阅读说明：本技术 人α干扰素亚型及受体结合相关位点突变体在制备防治新型冠状病毒感染药物中的用途 (Application of human alpha interferon subtype and receptor binding related site mutant in preparation of novel coronavirus infection prevention and treatment medicines ) 是由 袁正宏 陈捷亮 于 2020-07-01 设计创作，主要内容包括：本发明属医药和生物工程学技术领域,涉及防治新型冠状病毒(SARS-CoV-2)感染药物,具体涉及人类α干扰素及受体结合相关位点突变体在制备防治新型冠状病毒(SARS-CoV-2)感染药物和制剂中的用途。本发明通过对IFN-α2结合IFNAR1的位点第82、86、89和120位进行突变,经体外新冠病毒感染模型试验鉴定显示人α干扰素受体结合相关位点突变体IFN-α2-EIFK较IFN-α2具有更强的抗病毒活性,且在抗病毒浓度下无细胞毒效应。本发明所述的IFN-α14和IFN-α2受体结合相关位点突变体--IFN-α2-EIFK可进一步用于制备抗新型冠状病毒药物和制剂。(The present invention belongs to the field of medicine and biological engineering technology, and relates to medicine for preventing and treating SARS-CoV-2 infection, and is especially the use of human alpha interferon and receptor binding site mutant in preparing medicine and preparation for preventing and treating SARS-CoV-2 infection. The invention shows that the human alpha interferon receptor binding related site mutant IFN-alpha 2-EIFK has stronger antiviral activity than IFN-alpha 2 and has no cytotoxic effect under the antiviral concentration through carrying out mutation on the 82 th, 86 th, 89 th and 120 th sites of IFN-alpha 2 binding IFNAR1 through in vitro new coronavirus infection model test identification. The IFN-alpha 14 and IFN-alpha 2 receptor binding associated site mutant-IFN-alpha 2-EIFK can be further used for preparing anti-novel coronavirus medicaments and preparations.)

Use of IFN- α 14 for the preparation of a pharmaceutical preparation against a novel coronavirus infection.

2. The use of claim 1, wherein said IFN- α 14 subtype exhibits a half maximal inhibitory concentration against neocoronavirus 100-fold lower than IFN- α 2, and exhibits potent anti-neocoronavirus effects.

3. A mutant of human interferon alpha and interferon receptor IFNAR1 binding related site, which is obtained by the following method: the IFN-alpha 2 receptor binding related site mutant IFN-alpha 2-EIFK is obtained by mutating aspartic acid at position 82 of human IFN-alpha 2 into glutamic acid (E), threonine at position 86 into isoleucine (I), tyrosine at position 89 into phenylalanine (F) and arginine at position 120 into lysine (K);

the amino acid sequence of the IFN-alpha 2 recombinant protein is obtained from a human genome, and the sequence is SEQ ID NO. 1;

the sequence of the IFN-alpha 2-EIFK is SEQ ID NO. 2.

4. The use of the mutant of human interferon alpha and interferon receptor IFNAR1 binding-associated site in claim 3 for preparing a pharmaceutical preparation for resisting novel coronavirus infection, wherein the half effective inhibitory concentration of the IFN-alpha 2-EIFK mutant modified based on the mutations at positions 82, 86, 89 and 120 of the human interferon alpha and IFNAR1 binding-associated site is 100 times lower than that of IFN-alpha 2a/b, and is similar to the half effective inhibitory working concentration of IFN-alpha 14 against novel coronavirus.

5. Use according to claim 1, 2 or 4, wherein said new coronavirus is the new coronavirus SARS-CoV-2.

Technical Field

The invention belongs to the technical field of medicine and biological engineering, relates to a medicinal preparation for preventing and treating novel coronavirus infection, and particularly relates to application of human alpha interferon and receptor binding related site mutants in preparation of a medicament and a preparation for preventing and treating novel coronavirus (SARS-CoV-2) infection.

Background

According to epidemiological investigation, the novel coronavirus (SARS-CoV-2) is mainly transmitted by short-distance air droplets and close contact, infected persons mainly show symptoms of nature, strength and dry cough, severe persons about two-fold, dyspnea and/or hypoxemia can occur, severe persons can rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis and coagulation dysfunction, and the fatality rate is about 3%. The population is generally susceptible to SARS-CoV-2 and has strong infectivity. How to carry out emergency protection intervention on important people, particularly medical care personnel, patients who are closely contacted with confirmed patients and asymptomatic or slightly infected patients becomes one of key links for epidemic prevention and control. In addition, at present, a specific treatment method for diseases caused by novel coronavirus infection is not available, symptomatic support treatment is mainly carried out according to clinical conditions of patients, and IFN-alpha atomization inhalation can be adopted as a treatment intervention means in the scheme for treating pneumonia rash caused by novel coronavirus infection in the State Wei Jian Commission, but the real antiviral efficacy of interferon on the novel coronavirus is not clear.

The prior art discloses that Interferons (IFNs) are a class of cytokines encoded by the own genome, are named for their effect of interfering with viral infection replication, and play a central role in the anti-viral immune response of humans. Interferons are known to bind to surface receptors of cells in the body, thereby initiating downstream signaling pathways that induce the body to produce a series of antiviral protein molecules. These molecules can cut virus nucleic acid, inhibit virus protein synthesis, inhibit virus assembly, inhibit virus replication, and have the effects of early limiting virus replication and dissemination in infected cells and protecting uninfected cells against virus invasion. In addition, interferon also has strong immunoregulation function, and can activate immune cells such as natural killer cells and macrophages, and enhance the immune defense function of a host. Based on the different binding receptors, IFNs can be classified into different types, type I (IFN-I), type II (IFN-II) and type III (IFN-lambda), which differ in their function: the type I interferon comprises IFN-alpha, -beta, omega and the like, is mainly produced by infected cells and some professional cells, has limited manufacturing application on replication and dissemination in the early process of virus infection, and is an important component of host antiviral natural immune response; type II interferons are only one of IFN- γ, are synthesized and secreted mainly by Natural Killer (NK) cells, NKT cells, T cells, and the like, and are involved in initiating host antiviral adaptive immune responses. IFN-alpha is the biggest group in an interferon system and also comprises a plurality of subtypes, 13 human IFN-alpha subtypes are identified in succession, including alpha 1, alpha 2a/2b, alpha 4, alpha 1, alpha 5, alpha 6, alpha 7, alpha 8, alpha 10, alpha 13, alpha 14, alpha 16, alpha 17, alpha 21 and the like, encoding genes of the human IFN-alpha subtypes are located on the number 9 chromosome of a human, and the interferon-alpha subtypes have more similar structural domains, but about 30 percent of sequences are not conserved. Only interferon alpha 2a/2b is approved for clinical treatment of diseases caused by virus infection such as chronic hepatitis B virus infection since the last eighties, and can obtain a cure rate which cannot be achieved by common antiviral drug treatment.

Interferons are known to exert antiviral effects by inducing transcriptional expression of interferon-stimulated genes (ISGs) by initiating transduction of the downstream JAK-STAT signaling pathway through interferon receptors that specifically bind to cell surfaces. It has been shown that although different subtypes of IFN- α act by binding to the two subunits of the type I interferon receptor, IFNAR1 and IFNAR2, there are differences in the manner and extent to which each subtype of IFN- α activates the downstream classical or alternative signaling pathway due to the different binding affinities to the two receptor subunits. Research shows that IFN-alpha usually has high binding force to IFNAR2 and low binding force to IFNAR1 through affinity tests on interferon and an interferon receptor, wherein amino acid sites related to the binding of IFNAR1 and IFNAR2 in an IFN-alpha 2 amino acid sequence are basically resolved, and a basis is provided for modifying and optimizing the receptor affinity and biological effect of related interferon subtypes. In vitro studies on middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) have found that IFN-alpha and IFN-beta administered by nasal drip has inhibitory effect on SARS and MERS coronavirus infection. The research supports the potential application of interferon in the prevention and treatment of new coronavirus infection, but it is not clear which interferon subtype has the most efficient effect of resisting the new coronavirus and whether the effect of resisting the new coronavirus can be improved by mutating and modifying the type of the existing interferon.

Based on the foundation and the current situation of the prior art, the inventor of the application intends to provide a pharmaceutical preparation for preventing and treating novel coronavirus infection, in particular to the application of human alpha interferon and receptor binding-related site mutants in preparing medicaments and preparations for preventing and treating novel coronavirus (SARS-CoV-2) infection.

Disclosure of Invention

The invention aims to provide a pharmaceutical preparation for preventing and treating novel coronavirus infection based on the basis and the current situation of the prior art, and particularly relates to application of human alpha interferon and receptor binding related site mutants in preparation of a medicament and a preparation for preventing and treating novel coronavirus (SARS-CoV-2) infection.

The invention provides the use of IFN-alpha 2 interferon receptor binding related site mutants, the interferon is confirmed to have the effect of resisting new coronavirus by comparing the difference of the anti-new coronavirus effect of different types of human interferons, and the human interferon subtypes with stronger anti-new coronavirus effect than the currently clinically used IFN-alpha 2a and IFN-alpha 2b are screened and identified; the anti-new coronavirus effect of the IFN-alpha 2 and interferon receptor 1 subunit interaction site is improved by mutating amino acid of the interaction site. The invention lays a foundation for developing a novel means for preventing and treating the new coronavirus based on the novel IFN subtype and the IFN-alpha 2 modified mutant.

The experimental research shows that clinically approved interferon subtypes IFN-alpha 2a and IFN-alpha 2b have the function of resisting new coronary virus infection; different IFN subtypes have different effects on resisting the new coronavirus, wherein the effect of IFN-alpha 14 on resisting the new coronavirus infection is most obvious under the same action concentration; in addition, IFN-alpha 24 and IFNAR1 binding related amino acid sites to coding IFN-alpha 14 corresponding site amino acids, show that it has similar IFN-alpha 14 anti new crown virus effect.

The invention adopts a new coronavirus infection model based on Vero-E6 cells to screen and compare the anti-new coronavirus effect of a plurality of human IFN types of recombinant expression, and researches and incorporates representative human interferon subtypes comprising clinically approved IFN-alpha 2a and IFN-alpha 2b, IFN-alpha 1, IFN-alpha 14, IFN-beta, IFN-gamma, IFN-omega and IFN-lambda 1(IL-29), and results show that the anti-new coronavirus effect of the clinically approved IFN-alpha 2a and IFN-alpha 2b is relatively close, a plurality of other IFN subtypes have stronger anti-new coronavirus effect, the specific anti-new coronavirus activity is ranked as IFN-alpha 14> IFN-omega, IFN-gamma > IFN-alpha 1, alpha 2a, alpha 2b, IFN-beta > IFN-lambda 1(IL-29), the work concentration of IFN-alpha 14 against the new coronavirus is calculated to be about 100 times lower than that of the interferon used in the clinical application at present and about 5 times lower than that of IFN-omega; the results show that the lower the working concentration of the representative human interferon subtypes, the higher the antiviral efficacy, and the lower the potential adverse reactions and side effects are expected to be.

In the invention, the amino acid sites of IFN-alpha and IFNAR1 are analyzed by comparing the amino acid sequences of IFN-alpha 2 and IFN-alpha 14, 4 amino acid sites are found to have difference between the two, the 4 amino acid sites on the IFN-alpha 2 are mutated into the corresponding amino acid of IFN-alpha 14 (IFN-alpha 2-EIFK), and then the IFN-alpha 2 mutant is evaluated for antiviral function, and the result shows that the mutant has the anti-neocoronavirus effect similar to IFN-alpha 14. More specifically, on the basis of finding that 4 amino acid sites (82 th, 86 th, 89 th and 120 th) related to IFNAR1 binding are different in IFN-alpha of two subtypes, the invention mutates aspartic acid at the 82 th position of human IFN-alpha 2 into glutamic acid, mutates threonine at the 86 th position into isoleucine, mutates tyrosine at the 89 th position into phenylalanine, and mutates arginine at the 120 th position into lysine, so as to obtain an interferon mutant IFN-alpha 2-EIFK with improved affinity to IFNAR 1; then, in a new coronavirus infection model based on Vero-E6 cells, the anti-new coronavirus effects of human IFN-alpha and corresponding mutants are compared, the plaque formation number is detected, and the half effective inhibitory concentration is calculated, so that the result shows that the interferon mutant IFN-alpha 2-EIFK has a strong anti-new coronavirus effect similar to IFN-alpha 14, the effective working concentration is about 100 times lower than that of IFN-alpha 2, and no cytotoxicity exists under the working concentration.

In the invention, the amino acid sequence of the related IFN-alpha 2 recombinant protein is obtained from a human genome and has a sequence of SEQ ID NO. 1;

in the invention, the IFN-alpha 14 sequence which is subjected to amino acid sequence comparison with the IFN-alpha 2 is SEQ ID NO. 3;

in the present invention, the sequence of IFN-. alpha.2EIFK in which 4 IFNAR1 receptor binding-related amino acid sites of IFN-. alpha.2 were mutated is SEQ ID NO. 2.

The test result of the invention proves the application of the novel interferon subtype and the receptor binding related site mutant in resisting the new coronavirus infection, and particularly provides theoretical and technical basis for developing the specific interferon subtype and the modified mutant for preparing the medicine and the preparation for preventing and treating the new coronavirus.

The invention provides a new coronavirus infection resisting interferon, wherein a specific subtype IFN-alpha has a new coronavirus resisting effect superior to that of a currently clinically applied interferon subtype, and a new coronavirus infection resisting medicine and a preparation can be prepared by modifying a human interferon alpha receptor binding related site.

For the sake of understanding, the specific interferon subtypes and receptor binding site related mutants of the present invention are described in detail below with reference to the accompanying tables of the drawings as having superior activity against the novel coronavirus as compared to IFN-. alpha.2. It is noted that the drawings are for illustrative purposes only and that modifications, both individually and in the flow chart and the like, which are within the scope of the present invention, will be apparent to those of ordinary skill in the art from the description herein and are intended to be within the scope of the present invention.

Drawings

FIG. 1. Effect of each interferon subtype and mutant on the formation of Vero-E6 cell plaque caused by SARS-CoV-2 infection.

FIG. 2 Effect of individual interferon subtypes and mutants on cell viability in Vero-E6 cells.

FIG. 3 shows the purification of interferon by prokaryotic expression system and the evaluation of its purity;

wherein, A, human IFN-alpha 2, IFN-alpha 14 and IFN-alpha 2-EIFK mutant sequence alignment schematic diagram; b Coomassie Brilliant blue results of interferon purification with human IFN-. alpha.2 and IFN-. alpha.2-EIFK.

Detailed Description

Example 1 in vitro infection model of novel coronavirus and interferon treatment

(1) In the experiment, a novel coronavirus is separated from throat swabs of 1 Shanghai infected person by using a coronavirus-susceptible Vero E6 cell, and is named as nCoV-SH 01; sequencing the whole genome of the strain by adopting a first-generation Sanger and second-generation Illumina method, and finding that the homology of the strain and GenBank MN908947 is more than 99.99 percent; when nCoV-SH01 infects Vero E6 cells, typical syncytium lesions are caused, the cytopathic effect is obvious and rapid, and the nCoV-SH01 can be used for further establishing a SARS-CoV-2 cell infection model for evaluating the efficacy of antiviral drugs;

(2) spreading Vero-E6 cells to a 96-well plate at a conventional density, and culturing overnight;

(3) treating the above Vero-E6 cells with different concentration gradients of various interferon subtypes and IFN-alpha 2 mutants (IFN-alpha 2-EIFK), each group of duplicate wells, incubating for 16 hours;

(4) the treated cells were taken into a P3 laboratory, the medium was removed, nCoV-SH 01P 6 virus was inoculated at 100 PFU/100. mu.L/well (diluted with 2% FBS DMEM medium), and incubated at 37 ℃ for 1 hour;

(5) adding 4% FBS DMEM containing 2% methyl cellulose and 100uL of the methyl cellulose to each well, and culturing for 72h at 37 ℃;

(6) directly adding 100 mu L of 2% PFA for fixation for 6 hours, removing the culture medium and the like, adding 1% crystal violet, dyeing for 10 minutes, and washing for 5 times;

(7) taking a picture under a microscope (as shown in figure 1), the result shows that various interferons and mutants have anti-new coronavirus effects in different degrees, namely the number of plaques is reduced, but effective working concentrations of different interferon subtypes and mutants for completely inhibiting the plaque formation are obviously different, and the cell morphology is good under the treatment of various concentrations and various types of interferons, which indicates that no obvious cytotoxicity exists;

(8) counting the number of plaques (shown in table 1) and calculating the half effective inhibitory concentration (shown in table 2) of each interferon subtype, wherein the anti-new coronavirus efficacy is ranked as IFN-alpha 14> IFN-omega, IFN- > IFN-alpha 1, alpha 2a, alpha 2b, IFN-beta > IFN-lambda 1(IL-29), and the half effective inhibitory concentration of the IFN-alpha 14 against the new coronavirus is calculated to be about 100 times lower than that of the interferon clinically used at present and about 5 times lower than that of the IFN-omega;

(9) Vero-E6 cells treated by interferon for 16h are taken, culture medium is replaced and then cultured for 48h, and a CCK8 detection reagent kit is used for further cell viability evaluation, and the result shows that various interferon subtypes and mutants have no obvious cytotoxicity to Vero-E6 cells under the concentration of 25 ng/ml.

TABLE 1 number of plaque formation for SARS-CoV-2 in each interferon subtype and mutant treatment group

TABLE 2 half-effective concentration of each interferon subtype and mutant for inhibiting SARS-CoV-2

。

Example 2 preparation and purification of human IFN-. alpha.2 and IFN-. alpha.2-EIFK

Cloning a human IFN-alpha 2 gene coding sequence (SEQ ID No.1), an IFN-alpha 2-EIFK (SEQ ID No.2) interferon mutant sequence (shown in a figure 3A) and an IFN-alpha 14 sequence (SEQ ID No.3) on a prokaryotic expression vector after codon optimization, and then carrying out prokaryotic expression of recombinant protein;

(1) transforming the recombinant plasmid into E.coli BL-21, coating the recombinant plasmid on a solid LB culture medium containing interferon, selecting a single colony to 3-4ml LB for shaking after 16h at 37 ℃, inoculating the single colony into a large shake at a ratio of 1:100 after night, adding a bacterial solution into a 50ml centrifuge tube, centrifuging for 10min at 5000g and 4 ℃ after the protein induction expression is finished when the OD600 reading is between 0.5 and 0.6 and the IPTG final concentration is 10 mu M for induction when the temperature is 16 ℃ and the expression is 20 h, and discarding the supernatant;

(2) the centrifuged cells were resuspended in buffer A (20mM phosphate buffer, 0.5M sodium chloride, 20mM imidazole) at a volume 1/50-1/100 of the volume of the cells. Resuspending the pellet, after fully blowing it off, transferring it to a 2ml centrifuge tube, sonicating on ice: crushing for 10s in a high gear of an ultrasonic crusher, cooling for 10s, and circulating for 6 times; this step was repeated 3 times. 10000g, centrifuging at 4 ℃ for 25min, and collecting supernatant. Diluting the collected supernatant of the thallus lysate to 1/20 of the shake bacteria volume by using a buffer solution A, filtering the diluted sample, firstly passing through a filter membrane of 0.45 mu m, then passing through a filter membrane of 0.22 mu m, and placing the sample at 4 ℃ for later use;

(3) affinity purification was performed using GE AKTA avant machine in combination with GE Histrap HP (cat # 17524701), followed by ion exchange using GE AKRA avant machine in combination with GE Hitrap Q HP (cat # 17115401);

(4) the collected sample is stained with Coomassie brilliant blue, and the purity of the interferon is identified (as shown in FIG. 3B);

(5) concentrating the purified interferon by using an ultrafiltration tube, and replacing the buffer solution of the interferon with PBS;

(6) in order to eliminate the influence of endotoxin on the experimental result, the endotoxin in the interferon is removed by using Triton X-114, the interferon and 10 percent Triton X-114 are mixed according to the proportion of 9:1, the mixture is magnetically stirred for 60min at 4 ℃, fully and uniformly mixed, a metal bath at 30 ℃, the vibration at 1000rpm is carried out, and the vibration time is 20 min; the mixture is taken out, inverted and mixed evenly, and then the mixture is put into a metal bath at the temperature of 30 ℃ and shaken at 1000rpm for 20 min. Centrifuging at 25 deg.C and 14000g for 15min, removing the upper aqueous phase into tube, and repeating the above steps again;

(7) transferring the interferon into a pretreated dialysis bag, wherein a buffer solution on the outer side of the dialysis bag is precooled PBS, and dialyzing overnight;

(8) the purified interferon is diluted by different times, and BCA quantification is carried out. After all the interferon is finished, subpackaging the interferon and storing the interferon in a refrigerator at the temperature of minus 80 ℃;

(9) the differences in anti-neocoronaviruses of three interferons, human IFN-. alpha.2a/b, IFN-. alpha.2EIFK and IFN-. alpha.14, were evaluated in the same manner as in example 1, and the results showed that the interferon receptor binding-associated site mutant IFN-. alpha.2EIFK had a potent anti-neocoronaviruses effect similar to IFN-. alpha.14, with a half inhibitory concentration about 100-fold lower than that of IFN-. alpha.2 and no cytotoxicity at a working concentration of 25 ng/ml.

The experimental results show that the invention prepares the effect of mutant interferon on new coronavirus by comparing various wild-type interferon subtypes and cloning and purifying, shows that IFN-alpha 14 and IFN-alpha 2-EIFK (IFN-alpha 2 receptor binding site mutant) have high-efficiency new coronavirus resistance, and compared with IFN-alpha 2, the effective working concentration is about 100 times lower, and no obvious cytotoxicity exists; the novel interferon subtype and the interferon mutant after transforming IFN-alpha interferon receptor binding related sites provide technical support for developing novel interferon for resisting new coronavirus, and especially have good application potential and prospect in preparation of antiviral drugs in view of the fact that the novel interferon subtype and the interferon mutant related to the receptor binding sites have already mature bioengineering and clinical use experience of IFN-alpha 2 development and application.

Sequence listing

<110> university of Compound Dan

Application of <120> human alpha interferon subtype and receptor binding related site mutant in preparation of novel coronavirus infection prevention and treatment medicines

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