid into E.coli BL21 (DE 3) to obtain expression strain of mutant amino acid of tryptophan (W) mutated into histidine (H);
(3) Inoculating the expression strain into LB culture medium, culturing the strain to OD 600 Adding IPTG to 0.6-0.8, inducing expression overnight, collecting thallus, resuspending thallus, high-pressure crushing and centrifuging, adding supernatant containing target protein onto Ni-NTA affinity chromatographic column, then PPase enzyme cutting overnight, eluting target protein with eluting buffer,obtaining the high-purity mutant protein which does not need to be further purified, and naming the high-purity mutant protein as an N-NTD W132H mutant;
(4) The concentration and purity of the mutant protein N-NTD W132H are identified by polyacrylamide gel electrophoresis;
(5) After obtaining mutant protein N-NTD W132H, using a fluorescent kit Thermal Shift Dye Kit, detecting ROX fluorescence change by using a fluorescent quantitative PCR instrument Quantum studio 3 Real-Time PCR System to obtain a melting curve of fluorescence along with temperature rise, and using software Protein Thermal Shift TM The melting curve was analyzed by Software v1.0, and the denaturation temperature of the N-NTD W132H mutant protein was calculated, which revealed that the denaturation temperature was reduced from 48.4℃to 37℃in the wild-type, and it was confirmed that the mutant having reduced protein denaturation temperature was obtained.
2. A mutant having a reduced protein denaturation temperature, characterized in that: the mutants are respectively N-NTD W132T or N-NTD W108L with the denaturation temperature of 32-34 ℃; N-CTD W330N L W331 with denaturation temperature of 35-36 ℃; N-NTD W132H with denaturation temperature of 37 ℃; N-NTD W108H, nsp W24E Q F or Nsp 7W 24E Q W with denaturation temperature of 38deg.C; N-CTD W301F with denaturation temperature of 34-40 ℃; nsp 7W 24Q or Nsp 7W 24T with denaturation temperature of 41-42 ℃; nsp 7W 24E with denaturation temperature of 43-45 ℃; combining N-CTD W330N L W with denaturation temperature of 35-36 ℃, N-NTD W132H with denaturation temperature of 37 ℃ and Nsp 7W 24E Q F with denaturation temperature of 38 ℃; combining N-CTD W330N L W with denaturation temperature of 35-36 ℃, N-NTD W132H with denaturation temperature of 37 ℃ and Nsp 7W 24E Q W with denaturation temperature of 38 ℃; combining N-NTD W132T with denaturation temperature of 32-34 ℃, N-CTD W330N L331W with denaturation temperature of 35-36 ℃ and Nsp 7W 24E Q F with denaturation temperature of 38 ℃; combining N-NTD W132T with denaturation temperature of 32-34 ℃, N-CTD W330N L331W with denaturation temperature of 35-36 ℃ and Nsp 7W 24E Q W with denaturation temperature of 38 ℃; combining N-NTD W108L with denaturation temperature of 32-34 ℃, N-CTD W330N L331W with denaturation temperature of 35-36 ℃ and Nsp 7W 24E Q F with denaturation temperature of 38 ℃; combining N-NTD W108L with denaturation temperature of 32-34 ℃, N-CTD W330N L331W with denaturation temperature of 35-36 ℃ and Nsp 7W 24E Q W with denaturation temperature of 38 ℃; combining N-CTD W330N L W with denaturation temperature of 35-36 ℃, N-NTD W132H with denaturation temperature of 37 ℃ and Nsp 7W 24Q with denaturation temperature of 41-42 ℃; combining N-CTD W330N L W with denaturation temperature of 35-36 ℃, N-NTD W132H with denaturation temperature of 37 ℃ and Nsp 7W 24T with denaturation temperature of 41-42 ℃; combining N-NTD W132H with a denaturation temperature of 37 ℃, nsp 7W 24E Q F with a denaturation temperature of 38 ℃ and N-CTD W301F with a denaturation temperature of 34-40 ℃; combining N-NTD W132H with a denaturation temperature of 37 ℃, nsp 7W 24E Q W with a denaturation temperature of 38 ℃ and N-CTD W301F with a denaturation temperature of 34-40 ℃; combining N-NTD W108H with denaturation temperature of 38 ℃ and N-CTD W301F with denaturation temperature of 34-40 ℃; combining N-CTD W330N L W331 with denaturation temperature of 35-36 ℃ and N-NTD W108H with denaturation temperature of 38 ℃; combining N-NTD W132H with denaturation temperature of 37 ℃ and Nsp 7W 24E Q F with denaturation temperature of 38 ℃; combining N-NTD W132H with denaturation temperature of 37 ℃ and Nsp 7W 24E Q W with denaturation temperature of 38 ℃; combining N-CTD W330N L W331 with denaturation temperature of 35-36 ℃ and N-NTD W132H with denaturation temperature of 37 ℃; combining N-NTD W132H with denaturation temperature of 37 ℃ and N-CTD W301F with denaturation temperature of 34-40 ℃; combining Nsp 7W 24E Q F with denaturation temperature of 38 ℃ and N-CTD W301F with denaturation temperature of 34-40 ℃; the combination of Nsp 7W 24E Q W with denaturation temperature of 38℃and N-CTD W301F with denaturation temperature of 34-40 ℃.
Wherein, the N-NTD is the N-terminal domain of the novel coronavirus structural protein N-protein disclosed by the website https:// ww1.Rcsb. Org/structure/7CDZ, the N-CTD is the C-terminal domain of the novel coronavirus structural protein N-protein disclosed by the website https:// ww1.Rcsb. Org/structure/7C22, and the NSp7 is the nonstructural protein NSp7 of the novel coronavirus disclosed by the website https:// ww1.Rcsb. Org/structure/7 DCD; the mutant W132T was a mutation of tryptophan at position 132 to threonine, W108L was a mutation of tryptophan at position 108 to leucine, W330N L W was a mutation of tryptophan at position 330 to asparagine and leucine at position 331 to tryptophan, W132H was a mutation of tryptophan at position 132 to histidine, W108H was a mutation of tryptophan at position 108 to histidine, W24E Q F was a mutation of tryptophan at position 24 to glutamic acid and glutamine at position 31 to phenylalanine, W24E Q W was a mutation of tryptophan at position 24 to glutamic acid and glutamine at position 31 to tryptophan, W301F was a mutation of tryptophan at position 301 to phenylalanine, W24Q was a mutation of tryptophan at position 24 to glutamine, W24T was a mutation of tryptophan at position 24 to threonine, and W24E was a mutation of tryptophan at position 24 to glutamic acid.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5731168A (en) * | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
| CN103266105A (en) * | 2013-05-07 | 2013-08-28 | 暨南大学 | Method for improving protein soluble expression through rational translation pause sequence redesigning |
| CN108118043A (en) * | 2016-11-26 | 2018-06-05 | 华中科技大学 | The lipase mutant that a kind of thermal stability improves |
| CN113215123A (en) * | 2020-01-21 | 2021-08-06 | 华东理工大学 | S-type omega-transaminase high-activity mutant |
| WO2022080413A1 (en) * | 2020-10-14 | 2022-04-21 | 一般財団法人阪大微生物病研究会 | Beta coronavirus cold acclimatized strain and vaccine |
| CN114480462A (en) * | 2020-10-26 | 2022-05-13 | 南京吉芮康生物科技研究院有限公司 | Novel coronavirus vaccine antigen presentation system for secretion expression of NTD structural domain protein by attenuated salmonella and application thereof |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5278940B2 (en) * | 2007-11-12 | 2013-09-04 | 独立行政法人産業技術総合研究所 | Stable antibody binding protein |
| PT2635607T (en) * | 2010-11-05 | 2019-12-11 | Zymeworks Inc | Stable heterodimeric antibody design with mutations in the fc domain |
| CN102212511A (en) * | 2011-03-31 | 2011-10-12 | 山东大学 | Site directed mutagenesis thermoplasma acidophilum F3 factor recombinant protein and application thereof |
| EP2891664A1 (en) * | 2014-01-07 | 2015-07-08 | Technische Universität München | Novel methods for the stabilisation of immunoglobulin constant domains |
| WO2017017673A2 (en) * | 2015-07-28 | 2017-02-02 | Yeda Research And Development Co. Ltd. | Stable proteins and methods for designing same |
| CN106543274B (en) * | 2016-10-18 | 2020-10-27 | 天津大学 | Derivative of protein A structural domain Z with specific binding effect on antibody and application thereof |
| JP7061078B2 (en) * | 2016-12-14 | 2022-04-27 | タカラバイオ株式会社 | Thermostable reverse transcriptase mutant |
| CN113005100B (en) * | 2020-09-27 | 2022-02-25 | 湖北省农业科学院畜牧兽医研究所 | Genetic modification method for changing heat stability of enveloped virus, recombinant virus and application thereof |
| CN113293145B (en) * | 2021-02-01 | 2022-08-26 | 上海青赛生物科技有限公司 | New measles virus live vector corona vaccine |
-
2022
- 2022-08-19 CN CN202210997990.5A patent/CN115353552B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5731168A (en) * | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
| CN103266105A (en) * | 2013-05-07 | 2013-08-28 | 暨南大学 | Method for improving protein soluble expression through rational translation pause sequence redesigning |
| CN108118043A (en) * | 2016-11-26 | 2018-06-05 | 华中科技大学 | The lipase mutant that a kind of thermal stability improves |
| CN113215123A (en) * | 2020-01-21 | 2021-08-06 | 华东理工大学 | S-type omega-transaminase high-activity mutant |
| WO2022080413A1 (en) * | 2020-10-14 | 2022-04-21 | 一般財団法人阪大微生物病研究会 | Beta coronavirus cold acclimatized strain and vaccine |
| CN114480462A (en) * | 2020-10-26 | 2022-05-13 | 南京吉芮康生物科技研究院有限公司 | Novel coronavirus vaccine antigen presentation system for secretion expression of NTD structural domain protein by attenuated salmonella and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Crystal structure of the crenarchaeal ExoIII AP endonuclease SisExoIII reveals a conserved disulfide bond endowing the protein with thermostability;Zhou Yan等;《Biochemical and Biophysical Research Communications》;第490卷(第03期);第774-779页 * |
| 巴沙鱼皮胶原蛋白的提取、组成及变性温度研究;沈校等;《中国海洋药物》;第36卷(第04期);第41-46页 * |
| 新型冠状病毒核酸检测过程中灵敏度损失的定量分析;董宏杰等;《山东大学学报(医学版)》;第59卷(第01期);第1-7页 * |
| 点突变对枯草芽孢杆菌甘露聚糖酶结构和功能的影响;周海燕、吴永尧;《高等函授学报(自然科学版)》;第22卷(第01期);第9-12页 * |
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