CN106324244A - Key molecular characteristics of invasion with species specificity of CD81 mediated hepatitis C virus - Google Patents
Key molecular characteristics of invasion with species specificity of CD81 mediated hepatitis C virus Download PDFInfo
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Abstract
The invention discloses key molecular characteristics of invasion with species specificity of CD81 mediated hepatitis C virus. The invention provides applications of substances of intramolecular hydrogen bonds or salt bonds which maybe formed between 188th-196th amino acid residues of CD81-LEL protein in an animal to be detected to preparation of products for auxiliarily detecting or predicating whether the animal to be detected can be infected by HCV. Experiments prove that whether intramolecular hydrogen bonds or salt bonds which maybe formed between 188th-196th amino acid residues are used as one of sequence standards for judging whether HCV invasion is supported, and animal models which support HCV invasion are selected based on the characteristics. A HCV vaccine design is assisted by using the characteristics.
Description
Technical field
The present invention relates to biological technical field, the key particularly relating to CD81 mediation hepatitis C virus species specificity invasion is divided
Subcharacter.
Background technology
It is the main cause causing chronic hepatopathy that hepatitis C virus (HCV) infects, and finds so far from 1989, and HCV is entirely
Infect 1.7 hundred million person-times in the range of ball, and owing to lacking vaccine, there is also about the new infections of 300-400 ten thousand every year, it has also become
The primary cause of disease of liver transplantation is accepted in world wide.The standard treatments of HCV infection is Peg-IFN alpha-2b-α and Li Ba
Wei Lin drug combination, this Therapeutic Method can cause continued viral response (SVR).But there is obvious defect in the method,
Such as response rate less than 50%, significant side effect, poor resistance, terminal illness is failed to respond to any medical treatment.In order to preferably control
Treat HCV infection, directly act on antiviral drugs (DAAs) and arise at the historic moment.U.S. food Drug Administration (FDA) is
Closely have approved several protease inhibitor class DAAs, such as Telaprevir (VERTEX), Boceprevir (Merck),
Harvoni (Gilead Sciences) and Viekira Pak (AbbVie).This type of is introduced in standard treatments
DAAs makes therapeutic effect significantly improve, and the patient populations responding treatment substantially increases, and treatment cycle substantially shortens.But
This type of drug price is expensive, it is desirable to utilizes the method to control HCV completely and propagates and unrealistic, and the research and development gesture of HCV vaccine is must
OK.
HCV is single strand plus RNA virus, belongs to flaviviridae, and the genome translation of 9.6kb produces 3011 aminoacid of total length
Polyprotein, under the common effect of cell and virus protease, it is cut produce 3 structural protein (core protein,
E1, E2) and 7 non-structural proteins (P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B).
HCV surfaces of viral particles loads two kinds of surface protein E1 and E2, and the two is presented in heterodimer.Virus
Phagocytic process needs E1/E2 heterodimer to interact with host cell surface receptor.Participate in the cell table of HCV phagocytic process
Face receptor has a lot, wherein has conclusive evidence to show, glycosaminoglycans (GAG) and low density lipoprotein receptor (LDL-R) are diseases
Poison granule and host cell be initial stick necessary.Additionally, also 4 receptors determine participation poisoning intrusion process, it is respectively
Scavenger receptor B class 1 type (SR-B1), CD81, Claudin-1 and Occludin.
Cell surface protein CD81 participates in sticking of various physiological processes, such as immunocyte, form, activation, propagation, divides
Change.This molecule can mediate the invasion of multiple pathogens, including parasite, antibacterial, fungus and virus, the most of greatest concern
Be this molecule be HCV invade requisite receptor.CD81 belongs to tetratransmembrane albumen superfamily, and the CD81 of mammal leads to
Often being made up of 236 aminoacid, mainly by four cross-film districts, three intracellular rings and two born of the same parents' outer shrouds are in series, and wherein two
Individual born of the same parents' outer shroud is a utricle outer shroud (SEL) and big born of the same parents' outer shroud (LEL) respectively.Big born of the same parents' outer shroud in CD81 molecule
(LEL) it is the important area of its function, because this region is responsible for being combined with chaperone.It is reported, CD81-LEL can
To mediate self dimerization and directly in conjunction with HCV-E2.
HCV infection, HCV petty action can be supported owing to HCV infection has the strictest host specificity, only human and chimpanzee
The research and development of object model become retardance HCV correlational study, particularly one of bottleneck of vaccine research and development.The base of HCV host specificity
Plinth derives from the complexity of poisoning intrusion process.CD81 directly combines with HCV-E2, and the invasion of its mediation HCV has the strongest
Species specificity, the only CD81 of human and chimpanzee combines HCV-E2 with the highest affinity and supports that HCV invades, but its
Molecular mechanism is still not clear so far.Therefore the CD81-that mice and cercopithecus aethiops two kinds can not support the animal of HCV infection is resolved
The crystal structure of LEL, contrast finds difference present in itself and people's CD81-LEL crystal structure, will disclose CD81 and mediate HCV
The invasion specific molecular basis of kind, provides the theoretical foundation of screening for setting up of infectious animal model.
Summary of the invention
It is an object of the present invention to provide CD81-LEL albumin crystal the 188th amino acids residue and the in detection tested animal
The purposes of the material of intramolecular hydrogen bond or sat linkage can be formed between 196 amino acids residues.
CD81-LEL albumin crystal the 188th amino acids residue and the 196th bit amino in the detection tested animal that the present invention provides
Whether can the material that form intramolecular hydrogen bond or sat linkage between acid residue can quilt in preparation auxiliary detection or auxiliary prediction tested animal
Application in the product of HCV infection.
In above-mentioned application, described product is test kit.
In above-mentioned application, CD81-LEL albumin crystal the 188th amino acids residue and the 196th in described detection tested animal
Can the material that form intramolecular hydrogen bond or sat linkage between amino acid residue include following 1) and/or 2):
1), for the reagent of protein crystal；
2), record the readable carrier of XDS package software, the readable carrier of record Phaser software, COOT is soft in record
The readable carrier of part and the readable carrier of record Phenix software.
In above-mentioned application,
Described tested animal is behaved or mice or cercopithecus aethiops.
Another object of the present invention is to provide auxiliary detection or whether auxiliary prediction tested animal can be by HCV infection product.
The product that the present invention provides, for CD81-LEL albumen the 188th amino acids residue and the 196th in detection tested animal
Between can form the material of intramolecular hydrogen bond or sat linkage.
In the said goods,
Energy between CD81-LEL albumen the 188th amino acids residue and the 196th amino acids residue in described detection tested animal
The material of no formation intramolecular hydrogen bond or sat linkage includes following 1) and/or 2):
1), for the reagent of protein crystal；
2), record the readable carrier of XDS package software, the readable carrier of record Phaser software, COOT is soft in record
The readable carrier of part and the readable carrier of record Phenix software
In the said goods,
Described tested animal is behaved or mice or cercopithecus aethiops.
The 3rd purpose of the present invention is to provide CD81-LEL albumin crystal the 188th amino acids residue and the in detection tested animal
The purposes of the material of intramolecular hydrogen bond or sat linkage can be formed between 196 amino acids residues.
CD81-LEL albumin crystal the 188th amino acids residue and the 196th bit amino in the detection tested animal that the present invention provides
The application in identifying HCV infection animal model of the material of intramolecular hydrogen bond or sat linkage can be formed between acid residue.
The 4th purpose of the present invention is to provide promotion or suppression CD81-LEL albumin crystal the 188th amino acids residue and the 196th
The purposes of the material of intramolecular hydrogen bond or sat linkage is formed between amino acids residue.
The present invention provide promotion or suppression CD81-LEL albumin crystal the 188th amino acids residue and the 196th amino acids residual
The material of formation intramolecular hydrogen bond or sat linkage application in preparation HCV infection animal model or cell model between base.
In above-mentioned application, described promotion or suppression CD81-LEL albumin crystal the 188th amino acids residue and the 196th bit amino
The material forming intramolecular hydrogen bond or sat linkage between acid residue is: make CD81-LEL albumin crystal the 188th amino acids residue and/
Or the 196th amino acids residue produce sudden change material.
The experiment proves that, the present invention by resolve do not support HCV infection mice CD81-LEL (mCD81-LEL) and
The three-dimensional crystalline structure of cercopithecus aethiops CD81-LEL (agmCD81-LEL), by the people CD81-LEL with support HCV infection
(hCD81-LEL) contrastive Analysis of Structures finds: be respectively present 188-196 amino in mCD81-LEL and agmCD81-LEL
Define intramolecular hydrogen bond or sat linkage between acid residue, and similar intramolecular interaction is not exist in hCD81-LEL
's.In sum, the intramolecular hydrogen bond/sat linkage that whether can be formed between 188-196 amino acid residue discriminates whether to prop up for becoming
Hold one of sequence criteria of HCV invasion, can be characterized as according to the animal model selecting to support HCV invasion with this.This can be utilized special
Levy auxiliary HCV vaccine design.
Accompanying drawing explanation
Fig. 1 is sieve chromatography collection of illustrative plates.
Fig. 2 is the SDS-PAGE of mCD81-LEL and agmCD81-LEL solution.
Fig. 3 is the Rikagu diffraction patterns of mCD81-LEL and agmCD81-LEL.
Fig. 4 is the crystallogram of mCD81-LEL and agmCD81-LEL.
Fig. 5 is mCD81-LEL and hCD81-LEL Structure Comparison figure.
Fig. 6 is agmCD81-LEL and hCD81-LEL Structure Comparison figure.
Fig. 7 is E2-CD81-LEL binding tests.
Fig. 8 is CD81 disappearance Huh7.5.1 cell function detection
Fig. 9 is HCVpp infection experiment result.
Figure 10 is HCVcc infection experiment result
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Below example facilitates a better understanding of the present invention, but does not limit the present invention.
Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged.
Mice CD81 big born of the same parents outer shroud is also known as mCD81-LEL, and the sequence 1 of its aminoacid sequence such as sequence table is from N-terminal the 113rd
To shown in 202 amino acids residues；The encoding gene of mCD81-LEL albumen, also known as mCD81-LEL gene, its nucleotides sequence
Row are if the sequence 2 of sequence table is from shown in the 229th to 608 nucleotide of 5 ' end；
Cercopithecus aethiops CD81 big born of the same parents outer shroud is also known as agmCD81-LEL, and the sequence 3 of aminoacid sequence such as sequence table is from N-terminal
Shown in 113 to 202 amino acids residues；The encoding gene of agmCD81-LEL albumen, also known as agmCD81-LEL gene, its
Nucleotide sequence is if the sequence 4 of sequence table is from shown in the 229th to 608 nucleotide of 5 ' end.
Plasmid pET-28a (+): Novagen:69864-3.Escherichia coli Rosseta:Novagen:71402-4.
Embodiment 1, the big born of the same parents of mice CD81 are outer and the protein structure difference of cercopithecus aethiops CD81 big born of the same parents outer shroud
(1) preparation of mCD81-LEL and agmCD81-LEL albumen
One, the structure of recombiant plasmid
The sequence 2 of sequence table is inserted plasmid from the double chain DNA molecule shown in the 229th to 608 nucleotide of 5 ' end
PET-28a (+) Nde I and Xho I restriction enzyme site between, obtain recombiant plasmid pET28a-mCD81-LEL；
The sequence 4 of sequence table is inserted plasmid from the double chain DNA molecule shown in the 229th to 608 nucleotide of 5 ' end
PET-28a (+) Nde I and Xho I restriction enzyme site between, obtain recombiant plasmid pET28a-agmCD81-LEL.
Two, the preparation of mCD81-LEL and agmCD81-LEL albumen
1, recombiant plasmid pET28a-mCD81-LEL and pET28a-agmCD81-LEL step one obtained imports escherichia coli
The competent cell of Rosseta, obtains recombinant bacterium.
2, the monoclonal of recombinant bacterium step 1 obtained is seeded to 20ml containing 50mg/L kanamycin and 50mg/L chloromycetin
LB fluid medium, 37 DEG C, 220rpm shaken cultivation overnight.
3, take the bacterium solution that 20ml step 2 obtains, with the volume ratio of 1:50 be seeded to 1L containing 50mg/L kanamycin and
The LB fluid medium of 50mg/L chloromycetin, 37 DEG C, 200rpm cultivate place on ice to OD600nm=1.2；Place on ice
Add IPTG after half an hour and to make its concentration be 0.5mmol/L, 37 DEG C, 200rpm shaken cultivation 5 hours.
4, taking into the cultivating system of step 3,4000rpm is centrifuged 15min, collects thalline, (molten with 100ml lysate
Agent is the Tris-HCl buffer of pH8.5,50mM, containing 100mM NaCl) resuspended, ultrasonic disruption is then carried out on ice
(power 600W, effect 5s, interval 5s, 99 times), then 15000rpm is centrifuged 30min, takes precipitation i.e. inclusion body and enters
Row degeneration renaturation.
5, inclusion body adds 150mL degeneration liquid (8M Urea, 100mM NaH2PO4,10mM Tris-HCl pH
8.5) dissolving, stirring, to being completely dissolved, 4 DEG C, takes supernatant after 12000rpm, 30min are centrifugal, pours in bag filter and soak
Entering in 1L degeneration liquid, dropping 2L dialysis solution (50mM Tris-HCl pH 8.5,100mM NaCl), drips wherein
Carbamide final concentration position 2.67M in Cheng Houtong washing liquid；Pouring out 2L next day and add 2L dialysis solution, now carbamide is final concentration of
0.89M, all pours out and is added thereto to 2L dialysis solution, now carbamide final concentration of 0M next day, after gradient dialysis renaturation
To mCD81-LEL and the agmCD81-LEL albumen of solubility, after 12000rm, 30min are centrifugal, take supernatant.
6, the supernatant that step 5 obtains being splined on nickel post, first with the remove impurity liquid of 10 times of column volumes, (solvent is water, containing 100mM
NaCl, 50mM Tris-HCl, pH8.5) eluting is to remove foreign protein, then (solvent is water, containing 100mM with eluent
NaCl, 50mM Tris-HCl, 300mM imidazoles, pH8.5) eluting to collect destination protein, took the eluent after post.
7, nickel post eluent step 6 obtained adds 50 μ L Thrombin, pours dialysis excision N-end in bag filter into
6 × His tag, dialysis solution is 50mM Tris-HCl pH8.5,100mM NaCl.After dialysed overnight by solution in bag filter
Pouring out, 4 DEG C, 4000rpm, 30min are centrifuged off precipitation that may be present.
8, taking the solution that step 7 obtains, the ultrafiltration concentration pipe using molecular cut off to be 3KD is concentrated by ultrafiltration, and obtains
The concentrated solution of about 1ml.
9, the concentrated solution loading that step 8 obtained also carries out sieve chromatography.
Sieve chromatography uses selects Suerdex75 10/300 pillar to carry out the most isolated and purified, eluent: 50mM
Tris-HCl pH 8.5,100mM NaCl.Flow velocity is 0.5ml/min, and collecting retention volume is the mistake between 12mL-16mL
Eluent (Fig. 1) after post.
10, taking solution after the post excessively that step 9 obtains, it is dense that the ultrafiltration concentration pipe using molecular cut off to be 3KD carries out ultrafiltration
Contracting, obtains the solution (protein concentration is about 30mg/ml) of about 1mL, is respectively designated as mCD81-LEL (10KD, figure
2a) with agmCD81-LEL albumen (10KD, Fig. 2 b).
Use same method, preparation hCD81-LEL (10KD).
(2), the crystal of mCD81-LEL and agmCD81-LEL albumen resolves
(solvent is 50mM Tris-HCl, 100mM NaCl, pH's 8.5 to take the mCD81-LEL protein solution that () obtain
Buffer solution) and crystallization buffer (0.1M Sodium Citrate pH5.5,5%PEG1000,35%isopropano))
It is 1:1 mixing according to volume ratio, crystallizes at 22 DEG C 3 days and obtain mCD81-LEL crystal, and parsing obtains structure.
(solvent is 50mM Tris-HCl, 100mM NaCl, pH 8.5 to take the agmCD81-LEL protein solution that () obtain
Buffer solution) with crystallization buffer (5%Ethanol, 5%MPD, 0.1M Tris pH8.5,200mM Sodium
Chloride) it is 1:1 mixing according to volume ratio, crystallizes at 22 DEG C 2 days and obtain agmCD81-LEL crystal, and resolve
To structure.
(solvent is 50mM Tris-HCl, 100mM NaCl, pH's 8.5 to take the hCD81-LEL protein solution that () obtain
Buffer solution) with crystallization buffer (0.2M Magnesium acetate, 20%PEG3350) according to volume ratio be 1:1 mix
Even, crystallize at 22 DEG C 2 days and obtain hCD81-LEL crystal, and parsing obtains structure.
MCD81-LEL crystal and agmCD81-LEL crystal are respectively in Switzerland's synchrotron radiation (SLS) X06DA line station and this experiment
Collect on the domestic RigakuX x ray diffractometer x of room.After data being carried out preliminary process and integrating (XDS package), logical
The method analytic structure crossing molecular replacement (Phaser, CCP4package) generates cloud density figure, through essence progressively
Repair and optimize (COOT and Phenix) and obtain final structure.
The diffraction patterns of mCD81-LEL crystal and agmCD81-LEL crystal is shown in that (left figure is that mice CD81-LEL crystal spreads out to Fig. 3
Penetrate result；Right figure is cercopithecus aethiops CD81-LEL crystal diffraction result) and table 1.Crystal structure photo is shown in that (left figure is little to Fig. 4
Mus CD81-LEL crystal；Right figure is cercopithecus aethiops CD81-LEL crystal).
Table 1 is crystal data
Collect data after mCD81-LEL crystal and agmCD81-LEL crystal are passed through X-ray diffraction, data are carried out tentatively
Process with integrate after (XDS package), resolve knot by the method for molecular replacement (Phaser, CCP4package)
Structure generates cloud density figure, obtains final structure through refine progressively and optimization (COOT and Phenix), analyzes crystalline substance
Hydrogen bond or sat linkage in body.With hCD81-LEL crystal for comparison.
MCD81-LEL crystal testing result such as Fig. 5, left figure is the cloud density figure local of mCD81-LEL crystal structure, right
Figure is mCD81-LEL crystal and the contrast of hCD81-LEL crystal structure, and gold is mice, and blueness is people；
As shown in Figure 6, right figure is the cloud density figure of agmCD81-LEL crystal structure to agmCD81-LEL crystal testing result
Locally, left figure is agmCD81-LEL crystal and the contrast of hCD81-LEL crystal structure, and green is cercopithecus aethiops, and blueness is people.
It can be seen that
CD81-LEL is directly to mediate the Loop being combined with HCV-E2, and the aminoacid sequence from mice to people CD81-LEL is high
Degree is conservative, but the CD81 of only people and gorilla can combine HCV-E2 with nanomole level affinity and mediate HCV and enter
Invade.Being found by Multiple Sequence Alignment analysis, the sequence of CD81-LEL is identical in people and gorilla, and people,
Cercopithecus aethiops and mice also exist fine distinction (sequence homology of 81%-96%), wherein agmCD81-LEL and
The difference (residue 163,186,188 and 196) of only four amino acid residues between hCD81-LEL, CD81's Yu HCV
E2 combines the identification range of deciding factor and is limited in these four amino acid residues.Sequence analysis based on structure finds, this
Region residing for four amino acid residues is least guarded, wherein it is known that F186 is for most important directly in conjunction with E2,
And the contribution of other residues is still not clear.The multisequencing of CD81-LEL shows than result, it is possible to the CD81's of mediation HCV invasion
188 residues are the most electronegative, and 188 amino acids residues are the most electric in the CD81 that can not mediate HCV invasion
Neutral or positively charged.
The crystal of hCD81-LEL and mCD81-LEL is obtained for high-resolution through X-ray diffractionData,
Final models exhibit has gone out good stereochemical nature.The overall conformation of mCD81-LEL structure and hCD81-LEL class
Seemingly, all " mushroom-shaped " structure is formed by 5 alpha-helixs.The Structural superposition superposition of hCD81-LEL and mCD81-LEL
DrawR.m.s.d value be 87C alpha atom.Under the support of high resolution structures information, at atomic level
It is analyzed CONSTRUCTED SPECIFICATION finding, between Q188 and E196 of mCD81-LEL, there is an intramolecular hydrogen bond (Q188
Nε2-H:E196 Oε2,distance:angle:124).It should be noted that this hydrogen bond is at hCD81-LEL
In be non-existent, in hCD81-LEL, E188 and D196 of correspondence position is electronegative, its side chain due to electrostatic arrange
The effect scolded and far apart.Distance nearest between two residues is E188 O ε 2:D196 O δ 2,
Can be seen that from sequence in agmCD81-LEL, 188 is the lysine of a positively charged, in theory can be with
Forming sat linkage between D196, the Crystal X-Ray Diffraction resolution of agmCD81-LEL reachesIts structure display residue
Define sat linkage between K188 and residue D196, but this bond strength is more weak, it may be possible to owing to the side chain lengths of D196 is made not
Become.
Above-mentioned experiment shows,
The most there is not hydrogen bond between glutamic acid and the aspartic acid of 196 of 188 of people CD81-LEL and the most there is not sat linkage,
An intramolecular hydrogen bond, agmCD81-is there is between glutamine and the glutamic acid of 196 of 188 of mCD81-LEL
Sat linkage is formed between lysine and the aspartic acid of 196 of 188 of LEL.
Currently known people can with HCV infection, mice and cercopithecus aethiops cannot HCV infection, compare the difference of three CD81-LEL
Not, drawing can be according to the CD81-LEL albumin crystal structure of detection tested animal, between its 188 and 196 amino acid residue
Can form intramolecular hydrogen bond or sat linkage can predict tested animal be whether can by HCV infection, if the CD81-of tested animal
Intramolecular hydrogen bond or sat linkage can not be formed between 188th amino acids residue and the 196th amino acids residue of LEL, the most to be measured
Animal energy or candidate can be by HCV infection；
If molecule can be formed between the 188th amino acids residue and the 196th amino acids residue of the CD81-LEL of tested animal
Interior hydrogen bond or sat linkage, then tested animal can not or candidate can not be by HCV infection.
Intramolecular hydrogen can not be formed between CD81-LEL albumen the 188th amino acids residue and the 196th amino acids residue of people
Key can not form sat linkage, and people can be by HCV infection；CD81-LEL albumen the 188th amino acids residue of people is glutamic acid,
And the 196th amino acids residue be aspartic acid；
The intramolecular hydrogen formed between CD81-LEL albumen the 188th amino acids residue and the 196th amino acids residue of mice
Key, mice can not be by HCV infection；Mice CD81-LEL albumen the 188th amino acids residue is glutamine, and the 196th
Amino acids residue is glutamic acid；
The molecule formed between CD81-LEL albumen the 188th amino acids residue and the 196th amino acids residue of cercopithecus aethiops
Inner salt band, cercopithecus aethiops can not be by HCV infection；CD81-LEL albumen the 188th amino acids residue of cercopithecus aethiops is for relying ammonia
Acid, and the 196th amino acids residue is aspartic acid.
Whether auxiliary prediction or auxiliary detection tested animal are to be included detecting tested animal by being used for by the test kit of HCV infection
CD81-LEL the 188th amino acids residue and the 196th amino acids residue between can form intramolecular hydrogen bond or sat linkage
Material, material include crystallization buffer, record the readable carrier of XDS package software, record Phaser software readable
Carrier, the readable carrier recording COOT software and the readable carrier of record Phenix software.
Whether embodiment 2, auxiliary prediction or auxiliary detection tested animal are can be by HCV infection
One, the preparation of albumen
The method using embodiment 1, preparation people's CD81-LEL albumen (hCD81-LEL, aminoacid sequence is sequence 5), little
Mus CD81-LEL albumen and cercopithecus aethiops CD81-LEL albumen.
Two, protein crystal
Crystallize according to the method for embodiment 1, obtain people's CD81-LEL albumin crystal, mice CD81-LEL albumin crystal and non-
Continent grivet CD81-LEL albumin crystal.
Three, the hydrogen bond in detection albumin crystal or sat linkage
The method using embodiment 1, uses XDS package, Phaser, COOT and Phenix software analysis people CD81-
LEL albumin crystal, mice CD81-LEL albumin crystal and cercopithecus aethiops CD81-LEL albumin crystal, detect above-mentioned people CD81-
The 188th amino acids in LEL albumin crystal, mice CD81-LEL albumin crystal and cercopithecus aethiops CD81-LEL albumin crystal
Hydrogen bond or sat linkage whether is formed between residue and the 196th amino acids residue.
Result:
Be formed without between people's CD81-LEL albumin crystal the 188th amino acids residue and the 196th amino acids residue hydrogen bond or
Sat linkage, it was predicted that people can be by HCV infection；
It is formed without hydrogen between mice CD81-LEL albumin crystal the 188th amino acids residue and the 196th amino acids residue
Key, it was predicted that mice can not be by HCV infection；
It is formed without between cercopithecus aethiops CD81-LEL albumin crystal the 188th amino acids residue and the 196th amino acids residue
Sat linkage, it was predicted that cercopithecus aethiops can not be by HCV infection.
This can be by HCV infection with known people, and mice can not be consistent by HCV infection with cercopithecus aethiops, illustrates the present invention's
Method is correct.
Whether form hydrogen bond between embodiment 3, CD81-LEL albumin crystal 188-196 amino acid residue to go to detect whether by HCV
The checking infected
One, binding tests
For relation between intramolecular bond and HCV-E2 binding affinity, root between 188-196 residue in research CD81-LEL
According to structure design Mutagen nuclear expression CD81-LEL and a series of mutant, the method for Enzyme-linked Immunosorbent Assay (ELISA) is utilized to examine
Survey the binding ability between itself and recombinant soluble HCV-E2.
For people's CD81-LEL albumin crystal, then the 188th be glutamic acid and 196 be aspartic acid, it is impossible to form hydrogen bond
Or sat linkage, if sporting other, then it is likely to be formed hydrogen bond or sat linkage；
For mice CD81-LEL albumin crystal, then the 188th be glutamine and 196 be glutamic acid, hydrogen can be formed
Key, if sporting other, is then formed without hydrogen bond；
For cercopithecus aethiops CD81-LEL albumen, then the 188th be lysine and 196 be aspartic acid, salt can be formed
Key, if sporting other, is then formed without sat linkage；
Following CD81-LEL and mutant thereof be respectively as follows: hCD81-LEL (hCD81-WT, aminoacid sequence is sequence 5),
HCD81-LEL-E188K (for being lysine by the 196th glutamic acid mutation of sequence 5), hCD81-LEL-D196E are (for by sequence
The 196th Aspartic acid mutations of row 5 is glutamic acid), hCD81-LEL-D196Q is (for by the 196th aspartic acid of sequence 5
Sport glutamine), hCD81-LEL-D196K (for being lysine by the 196th Aspartic acid mutations of sequence 5),
HCD81-LEL-D196R (for being arginine by the 196th Aspartic acid mutations of sequence 5), hCD81-LEL-D196E (are will
The 196th Aspartic acid mutations of sequence 5 is glutamic acid), hCD81-LEL-D196E-E188K is (for by sequence 5 the 196th
Position Aspartic acid mutations be glutamic acid, and be lysine by the 188th glutamic acid mutation), mCD81-LEL (mCD81-WT),
MCD81-LEL-L186F (for the 186th leucine of sequence 1 is sported phenylalanine), mCD81-LEL-L186F-Q188E
(for the 186th leucine of sequence 1 is sported phenylalanine, the 188th glutamine sports glutamic acid), mCD81-
(for the 186th leucine of sequence 1 is sported phenylalanine, the 196th glutamic acid mutation is sky to LEL-L186F-E196D
Winter propylhomoserin), mCD81-LEL-L186F-Q188A (for the 186th leucine of sequence 1 being sported phenylalanine, the 188th
Position glutamine sports alanine), mCD81-LEL-L186F-Q188E E196D is (for by the 186th leucine of sequence 1
Sporting phenylalanine, the 188th glutamine sports glutamic acid, and the 196th glutamic acid mutation is aspartic acid),
AgmCD81-LEL (agmCD81-WT), agmCD81-LEL L186F are (for sport benzene by the 186th leucine of sequence 3
Alanine), agmCD81-LEL L186F-K188A (for the 186th leucine of sequence 3 is sported phenylalanine, 188
Position lysine mutation is alanine), agmCD81-LEL L186F-E196D is (for by the sudden change of the 186th leucine of sequence 3
For phenylalanine, 196 glutamic acid mutation are aspartic acid), agmCD81-LEL L186F-K188E-E196D is (for by sequence
The 186th leucine of row 3 sports phenylalanine, 188 lysine mutations be 196 glutamic acid mutation of glutamic acid be Radix Asparagi
Propylhomoserin).
Specific experiment operation is as follows:
In this test, CD81-LEL and mutant thereof are fixed on plank (1 μ g/well) as capture antibody, slow with being coated
Rush liquid (PBS, 137mM NaCl, 8.1mM Na2HPO4,1.5mM KH2PO4, 2.7mM KCl, pH 7.4) and by albumen
Being diluted to 10 μ g/mL, add 100 μ L in every hole, after shrouding, 4 DEG C overnight.Every hole adds 250 μ L confining liquids (5%
Non-fat miLk in PBST) after incubated at room 2h, wash plate once with PBST solution (0.05%Tween-20).?
Recombinant soluble E2 albumen (sequence 6) is diluted to 500ng/mL and joins in hole, after incubated at room 2h, molten with PBST
Liquid (0.05%Tween-20) washes plate 3 times.Add E2 antibody (0.5 μ g/mL, 100 μ L/weLL) incubated at room 2
h.After again washing plate 3 times with PBST solution (0.05%Tween-20), adding the two of HRP labelling and resist in hole, room temperature is incubated
Educate 1h.TMB (SoLarbio science&technology co., LTD) is used to detect as substrate, all
Test parallel being repeated 3 times.
Result is as it is shown in fig. 7, in the wild type and mutant of all CD81-LEL, hCD81-LEL Yu E2 has the highest
Binding affinity, and the binding affinity of mCD81-LEL and agmCD81-LEL and E2 is nearly no detectable.Such as hCD81-
LEL-D196E is the conservative variants carrying out D196E in hCD81-LEL, can not be formed between 188-196 residue hydrogen bond or
Sat linkage, then it is almost no impact by this sudden change with the binding affinity of E2.The simple point mutation of hCD81-LEL-E188K
Make 188 electrical upsets, mutant can be formed between K188-D196 faint sat linkage (pairing of K-D by
The structure of agmCD81b-LEL is inspired), this mutant and E2 obtain binding affinity and decline about 20%.And hCD81-LEL-
In the double mutant of D196E-E188K, the D of 196 is mutated into E and extends side chain lengths, and keeps dashing forward of E188K simultaneously
The electrical upset become, promotes to form stable K-E match type sat linkage between 188-196 site, this mutant and the knot of E2
Close affinity and have dropped about 80%, be similar to mediate the CD81-LEL of HCV invasion.Result data display K-D pairing
Sat linkage is not enough due to side chain lengths, and the sat linkage of formation is not sufficiently stable, it is impossible to produce the conformation conformational flexibility of spiral-D enough
Constraint effect, so still there is certain adhesion with E2.By comparison, the sat linkage of K-E match type is sufficiently stable, permissible
The conformational flexibility of spiral-D is produced strong constraint effect, so its binding affinity with E2 is remarkably decreased.As
Expection is expected, D196K and D196R these type of 196 occurs the mutant of electrically upset, and it is the most aobvious with the binding affinity of E2
Writing and reduce, this result confirms that between 188-196 site, K-E and E-R pairing the most enough forms stable sat linkage, to spiral-D
Conformational flexibility produce constraint effect thus weaken the binding affinity of itself and E2.
Meanwhile, the sudden change of mCD81-LEL-D196Q can promote to be formed between Q196-E188 hydrogen bond, this mutant and mCD81-
The hydrogen bond that observed Q188-E196 pairing in LEL structure is in opposite direction, and result also demonstrates that this sudden change result in it and ties with E2
Being remarkably decreased of conjunction ability.
The hCD81-LEL-D196E-E188K mutant of design, is equal to introduce in hCD81-LEL and mCD81-LEL
The binding affinity of the intramolecular hydrogen bond that middle Q188-E196 pairing is identical, this mutant and E2 have decreased to the level of background,
Although now F186 remains existence.
Result confirms that induction hCD81-LEL 188-196 interdigit forms hydrogen bond or sat linkage, can weaken it affine with the combination of E2
Power；Next devise the different mutants of mCD81-LEL and agmCD81-LEL, destroy its intramolecular naturally occurring
Hydrogen bond or sat linkage between 188-196, analyse whether to strengthen its binding ability with HCV-E2.Studies have reported that before,
F186 site is most important with the combination of E2 for it, and the first contribution to L186F simple point mutation is studied, and result shows
After the simple point mutation of L186F, its binding ability with HCV-E2 there is no and is remarkably reinforced.It follows that keeping L186F sudden change
Meanwhile, Q188E, Q188A, E196D, the Q188E/E196D (188-of a similar hCD81 are introduced at mCD81-LEL
196 amino acid whose combinations) different sudden changes, upset the chemical bond between 188-196, result show and L186F simple point mutation
Comparing with wild type mCD81-LEL, these mutants have been improved with the binding affinity of E2.AgmCD81-LEL draws
The simple point mutation entering K188A or K188E destroys the chemical bond between 188-196, it was similarly observed that similar result, with
L186F single-point mutants is compared with wild type agmCD81-LEL, and it has had improvement with the binding affinity of E2.?
The design of the sudden change introducing E196D in agmCD81-LEL is to be shortened by carboxylic side-chain to weaken sat linkage between K188-D196,
This sudden change improves the combination of its E2, again demonstrates K-D match type sat linkage and is not sufficiently stable, it is impossible to the conformation spirit to spiral-D
Activity produces enough constraint effects, and weakens the binding ability of itself and E2.
The above results shows, is formed without hydrogen bond or sat linkage can be tied between the 188th and 196 amino acids of CD81-LEL
Close HCVE2 albumen, if forming hydrogen bond or sat linkage, then can not be in conjunction with HCVE2 albumen.
Two, poisoning intrusion test
Above-mentioned Binding experiment proves further, and the 188-196 intramolecular bond found in CD81-LEL affects itself and HCV-E2's
Binding ability, but whether this intramolecular bond in physiological conditions on the knees of the gods affects the function of CD81 mediation HCV invasion, utilizes
It is furtherd investigate by poisoning intrusion test.
1, CD81 deficiency Huh7.5.1 cell line is built
Before carrying out poisoning intrusion test, establish CD81 deficiency first with CRISPR/Cas9 gene editing technology
Huh7.5.1 cell line (Huh7.5.1CD81-)。
Specific as follows:
For reducing effect of missing the target, two sgRNA of this EXPERIMENTAL DESIGN, sequence following CD81-sgRNA1-F:CAC CGG CGC CCA
ACA CCT TCT ATG T；CD81-sgRNA1-R:AAA CAC ATA GAA GGT GTT GGG CGC C；CD81-
sgRNA2-F:CAC CGC ATG ATG TTC GTT GGC TTC C；CD81-sgRNA2-R:AAA CGG AAG CCA ACG
AAC ATC ATG C.First sgRNA is building up on the good lentiCRSPER of linearisation (addgene, 49535) carrier.
Transfect first 18～24 hours with the inoculated and cultured 293T cell in 10cm plate of the culture medium containing 10%FBS, 37 DEG C, CO2
(5%) constant temperature culture is overnight, next day cell density to reach 80% be suitable.TransMAXi transfection reagent is used after 18～24 hours
The recombinant vector that (Beijing North Jian Te bio tech ltd) cotransfection builds and pVSVg (addgene, 8454) matter
Grain and psPAX2 (addgenen, 12260) plasmid, the slow virus of 293T cell intermediate package process LAN sgRNA.48～
Supernatant is collected after 72 hours, centrifugal, subpackage.-70 DEG C of Refrigerator stores, standby.
The slow virus infection of sgRNAHuh7.5.1 cell (given by Dr.Francis Chisari, be documented in as
In Publication about Document, Zhong J, et al.Proc Natl Acad Sci U S A 2005；102:9294-9299.), infect
After 48h, Huh7.5.1 cell is passed on, containing 10% hyclone and the DMEM culture fluid of 1 μ g/mL puromycin
Middle cultivation.After two weeks, cell clone Huh7.5.1 that isolated is stableCD81-。
Utilize Trizol reagent to collect part cell, extract cell total rna, with total serum IgE as template, use QuantiFast
SYBR Green fluorescence real-time quantitative RT-PCR test kit (being purchased from TakaRa company) is to intracellular host gene CD81's
MRNA level in-site quantitative determines.Additionally, infect Huh7.5.1 with HCVccCD81-Cell, viral to HCV after infecting 48h
The rna level of genome carries out absolute quantitation detection.In native system, reverse transcription and PCR complete in same reaction system,
Reaction system includes (20uL): upstream and downstream primer (10uM) each 0.6uL, RNA template 1uL, and (RNA inverts enzymatic mixture
Record enzyme and archaeal dna polymerase) 0.2ul, 2xQuantiFastSYBR GreenRT-PCRMaster Mix 10uL, RNA-free
Water 7.6ul.Reaction condition is 50 DEG C, 10min；95 DEG C, 5min；40 circular response, cycling condition is 95 DEG C,
10s, 60 DEG C, 30s；Solubility curve 65～95 DEG C are continuously.
RT-PCR primer sequence:
CD81: upstream 5 '-TGTTCTTGAGCACTGAGGTGGTC-3 '
Downstream 5 '-TGGTGGATGATGACGCCAAC-3 '；
HCV 5 ' UTR: upstream 5 '-GTCTAGCCATGGCGTTAGTA-3 '
Downstream 5 '-CTCCCGGGGCACTCGCAAGC-3 '
As shown in Figure 8, (a) builds Huh7.5.1 to resultCD81-Principle be use lentivirus mediated CRISPR/Cas9 system
System.B () uses qRT-PCR to detect respectivelyWith endogenous CD81RNA in CD81 deficiency Huh7.5.1 cell
Level；C () uses the coated HCVpp of envelope protein of genotype H77 to infect respectivelyWith CD81 deficiency
Huh7.5.1 cell, is carried out quantitatively by luciferase plain gene reporting system；D () uses HCVcc to infect respectivelyAnd CD81
Deficiency Huh7.5.1 cell, uses the method for qRT-PCR (d) and In-Cell Western assay (e) to cell respectively
The copy number of interior HCV geneome RNA and the quantity of focus of infection carry out quantitatively, with the naive Huh7.5.1 cell phase before disappearance
Ratio, is nearly no detectable the mRNA of endogenous CD81 in Huh7.5.1CD81-cell.Additionally, when Huh7.5.1 cell
The when of infection by HCVcc, the level of the RNA of intracellular HCV is decreased obviously.Result above shows Huh7.5.1CD81-Carefully
Born of the same parents can study CD81 function in the environment when virus infects as a kind of good model.
2, amplification HCVpp and HCVcc virion
1) preparation of pseudovirus
Transfect first 18～24 hours inoculated and cultured 293T cells in 10cm plate, 37 DEG C, CO2 (5%) constant temperature culture mistake
Night, by TransMAXi transfection reagent cotransfection equivalent PNL4-3.Luc.R-E-respectively (by Dr.F. after 18～24 hours
Cosset give, Bartosch B, et al.J Exp Med 2003；197:633-642.) and empty carrier, equivalent
PNL4-3.Luc.R-E-and PCMV-E1E2 (is given McKeating JA, et al.J Virol by Dr.Linqi Zhang
2004；78:8496-8505) and equivalent PNL4-3.Luc.R-E.-and VSV-G, Env-PP, HCV pseudovirus is produced respectively
Grain HCVpp and VSV-G vacation type slow virus VSVpp.Supernatant is collected after 48～72 hours, centrifugal, subpackage.-70 DEG C of refrigerators are protected
Deposit, standby.The polybrene of 4～8 μ g/ml can be added wherein to keep viral viability.
2) preparation of JFH1HCVcc
With laboratory original JFH1HCVcc (J Virol.2008Jul；82(14):7034-46.doi:
10.1128/JVI.00118-08.Epub 2008May 14.Efficient trans-encapsidation of
hepatitis C virus RNAs into infectious virus-like particles.Steinmann E1,Brohm
C, Kallis S, Bartenschlager R, Pietschmann T.) infect Huh7.5.1 cell, by constantly passing on
Cultivate, collect the cell conditioned medium containing virion.General 1 cells and supernatant of collection after 2 days.To the supernatant collected
First carrying out 450 × g to be centrifuged 5 minutes, to remove the precipitate such as cell debris therein, then 5ml/15ml centrifuge tube divides
Dress ,-80 DEG C frozen standby.
3, HCVpp infection experiment
In order to reduce the impact on test of transfection efficiency and protein expression level, use immunoblotting analysis (Western Blotting)
Expression to external source CD81 wild type and mutant recombiant plasmid is carried out quantitatively.
CD81 in above-mentioned two and mutant code gene thereof are inserted respectively pcDNA3.1-myc (Invitrogen) and expresses load
BamHI and the XhoI site of body, obtains the different recombinant vectors expressing different albumen.
The different recombinant vectors expressing different albumen is proceeded to Huh7.5.1 respectivelyCD81-, obtain transfecting the transgenic of different CD81
Cell.
The transgenic cell of the different CD81 of transfection uses the lysate that with the addition of protease inhibitor (Sigma or Pierce)
(50mMTris-HCl, pH7.5,150mM NaCl, 1%Nonidet P40,50mM NaF, 1mM Na3VO4,5mM
β-glycerophosphate, 1mM dithiothreitol, 1mM phenylmethylsulfonyL fluoride)
On ice after cracking, 14,000 × g 20min is centrifuged.Sample adds 2 × SDS sample-loading buffer (Loading buffer)
After boil process, use 9-12%SDS-PAGE glue to carry out electrophoresis.Afterwards, by the protein delivery of separation to nitrocellulose filter
Upper (Bio-Rad, Hercules, CA), 10% milk is closed 1h, is hatched one for 4 DEG C and resist overnight.Two anti-uses time
The anti-IgG antibody of the HRP label of 1:2000 dilution.ECL reagent (Amersham Biosciences, Piscataway,
NJ) as detection substrate.Test result indicate that the expression of CD81 wild type and all mutants is on close level.
The transgenic cell of the different CD81 of transfection, after transfection 24h, adds 200 μ L and comprises HCVpp, VSV-Gpp in every hole
Or the supernatant of bald virus, 8 μ g/mL polybrenes and 2 μ L 2M HEPES (pH 7.55), at desk centrifuge
Middle rotation infects 1.5h (2500rpm, 30CeLsius degree), then hatches 1.5h in CO2 gas incubator.
Rotate 48h after infecting, cell lysis, use ModuLus MicropLate Luminometer (Turner
BioSystems, Sunnyvale, CA, USA) to luciferase element detecting system (Luciferase Assay System,
Promega, Madison) detect.All of test is in triplicate.Generally count range is 10,000-900,000,
And the signal as background that bald virus infects is usually less than 100.All tests are in triplicate.
Result invades result of the test as it is shown in figure 9, a is HCVpp, and b is western-blot result of the test, and HCVpp feels
Dye test is identical with the HCV-E2 binding tests result of ELISA detection.D196E conservative variants, HCVpp is introduced in hCD81
Invasion efficiency almost without any loss；And if in hCD81 introduce D196Q, D196K, D196R,
The sudden change of chemical bond (hydrogen bond or sat linkage) between these beneficially formation 188-196 of E188Q/D196E and E188K/D196E, then
Being remarkably decreased of HCVpp invasion efficiency can be caused.The sudden change of E188K can promote to be formed between 188-196 a more weak K-D
Type sat linkage, HCVpp invasion efficiency impact can be ignored by this key substantially.MCD81 is increased destroying intramolecular bond between 188-196
Or before the efficiency of agmCD81 mediation HCVpp invasion, first have evaluated the impact of L186F simple point mutation, as it can be seen,
Introducing the simple point mutation of L186F in mCD81 and agmCD81, the invasion efficiency of HCVpp has slight rising.If retained
While L186F, the sudden change Q188E, Q188A, E196D, and the Q188E/E196D that introduce other in mCD81 break
Chemical bond between bad 188-196, the invasion efficiency of HCVpp has had bright compared with natural mCD81 and L186F simple point mutation
Aobvious lifting.In agmCD81, obtained similar result, introduce while retaining L186F sudden change K188A, K188E and
The sudden change of K188E/E196D destroys 188-196 intramolecular bond so that the invasion efficiency of HCVpp has had significant raising.?
Introducing the sudden change of E196D in agmCD81, can promote to be formed between 188-196 the weak sat linkage of K-D type, it is functionally equivalent to cut
Intramolecular bond between weak 188-196, therefore the invasion efficiency of HCVpp can also be improved.
It can be seen that hydrogen bond cannot be formed between the 188th and 196 amino acids of CD81-LEL or sat linkage can improve HCVpp
Invasion efficiency, if forming hydrogen bond or sat linkage, then HCVpp can not invade.
4, HCVcc infection experiment
The method detection using JFH1 HCVcc and In-CeLL Western (ICW) expresses different CD81's
The efficiency of infection of HCVcc in Huh7.5.1CD81-.Huh7.5.1CD81-cell is inoculated in 96 orifice plates with 1 × 104 density
In, in cell, transfect CD81 wild type next day and sudden change becomes expression plasmid (by people, mice and cercopithecus aethiops CD81 total length base
Cause and mutant gene thereof are inserted in BamHI and the XhoI restriction enzyme site of pcDNA3.1-myc carrier (Invitrogen)
The plasmid obtained).
After 24h, use HCVcc (MOI=0.01) that cell is infected.After infecting 72h, add the anti-of immunostaining
The antibody (1:400 dilution) of-D8L and IRDye bis-anti-(1:1000 dilutes, Li-Cor, Nebraska,
USA), use paraformaldehyde by fixing for cell afterwards in archioporus, use Triton X-100 infiltration.Eventually through
Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE, USA) obtains data picture.
Huh7.5.1CD81-The immunostaining of cell uses the antibody of HCV-Ab IgG core protein, uses Leica SPW5 Laser Scanning Confocal Microscope
Obtaining data picture uses DAPI to carry out nucleic acid staining.
As shown in Figure 10, a is that HCVcc invades result of the test to result, and b is that the confocal immunofluorescence microscopy of HCVcc invasion is micro-
Mirror display pattern, the result that result of the test invades efficiency test with E2 binding tests and HCVpp is basically identical.In brief,
The D196E sudden change that can not induce bonding between 188-196 and the E188K that can promote to be formed the weak sat linkage of K-D is introduced in hCD81
Suddenly change the least on HCVcc invasion impact, and the E188K/D196E sudden change that formation between 188-196 can be induced to stablize sat linkage can be led
Cause HCVcc invasion substantially to reduce.After introducing L186F simple point mutation in mCD81 and agmCD81, HCVcc invasion is without substantially increasing
High.In mCD81 and agmCD81, keep L186F sudden change, more respectively additionally introduce destruction or weaken 188-196 it
Between the sudden change of hydrogen bond, these mutants make the invasion of HCVcc significantly carry compared with L186F simple point mutation and wild type CD81
High.Immunofluorescence (immunefluorescent) is used to colour to D8L and use co-focusing imaging
(confocal imaging) detects, and have also been obtained similar result.
Above-mentioned all proving, forming intramolecular hydrogen bond/sat linkage between the 188-196 amino acid residue of the CD81-LEL of tested animal can
To judge that this tested animal can be by HCV infection.
Claims (10)
1. in detection tested animal CD81-LEL albumin crystal the 188th amino acids residue and the 196th amino acids residue it
Between can form the material of intramolecular hydrogen bond or sat linkage whether can be by HCV infection in preparation auxiliary detection or auxiliary prediction tested animal
Product in application.
Application the most according to claim 1, it is characterised in that: described product is test kit.
Application the most according to claim 1 and 2, it is characterised in that:
In described detection tested animal CD81-LEL albumin crystal the 188th amino acids residue and the 196th amino acids residue it
Between can form the material of intramolecular hydrogen bond or sat linkage and include following 1) and/or 2):
1), for the reagent of protein crystal；
2), record the readable carrier of XDS package software, the readable carrier of record Phaser software, COOT is soft in record
The readable carrier of part and the readable carrier of record Phenix software.
4. according to described application arbitrary in claim 1-3, it is characterised in that:
Described tested animal is behaved or mice or cercopithecus aethiops.
5. whether auxiliary detection or auxiliary prediction tested animal can be by HCV infection products, for CD81-LEL in detection tested animal
The material of intramolecular hydrogen bond or sat linkage can be formed between albumen the 188th amino acids residue and the 196th.
Product the most according to claim 5, it is characterised in that:
Energy between CD81-LEL albumen the 188th amino acids residue and the 196th amino acids residue in described detection tested animal
The material of no formation intramolecular hydrogen bond or sat linkage includes following 1) and/or 2):
1), for the reagent of protein crystal；
2), record the readable carrier of XDS package software, the readable carrier of record Phaser software, COOT is soft in record
The readable carrier of part and the readable carrier of record Phenix software
7. according to the product described in claim 5 or 6, it is characterised in that: described tested animal is behaved or mice or African green
Monkey.
8. in detection tested animal CD81-LEL albumin crystal the 188th amino acids residue and the 196th amino acids residue it
Between can form the application in identifying HCV infection animal model of the material of intramolecular hydrogen bond or sat linkage.
9. promote or shape between suppression CD81-LEL albumin crystal the 188th amino acids residue and the 196th amino acids residue
The application in preparation HCV infection animal model or cell model of the material of one-tenth intramolecular hydrogen bond or sat linkage.
Application the most according to claim 9, it is characterised in that: described promotion or suppression CD81-LEL albumin crystal the
The material forming intramolecular hydrogen bond or sat linkage between 188 amino acids residues and the 196th amino acids residue is: make CD81-LEL
Albumin crystal the 188th amino acids residue and/or the 196th amino acids residue produce the material of sudden change.
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