Suppress polypeptide and its application of human coronary virus's infection wide spectrum
Technical field
The invention belongs to biomedicine field, it is related to the polypeptide for suppressing human coronary virus's infection, and in particular to can wide spectrum
Ground suppresses polypeptide and its application of human coronary virus's infection.
Technical background
Data is shown, finds a kind of new pathogen first in GuangZhou, China within 2002, it causes the serious acute of people to exhale
Inhale trace integration and levy (Severe Acute Respiratory Syndrome, SARS), be otherwise known as severe acute respiratory syndrome,
The pathogen is referred to as severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome
coronavirus,SARS-CoV).By in July, 2003, the illness has involved 29, whole world countries and regions, accumulative to make a definite diagnosis
Case load is more than 8000 people, and average mortality is more than 10%.In 2012, another novel coronavirus was in the quick-fried of Middle East
Hair, i.e. Middle East respiratory and enteric coronavirus (Middle East respiratory Syndrome coronavirus, MERS-
CoV), according to the World Health Organization (WHO), by January 20th, 2016, the case made a definite diagnosis was up to 1626, and death
Rate is up to 36%.At present, can infect the coronavirus of the mankind also includes human corona virus 229E (HCoV-229E), the coronal disease of people
Malicious NL63 (HCoV-NL63), human corona virus OC43 (HCoV-OC43), the type human corona virus (HCoV-HKU1) of Hong Kong I.The mankind
The existence for existing for the mankind of coronavirus brings serious threat with health, and the economic development to the whole world brings serious
Influence.In addition, it has now been found that some class SARS viruses, including RsSHC014-CoV or RsW1V1-CoV, the base with SARS-CoV
Because sequence is highly similar.Relevant research shows, RsSHC014-CoV equally has the potentiality of the infection mankind, and SARS-CoV
Specificity does not have inhibitory action (bibliography to it for antibody drug:Menachery,V.D.,B.L.Yount,Jr.,
K.Debbink,S.Agnihothram,L.E.Gralinski,J.A.Plante,R.L.Graham,T.Scobey,X.Y.Ge,
E.F.Donaldson,S.H.Randell,A.Lanzavecchia,W.A.Marasco,Z.L.Shi,and
R.S.Baric.2015.A SARS-like cluster of circulating bat coronaviruses shows
potential for human emergence.Nat Med 21:1508-1513.);Thus, the presence of this viroid, equally
Carry out serious hidden danger for the existence and health care belt of the mankind.
Research shows that human corona virus's virus is during target cell is infected, and its envelope glycoprotein (S protein) is played
Key effect;S protein can be divided into S1 subunits and S2 subunits, S1 subunits be responsible for and its target cell surface receptors combination,
Then cause after a series of molecular events, the fusogenic peptide of S2 albumen n ends is exposed, can insert in target cell membrane;Afterwards
A series of conformational changes occur for S2 albumen, its N- ends heptapeptide duplicate domain (HR1 or NHR) formation tripolymer, and with its C- ends heptapeptide
Duplicate domain (HR2 or CHR) interacts, and is formed " fusion core texture " --- " Six helix bundle " (6HB) so that viromembrane and target
Cell membrane is furthered so as to occur film fusion, and viral genetic stew is by merging hole into the new disease of duplication generation in target cell
Malicious particle.
At present, about in the research and development of the inhibitor of the viroid, the S1 regions and S2 regions of S protein are two important targets
Point, wherein, the entry inhibitor in S1 regions is directed to based on antibody, although during this antibody-like has well to specific virus
And activity, but do not have cross-protection to other human corona viruses;There are some researches show, although SARS-CoV is similar with its
Viral RsSHC014-CoV genetic homology is higher, and acceptor is identical, but SARS-CoV specific antibody still lacks
To RsSHC014-CoV neutralization activity (bibliography:Menachery,V.D.,B.L.Yount,Jr.,K.Debbink,
S.Agnihothram,L.E.Gralinski,J.A.Plante,R.L.Graham,T.Scobey,X.Y.Ge,
E.F.Donaldson,S.H.Randell,A.Lanzavecchia,W.A.Marasco,Z.L.Shi,and
R.S.Baric.2015.A SARS-like cluster of circulating bat coronaviruses shows
potential for human emergence.Nat Med 21:1508-1513.);Target the entry inhibitor in S2 regions
Based on polypeptide drug, this kind of entry inhibitor multi-source is in viral S2 eggs heptad repeat 2 (HR2) amino acid sequence;
HR2 polypeptides can be combined with heptad repeat 1 (HR1) tripolymer of virus, formed stable heterologous 6HB, prevented virus
HR2 combined with the HR1 tripolymers of virus, prevent them from forming homologous 6HB, so as to inhibit viromembrane and target cell membrane
Fusion, the gene that also may refrain from virus is replicated (as shown in Figure 1) into target cell membrane;At present, with phase same-action machine
The polypeptide entry inhibitor of system has been played an important role in terms of AIDS virus (HIV) treatment, for example, T-20
(Enfuvirtide, Fuzeon, also known as T20) is first polypeptide HIV entry inhibitor in the world of U.S. FDA approval, its
It is currently used for the clinical treatment of AIDS, having produced the drug-fast patient of hiv reverse transcriptase inhibitor to those has well
Curative effect;One of present inventor-ginger generation is suddenly that (U.S. is special by the inventors of T20 medicine prototype polypeptides (polypeptide 637-666)
Profit number:5,444,044).
The early-stage Study of the application is found, in the S2 regions of human coronary virus, the amino acid of heptapeptide duplicate domain (HR)
The similarity of sequence is higher, and it is very similar by the mechanism for forming six spiral mediated fusions, and this is the people of research and development wide spectrum
Class coronavirus polypeptide class inhibitor provides important theoretical foundation;But the design of current polypeptide entry inhibitor
In, it is based only on the virus HR2 of itself amino acid sequence or six helical structures and designs, still lacks the design reason of broad spectrum activity
Read, thus the human corona virus's peptide inhibitor reported at present often lacks the antiviral effect (bibliography of wide spectrum:
Lu,L.,Q.Liu,Y.Zhu,K.H.Chan,L.Qin,Y.Li,Q.Wang,J.F.Chan,L.Du,F.Yu,C.Ma,S.Ye,
K.Y.Yuen,R.Zhang,and S.Jiang.2014.Structure-based discovery of Middle East
respiratory syndrome coronavirus fusion inhibitor.Nat Commun 5:3067. and Gao, J.,
G.Lu,J.Qi,Y.Li,Y.Wu,Y.Deng,H.Geng,H.Li,Q.Wang,H.Xiao,W.Tan,J.Yan,and
G.F.Gao.2013.Structure ofthe fusion core and inhibition of fusion by a heptad
repeat peptide derived from the S protein of Middle East respiratory syndrome
coronavirus.J Virol87:13134-13140.)。
Present situation based on prior art, present inventor intended by showing that the HR regions based on virus are more guarded
As according to the design feature of a variety of human corona virus HR2 sequence characteristic and six spirals, and using to a variety of biochemistry means
The activity of polypeptide is predicted and assesses that there is provided can be to inhibitory action of the infection with wide spectrum of two or more human coronary virus
Polypeptide, the application will be to be significant in terms of human corona virus's prevention and control and treatment, can be at present still in popular people
Class coronavirus and the following novel human coronavirus for being possible to outburst provide the drug candidate effectively prevented and treated.
The content of the invention
The purpose of the present invention is that the defect for the presence for overcoming prior art is felt wide spectrum there is provided that can suppress human coronary virus
The polypeptide of dye.
HR region of the application based on virus more conservative phenomenon, according to a variety of human corona virus HR2 sequence characteristic
There is provided can infect human coronary virus the polypeptide with wide spectrum suppression function into suppression with the design feature of six spirals
Agent.Described wide spectrum polypeptide entry inhibitor can be (including currently popular with human coronary virus of more than two kinds
MERS-CoV) the HR1 regions in S2 albumen are combined, the forming process of the spiral of viral interference itself six, so as to suppress melting for virus
Close course of infection.
Its sequence of polypeptide of the present invention is (or referring in peptide sequence table) as shown in table 1, or by amino therein
Acid sequence is by modification for example by substitution, the amino acid sequence for lacking or adding one or more amino acid composition, these polypeptides
Common characteristic for by suppressing I type transmembrane glycoprotein -- the fusion process that S protein is mediated by human coronary virus surface
Play the inhibitory activity to virus infection.
Table 1,
Polypeptide of the present invention its there is " broad spectrum activity " of polypeptide antiviral activity;Practice display, for the coronal disease of people
The prevention and control of poison can not be only for single viral species, and to find its " general character " and be prevented and treated, and so just can preferably prevent
Control the outburst of currently a popular and following new human coronary's disease;Using a variety of biochemistry means to described in the present invention
The activity of polypeptide be predicted assessment, it was demonstrated that described polypeptide can have wide spectrum to the infection of two or more human coronary virus
Inhibitory action;Especially to severe acute respiratory syndrome coronavirus (Severe Acute Respiratory
Syndrome Coronavirus, SARS-CoV), class sars coronavirus (RsSHC014-CoV or RsW1V1-CoV), the Middle East
Respiratory and enteric coronavirus (Middle East Respiratory Syndrome Coronavirus, MERS-CoV) and people are coronal
Inhibitory action of the infection with wide spectrum of two or more human coronary virus in viral 229E (HCoV-229E), the present invention will be to people
Coronavirus prevention and control and treatment face are significant, can be for still have at present in popular human coronary virus and future can
The novel human coronavirus that can be broken out provides the drug candidate effectively prevented and treated.
In the present invention, by setting up the Cell-Cell Fusion system mediated respectively by MERS-CoV and SARS-CoV S proteins
System carries out the detection serial polypeptide to the inhibitory activity (as shown in Figure 2) of corresponding viral fusion process, a reality of the invention
Apply Cell-Cell Fusion system (the lu l. (one of land route, inventor of this patent) etc. in 2014 using MERS-CoV in example
People constructs MERS-CoV Cell-Cell Fusion system by the method that the S protein and GFP of virus are co-expressed, and herein
In system, successfully design and have evaluated a series of activity preferably MERS-CoV polypeptide entry inhibitors, the achievement is delivered
On Nature periodicals Nature Communications), and have detected respectively using identical method described herein
Polypeptide is entered with the best MERS-CoV polypeptides of activity at present and pressed down to MERS-CoV and SARS-CoV fusion inhibition
Preparation HR2P-M2 is used as control (bibliography:Lu,L.,Q.Liu,Y.Zhu,K.H.Chan,L.Qin,Y.Li,Q.Wang,
J.F.Chan,L.Du,F.Yu,C.Ma,S.Ye,K.Y.Yuen,R.Zhang,and S.Jiang.2014.Structure-
based discovery of Middle East respiratory syndrome coronavirus fusion
inhibitor.Nat Commun5:3067.), as a result show, in MERS-CoV Cell-Cell Fusion experiment, HCoV-EK
Serial polypeptide and HR2P-M2 polypeptide shows preferable inhibition, and the serial polypeptide inhibitory activity of HCoV-EK is substantially excellent
In shown in HR2P-M2 polypeptides (as shown in Figure 2 a);Equally, the cell that the serial polypeptides of HCoV-EK are mediated to SARS-CoV S proteins-
Cell, which melts, equally has preferable inhibitory activity, and polypeptide HR2P-M2 does not have obvious inhibition at 5 μM (such as Fig. 2 b institutes
Show).
In the present invention, further experiment verifies broad spectrum activity of the serial polypeptides of the HCoV-EK to the suppression of human corona virus,
The Cell-Cell Fusion system of HCoV-229E and class SARS virus (RsSHC014-CoV or RsW1V1-CoV) is set up respectively, and
The broad spectrum activity of the serial polypeptide is detected by representative of HCoV-EK1 polypeptides;As a result show, HCoV-EK1, can be right at 10 μM
MERS-CoV, SARS-CoV, HCoV-229E, and the mediation of class SARS virus (RsSHC014-CoV or RsW1V1-CoV) S protein
Cell-Cell Fusion fusion process reach 100% inhibition (as shown in Figure 3 a);Specifically show in fig 3b
Inhibitory activity of the HCoV-EK1 polypeptides in above-mentioned a variety of viral Cell-Cell Fusion experiments, its half-inhibition concentration
(IC50) below 1 μM, so as to further show inhibition of the described serial polypeptide to human corona virus's high-efficiency broad spectrum;
In table 2, IC50 of the serial polypeptides of HCoV-EK in different Fusion Models is further systematically presented.
The suppression for the Cell-Cell Fusion that the serial polypeptide of table 2, HCoV-EK is mediated to the S protein of a variety of human corona viruses
Activity:
It is viral for representative with the MERS-CoV at present still in popular state in the present invention, the serial polypeptides of detection HCoV-EK
To the inhibition of its pseudovirus, as a result show, HCoV-EK series polypeptides have to MERS-CoV pseudovirus preferably to be suppressed to imitate
Really, or even the antiviral activity than polypeptide HR2P-M2 has 6-10 times of a raising (as shown in Figure 4), the result and above-mentioned MERS-
CoV Cell-Cell Fusion experimental results are more consistent.
In the present invention, the activity of anti-human coronavirus of high-efficiency broad spectrum is shown in the serial polypeptides of the HCoV-EK simultaneously,
It is preferred that, the specific Antiviral Mechanism of the serial polypeptide by representative of HCoV-EK1 polypeptides has obtained clear and definite displaying;As schemed
In Native Page experiments shown in 5a, HCoV-EK1 polypeptides can be with the polypeptide (bag derived from a variety of human corona virus HR1
Include SARS-CoV-HR1P, MERS-CoV-HR1P, HcoV-229E-HR1P) mixing after, generate a new band, this
As doubtful six helical strips of band are (due to SARS-CoV-HR1P's and RsSHC014-CoV-HR1P or RsW1V1-CoV-HR1P
Amino acid sequence is identical, does not just do repetition again here);In addition, experimental result also shows:HCoV-EK1 polypeptides and polypeptide
After HCoV-NL63-HR1P or polypeptide HCoV-OC43-HR1P mixing, same phenomenon can occur, show that HCoV-EK series is more
Peptide also has the activity for suppressing HCoV-NL63 and HCoV-OC43;And shown in the secondary structure detection of polypeptide, work as HCoV-
After EK1 polypeptides and SARS-CoV-HR1P, MERS-CoV-HR1P or HcoV-229E-HR1P mixing, there is the spiral knot of height
Structure, the result meets the CD spectrum of six helical core structures, and its Tm value represents its degree of stability (as shown in Figure 5 b);Thus may be used
See, the Antiviral Mechanism of HCoV-EK series polypeptides is mainly to be combined by the HR1 regions with virus, viral interference itself 6HB's
Formed, so that the fusion for suppressing virus enters process.
The present invention above-mentioned test result indicates that, the serial polypeptide of described HCoV-EK has very wide spectrum and efficient disease-resistant
Cytotoxic activity, can have good inhibition to human corona virus of more than two kinds.This will provide one for the prevention and control of human coronary virus
New strategy is planted, while also the research and development of the polypeptide small molecule medicine to accelerate the anti-human coronavirus of wide spectrum provide new theory
Foundation;Further, the serial polypeptides of HCoV-EK clearly Antiviral Mechanism, be its application security and Optimized Approaches it is bright
True property, which is provided, to be ensured.
Another object of the present invention is to provide suppresses function with having wide spectrum to human coronary virus's infection infection
Recombinant protein, the nucleus of the albumen is that involved having suppresses a variety of human coronary virus's infection abilities in the application
Peptide sequence.
The invention further relates to one group of nucleic acid molecules, it encodes the peptide of separation of the present invention, including codon optimization is spread out
It is biological.
The invention further relates to one group of expression vector, it includes the nucleic acid molecules of separation of the present invention.
The invention further relates to the recombinant cell of one group of separation, it includes expression vector of the present invention.
Medicine is used as the invention further relates to nucleic acid molecules, expression vector or the recombinant cell of the peptide of described separation, separation
Application.
The present invention relates to one group of pharmaceutical composition, its polypeptide comprising the present invention, nucleic acid molecules, expression vector and/or again
Group cell and the pharmaceutically acceptable carrier or excipient being optionally present.
The invention further relates to polypeptide of the present invention, nucleic acid molecules, expression vector or recombinant cell for preparing
Suppress the application in a variety of human coronary virus's infection medicine compositions.
Suppress or prevent the method for a variety of human coronary viruses infection infection the invention further relates to a kind of, including give by
A variety of human coronary virus's infection infection have the object by a variety of human coronary viruses infection infection risk to apply institute of the present invention
Polypeptide, nucleic acid molecules, expression vector, recombinant cell or the pharmaceutical composition stated.
The present invention is shown through experiment, successfully obtains " general character " of human coronary virus, i.e., similar HR regions are situated between with it
The identical syncretizing mechanism led, and the serial polypeptides of HCoV-EK are provided as point of penetration, it is described many too not only to currently a popular
Some human corona viruses have preferable inhibition, and to being possible to infect the class SARS virus (RsSHC014-CoV of the mankind
Or RsW1V1-CoV) equally there is good inhibitory activity.In addition, beneficial effects of the present invention are also embodied in human corona virus
The research and development field of polypeptide entry inhibitor has great importance, all the time, related research all show MERS-CoV and
There is no the protecting effect intersected between SARS-CoV polypeptide entry inhibitor, it is thus regarded that human corona virus's polypeptide enters
Inhibitor is similarly subjected to the limitation (bibliography of virus-specific:Lu,L.,Q.Liu,Y.Zhu,K.H.Chan,L.Qin,
Y.Li,Q.Wang,J.F.Chan,L.Du,F.Yu,C.Ma,S.Ye,K.Y.Yuen,R.Zhang,and
S.Jiang.2014.Structure-based discovery of Middle East respiratory syndrome
coronavirus fusion inhibitor.Nat Commun5:3067. and Gao, J., G.Lu, J.Qi, Y.Li, Y.Wu,
Y.Deng,H.Geng,H.Li,Q.Wang,H.Xiao,W.Tan,J.Yan,and G.F.Gao.2013.Structure of
the fusion core and inhibition of fusion by a heptad repeat peptide derived
from the S protein of Middle East respiratory syndrome coronavirus.J Virol
87:13134-13140.), however the present invention will break this traditional view first, this has great to human coronary's prevention and control
Meaning.
Brief description of the drawings
Fig. 1, human coronary virus infect the ideograph of target cell, and the serial polypeptides of HCoV-EK suppress human coronary's disease
The mechanism of action that malicious S protein mediate retroviral is merged with target cell.
Fig. 2, HCoV-EK series polypeptide to SARS-CoV, the Cell-Cell Fusion that MERS-CoV S protein is mediated
Inhibitory activity.
Fig. 3, HCoV-EK1 represent the broad spectrum activity of the anti-human coronavirus of polypeptide.
Inhibitory activity of Fig. 4, HCoV-EK series polypeptide to MERS-CoV pseudovirus.
Fig. 5, HCoV-EK1 represent the antiviral mechanism of action of polypeptide.
Embodiment
The present invention is further elucidated by following embodiments, but any embodiment or its combination are not construed as to this hair
The limitation of bright scope or embodiment.It is intended that the scope of the present invention be defined by the claims appended hereto, with reference to this specification and ability
Domain common sense, those of ordinary skill in the art can be clearly understood that claims limited range.Without departing from this
On the premise of the spirit and scope of invention, those skilled in the art can carry out any modification or change to technical scheme
Become, this modifications and changes are also contained in the scope of the present invention.
Involved experiment material:
Polypeptide:
By by the homology alignment of HR1 and HR2 sequences in people's type coronavirus S2 albumen in the application, and based on phase
The crystal structure of pass, the artificial polypeptide that design three is derived from HR2 regions, is closed by Shanghai Jie Tai bio tech ltd
Into and purifying, its purity>95%.
Embodiment 1 suppresses the Cell-Cell Fusion experiment (cell-cell of human coronary virus's S protein mediation
fusion)
(1), experimental method:
1.293T cell transfectings cultivate 36-48h after encoding the plasmid of mankind's coronavirus S protein, as effector cell,
For example, use can encode the plasmid of MERS-CoV S proteins -- the 293T cells of pAAV-IRES-GFP-MERS-S transfections are referred to as
293T/MERS/EGFP cells, 293T cell transfecting empty carrier plasmids -- it is 293T/EGFP cells after pAAV-IRES-GFP,
It is used as negative control cell;
2. above-mentioned cell is digested using 0.02% EDTA, centrifugation uses fresh 10%FBS DMEM culture mediums
Cell is resuspended, and adjusts cell concentration to 2 × 105Individual/mL, takes out 50 μ L and is added in the medicine of gradient dilution (50 μ L),
37 DEG C, it is incubated 30min;
3. taking 100 μ L cells/medicine mixed liquor to be added to, bed board is on the target cell (Huh-7) of 96 orifice plates, and 5%
CO2,37 DEG C are cultivated 2-4 hours, and the fusion situation of cell is observed and recorded with the green fluorescence channel of fluorescence microscope;
As a result show:
1. such as Fig. 2 a, shown in b, it is thin that HCoV-EK series polypeptides are mediated to SARS-CoV and MERS-CoV S protein
Born of the same parents-cell fusion has good inhibition, it is shown that HCoV-EK series polypeptides can suppress the wide of two kinds of human corona viruses simultaneously
Spectrality, wherein HCoV-EK1 polypeptides antiviral activity are best;
2. using HCoV-EK1 polypeptides as representative, further the antiviral broad spectrum activity of the serial polypeptide of detection, as a result shows, thin
On born of the same parents-cell fusion system, HCoV-EK1 polypeptides not only can be inhibited to SARS-CoV and MERS-CoV, and also
The fusion that class sars coronavirus (RsSHC014-CoV or RsW1V1-CoV) and HCoV-229 S protein are mediated can be suppressed
Process (as shown in Figure 3), in table 2, system illustrates the wide of in Cell-Cell Fusion experiment serial polypeptides of HCoV-EK
The antiviral effect of spectrum, IC50 in nanomolar range, comprehensively illustrates the antiviral work of the high-efficiency broad spectrum of serial polypeptide
The feature of property.
The polypeptide of embodiment 2 is determined to the inhibitory activity of human coronary virus's pseudovirus
(1), experimental method:
1. dissolve polypeptide using DMSO and determine peptide concentration;
2. target cell (HuH7) cell suspension of expression human coronary virus's acceptor is prepared, after adjustment cell concentration in every hole
Add 104Individual cell;
2 times of gradient dilution polypeptide drugs of DMEM nutrient solutions containing 10%FBS are used in 3.96 orifice plates, per the μ L of hole 50;
4. the MERS-CoV pseudovirus of certain titre is added into 50 μ L/ in drug dilution plate to add per hole.Room temperature is acted on
30min, makes medicine and virus fully effect.Medicine is added in per hole the target for removing supernatant is added to the virus μ L of mixed liquor 100
In cell, the fresh DMEM medium containing 10%FBS is changed after 37 DEG C of culture 12h.Luciferase is determined after 5.72h, is calculated
Inhibiting rate curve is made, and calculates the half-inhibition concentration (IC50) of medicine;
(2), result is shown,
1. at present still in popular coronavirus MERS-CoV as representative, to be detected again in its pseudovirus system
HCoV-EK series polypeptide its antiviral activity, as a result as shown in figure 4, HCoV-EK series polypeptides equally show it is preferably disease-resistant
Cytotoxic activity, and it is more consistent with the result of Cell-Cell Fusion experiment before;
2. compared with the MERS-CoV polypeptide entry inhibitors HR2P-M2 of recent report, HCoV-EK series polypeptides it is anti-
MERS-CoV activity is significantly improved, and shows that the serial polypeptides of the HCoV-EK in the present invention are obtaining its antiviral " wide spectrum
Property " while, not to sacrifice its antiviral activity as cost.
Embodiment 3, HR2 polypeptide Antiviral mechanism research experiments
Using HCoV-EK1 as representative, the serial polypeptide of detection and the polypeptide derived from different human corona virus HR1 regions
(HR1Ps) interaction between, so as to further explore the Antiviral Mechanism of the serial polypeptides of HCoV-EK;Due to SARS-CoV
It is completely the same with the sequence of class SARS virus (RsSHC014-CoV or RsW1V1-CoV) in HR1 regions, thus here can be same
When explain the serial polypeptide anti-RsSHC014-CoV or RsW1V1-CoV of HCoV-EK mechanism;This part research reference literature is carried out.
(Liu,S.,G.Xiao,Y.Chen,Y.He,J.Niu,C.R.Escalante,H.Xiong,J.Farmar,A.K.Debnath,
P.Tien,and S.Jiang.2004.Interaction between heptad repeat 1 and 2 regions in
spike protein of SARS-associated coronavirus:implications for virus fusogenic
mechanism and identification of fusion inhibitors.Lancet 363:938-947.)
(1), experimental method:
1st, non denatured polyacrylamide gels (N-PAGE):The serial polypeptides of HR1 are mixed respectively at the serial polypeptides of HR2
(each 30 μM of final concentration), 37 DEG C are incubated 30 minutes, take 5 × high ph-values sample-loading buffer, are mixed with the peptide mixer of 4 times of volumes,
Then point sample (per μ l of hole 25) (Beijing day bounties) in 18% non denatured glue, 125V constant pressures electrophoresis 2 hours, examine horse at room temperature
This light blue is dyed, and is as a result shown, HCoV-EK1 with the serial polypeptides of HR1P after mixing, and it is probably six spirals to generate one
The new band (as shown in Figure 5 a) of compound;
2nd, circular dichroism spectra determines polypeptide secondary structure:With 50mmol/L, pH 7.2 phosphate buffer by free polypeptide or
Mixtures of polypeptides is diluted to final concentration of 10 μm of ol/L, is determined using light splitting polarimeter (J-815 types, Jasco Inc, Japan)
Circular dichroism spectra.4 DEG C of detection temperature, bandwidth 5.0nm, resolution 0.1nm, optical path 0.1cm, reaction time 4.0s, sweep speed
50nm/min.In 222nm with the thermal denaturation of 5 DEG C/min thermograde variation monitoring polypeptide, the blank pair of cushioning liquid is subtracted
According to spectrum is corrected, smoothing techniques are carried out to melting curve, and the neutral temperature that thermal dissociation changes, i.e. Tm are calculated with Jasco
Value.
(2), result is shown:
1. the polypeptide as shown in Figure 5 a, derived from a variety of human corona virus HR1, including SARS-CoV-HR1P
(RsSHC014-CoV-HR1P or RsW1V1-CoV-HR1P), MERS-CoV-HR1P, HcoV-229E-HR1P, with HCoV-EK1
Polypeptide mixing after, generate one be probably six helix complex new band;In addition, test result indicates that HCoV-EK1 is more
After peptide is mixed with HCoV-NL63-HR1P polypeptides or HCoV-OC43-HR1P polypeptides, same phenomenon can occur, point out HCoV-
EK series polypeptides also have the activity for suppressing HCoV-NL63 and HCoV-OC43;
2. as shown in Figure 5 b, single polypeptide is low helical structure, as HCoV-EK1 polypeptides and SARS-CoV-HR1P
After (RsSHC014-CoV-HR1P or RsW1V1-CoV-HR1P), MERS-CoV-HR1P or HcoV-229E-HR1P mixing, occur
The helical structure of height, the result meets the CD spectrum of six helical core structures;And its Tm value represents its degree of stability.
The application above-mentioned test result indicates that, the serial polypeptides of the HCoV-EK have very wide spectrum and efficient antiviral
Activity, can have good inhibition, including SARS-CoV, MERS-CoV, HCoV- to human corona virus of more than two kinds
229E and class SARS virus (RsSHC014-CoV or RsW1V1-CoV);The major antiviral machine of HCoV-EK series polypeptides
System is combined by the HR1 regions with virus, forms stable heterologous 6HB, prevents viral HR2 and the HR1 tripolymer of itself
With reference to preventing them from forming homologous 6HB, and then merging between viral interference and cell membrane, blocking virus infection.
SEQUENCE LISTING
<110>Fudan University
<120>Suppress polypeptide and its application of human coronary virus's infection wide spectrum
<130> 16
<160> 3
<170> PatentIn version 3.3
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