CN1733798A - Hepatitis B virus surface L protein related peptide - Google Patents

Abstract

The invention relates to peptides derived from HBV L protein for the prevention and treatment of Hepatitis B virus (HBV) infection, screening method for removing antigenicity of these peptides and the deantigenic derivative peptide screened through the method, wherein the HBV surface L protein pre-S1 region contains key amino acid sequence of the virus adhesion cell surface acceptor and antigenic amino acid sequence bonding with the anti-L protein antibody. The polypeptides at the HBV surface L protein pre-S1 region can suppress HBV infection to cells. The invention also provides the screening method for removing the antigenicity of these polypeptides.

Description

Hepatitis B virus surface L protein related peptide

Technical field

The present invention relates to be used for prevention, treatment hepatitis B virus (human hepatitis B virus, HBV) the HBV surface L protein derived peptide of Gan Raning, remove these polypeptide antigens screening method and by this method screen obtain go the antigenicity derived peptide.Particularly the present invention relates to derive from the L albumen pre-S1 district polypeptide of B genotype adw serotype, C genotype adr serotype and D genotype ayw serotype HBV.Equally especially, the present invention relates to remove the antigenic screening method of HBV surface L protein derived peptide and the HBV surface L protein that infects but not with antibodies by the HBV capable of blocking that this method obtains goes the antigenicity derived peptide.This goes the antigenicity derived peptide to infect with the collaborative HBV of inhibition of anti-HBV surface L protein antibody.

Background technology

1.HBV epidemiology

It is one of the most serious public health problem in the whole world that HBV infects, and is the 3rd common disease that is only second to venereal disease and varicella.The whole world has 2,000,000,000 people to infect HBV approximately, and 75% world population is lived in high incidence of hepatitis b, and the chronic HBV infection person surpasses 3.5 hundred million.The annual death toll relevant with the HBV infection is up to 1,000,000 (1), and annual newly-increased the infection estimates it is that HIV increases 2.5～4 times that infect newly.75% HBV chronic infection person concentrates on Asia (being about 2.87 hundred million) in the world, China is the high popular district of hepatitis B, the sick investigation data of learning of the end of the seventies and the beginning of the nineties twice national hepatitis epidemic shows, China infected HBV person 6.9 hundred million, infection rate is 57.6%, HBV person 1.2 hundred million is carried in the whole nation for a long time, HBV surface antigen carrying rate 9.75%, surplus the existing chronic hepatitis B patient 2,000 ten thousand, and begin from nineteen ninety-five, Chinese population hepatitis B morbidity ratio is the trend that raises year by year, and therefore hepatitis B having been listed in China in Ministry of Health's " sanitary work main points in 2005 " needs one of four big diseases of emphasis control.

2.HBV prevention of infecting and treatment present situation

The importance of HBV obtains worldwide already extensively generally acknowledging that its prevention and treatment also are placed in precedence.But still lack the medicine that can remove HBV in the body so far, the definite relatively antiviral of curative effect mainly is interferon-alpha and nucleoside analog (lamivudine, Adefovir, Entecavir and Te Bifuding etc.).Interferon-alpha is mainly by immunomodulatory with induce in the target cell antiviral protein and bring into play antivirus action; Nucleoside analog then acts on the HBV reversed transcriptive enzyme, suppresses the synthetic of viral DNA chain, thereby suppresses duplicating of HBV DNA.Standard course of therapy through 1 year, about 33% interferon-alpha treatment patient and Yue 16% lamivudine therapy patient can obtain thoroughly or the part serology is replied (HBsAg turns out cloudy, HBeAg all turns out cloudy, occur or do not occur resisting-HBe antibody), virusology is replied (HBV DNA turns out cloudy) and biochemical responses (alanine aminotransferase interval continuous 2 detections in 1 month are all normal, and liver function is normal again) completely.

After Krugman in 1970 obtained the earliest hepatitis B vaccine, various countries utilized asymptomatic HBsAg carrier's blood plasma to extract haematogenous HBsAg to prepare the HBV vaccine in succession.Haematogenous HBV vaccine is proved to be safe and effective through life-time service, but because its source is limited, preparation cost is high, is replaced by gene recombination HBV vaccine.Main recombiant vaccine comprises the reconstituted hepatitis B vaccine of yeast and Chinese hamster ovary cell expression at present.These vaccine immunogenicities are strong, and the omnidistance immunity of 3 pins back anti-HBsAg antibody male rotary rate is not less than 85%.But it is slower that its main drawback is an antibody response, and HBV infects newborn infant's initial immunity of puerpera if surpass more than 7 days, then loses the HBV vaccine is blocked vertical transmission to the newborn infant effect.And 10%～15% the inoculator do not produce and reply or low replying, this crowd still can be infected by HBV.

Hepatitis B immune globulin (HBIG) is to use behind the healthy people of hepatitis b vaccination, the tire immunoglobulin preparation of blood plasma or the preparation of serum separation and Extraction of the height of collection, and its antibody titer is more than 100IU/ml.Be applicable to that burst inadvertent contamination crowd, immunologic hypofunction person and HBV infect the neonatal passive immunization prevention of puerpera.The chronic HBV infection person of China has 30%-50% approximately by mother-to-baby transmission; be blocking-up HBV mother and baby perinatal transmission; generally with HBIG and Hepatitis B virus vaccine combined utilization; though protection ratio can reach more than 80%; but the blocking-up that HBIG provides contribution is also not obvious; combined utilization is only than the blocking-up rate of Hepatitis B virus vaccine list with raising 5%～10%, and the still more healthy puerpera newborn infant of HBV infection puerpera newborn infant's HBV infection rate is high 4 times after the combined utilization.In recent years find that intrauterine infection may be the important channel of mother-to-baby transmission, the HBV infection sign recall rate that HBV infects pregnant woman's induced labor foetus liver or blood can reach 40%, the positive mother's of HBsAg baby's intrauterine infection rate about 16%.Therefore domestic some hospital infects the HBIG of pregnant woman's shot in every month greater than 200IU in last 3～4 middle of the month of gestation to HBV, in the hope of prevention fetus HBV intrauterine infection.But this method Intrauterine Transmission barrier effect is unsatisfactory, and may cause the appearance and the immunoreactive generation of immunocomplex pathology of HBV S district variant.

HBIG also is widely used in the prevention of the relevant after liver transplantation HBV infection and recurrence of hepatitis B.Do not having under the situation of preventive measures, in the relevant after liver transplantation of hepatitis B 6 months HBV again infection rate up to about 40%, 2 year in again infection rate up to about 60%.Majority cases of infection is again gone through acute, chronic hepatitis and is developed into liver cirrhosis, liver failure, and long-term prognosis is not good, needs liver retransplantation.Adopt separately lamivudine prevention recurrent HBV after liver transplantation, its Long-term HBV-DNA, HBeAg and HBsAg negative conversion rate are about 60～70%, and the HBV transgenation easily takes place, and YMDD resistance aberration rate is up to 21%.How long the phase was used antiviral before the patient transplanted in addition, and antiviral opposing phenomenon appears in majority, has increased the difficulty of transplanting back virus infection recurrence rate and prevention.Therefore state adds with heavy dose of HBIG with the relevant after liver transplantation hepatitis B recurrence of prevention hepatitis B.After liver transplantation is not accepted to occur the HBsAb of different titers in the early stage body of postoperative among the receptor of HBIG treatment, and serum HBsAb fades away subsequently, and uses the higher titre of patients serum HBsAb energy long term maintenance of HBIG.High dosage unrestriction HBIG single therapy can stop 65%～80% patient recurrence, but HBIG costs an arm and a leg, and expense every year is up to about 1,300,000 yuans.Though it is good that domestic employing lamivudine and low dose of HBIG combined utilization stop the curative effect of transplanting back HBV infection and recurrence to show, confirms without the large sample clinical trial.And the immune pressure that prolonged application HBIG brings can cause HBV genovariation and the immunologic escape phenomenon occur, makes HBIG be used for the prevention that the relevant after liver transplantation HBV of hepatitis B infects again and is very limited.

In sum, be used to prevent and treat the active passive immunity that means that HBV infects mainly are limited to nucleotide analog inhibition, antiviral cell factor and neutrality antibody at present, lack that other infect the inhibition means of links to HBV virus.HBV surface L protein involved in the present invention goes the antigenicity derived peptide then can directly act on the initial infection link of HBV and surface of hepatocytes receptors bind, and directly blocking virus is to hepatocellular infection, and treatment and the prevention infected for HBV provide new means.

3.HBV virusology

1) general introduction

HBV belongs to the molten broken venereal disease poison of Hepadnaviridae acellular.Its genome is partially double stranded cyclic DNA, only is about 3.2kb, is the dna virus of known minimum.4 gene regions codings at least 8 kinds of albumen, i.e. L, the M of S district genes encoding, S surface antigen HBs of high superposed; The pre-C of C genes encoding, HBc, HBe; The HBx of the polysaccharase of P genes encoding and X gene coding.The infection of HBV generally and duplicating does not cause that the tangible pathologic of liver cell changes, and the virion of generation discharges in the secretion mode.The relevant particle that HBV produces mainly contains 3 kinds of forms, comprises that Dane particle, the abundance of diameter 42-47nm is higher than Dane particle 10,000-1, the filamentary fibers that 000,000 times 20nm spherical particle and a small amount of diameter 20nm are uneven in length.Wherein having only the Dane particle to be made of genomic dna, viral dna polymerase, nucleocapsid and tunicle, is the unique viral particle with infection activity of HBV.

2) HBV genotype, serotype and epidemiology thereof distribute

There are two kinds of different somatotype systems of serotype and genotype in HBV at present.Lebouvier in 1972 etc. are divided into adw with HBV, adr, four serotypes of ayw and ayr according to the common antigenic determinant a of HBV surface protein HBsAg and two couples of antigenic determinant d/y that repel mutually and w/r.Courouce Pauty in 1978 etc. are divided into antigenic determinant w w1～4 again, add newfound antigenic determinant q, HBV are further divided into ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4,9 serotypes (2) such as adrq+ and adrq-.Since 1988,, be the somatotype standard, HBV is divided into 8 genotype (A～H) (3,4) with full gene nucleotide heterology 〉=8% according to the result of the full genic system evolutionary analysis of HBV.Combination between each genotype also can produce 1 different recombinant chous such as A/D, B/C, Ba and Bj.Corresponding relation between HBV serotype and the genotype sees Table 1.

Though HBV infects in various degree popular arranged all over the world, between country and domestic interzone have very evident difference (table 1).Genotype HBV such as China A, B, C, D infect the existence that all is in various degree, northern area is based on the C genotype, increased gradually by north to southern B genotype infection rate, Shenzhen B genotype is suitable with C genotype infection rate ratio, and minority area D genotype such as Tibet have higher infection rate.Serotype HBV such as China adr, adw2, ayr, ayw1, ayw2 and ayw3 all have distribution, but are advantage serotype with adr and adw2.There is obvious corresponding relation in China B genotype and adw2 serotype, C genotype and adr serotype.

Table 1.HBV genotype and serology hypotype, HBV albumen, PC/BCP

The relation and the main popular region of sudden change

Genotype	The serology type	Genome length (nt)	HBVsAg Pre-S1 amino acid no	Main popular region
					A	adw2， ayw1	3221	119	West Europe, Northern Europe, North America, middle Africa, India
B	adw2， ayw1	3215	119	South East Asia, China, Japan
					C	ayr，adrq+ adrq-，adr adw2	3215	119	South East Asia, China, Japan, Australia, the U.S.
D	ayw2， ayw3 ayw4	3182	108	Mediterranean sea region, Russia, India, the U.S.
					E	ayw4	3212	118	Xi Feizhou
F	ayw4，adw2 adw4q-	3215	119	Central America, South America, Polynesia
					G	adw2	3248	108	Central America, the U.S., France, Germany
H	adw4	3215	119	Central America, South America, the U.S.

4. the surface L protein key amino acid sequence of mediation HBV infection and derived peptide thereof suppress the mechanism of virus infection

The HBV peplos contain greatly (L), in (M), little (S) three kinds of surface antigen proteins, these albumen are by the single open reading frame coding of the S district gene with 3 different translation initiation sites, i.e. L (Pre-S1+Pre S2+S), M (Pre S2+S) and S (S).6 amino acid of 801 alkali yl coding S of HBV S gene regions protein 26,55 amino acid that Pre-S2 district 165 alkali yl coding M albumen is other.The Pre-S1 district is 357 bases in A, B, C, F and H genotype, 119 other amino acid of coding L albumen.D, G genotype lack 33 and 3 bases respectively in this district, 108 and 118 amino acid of the L protein N terminal of encoding respectively.The expression amount difference great disparity of three kinds of surface proteins, and distribution mode is significantly different.S expressing quantity height is the major protein of HBs in the HBV infected patient circulation blood, and M and L albumen then content are very little, only account for whole HBs proteic 5～15% and 1～2% respectively.The envelope protein of 20nm spherical particle and filamentary fibers mainly is made up of the M albumen that S albumen and quantity do not wait, and contains L albumen hardly, and the albumen of Dane particle coating then is mainly L albumen.

The L albumen Pre-S1 district of HBV exists and liver cell specific receptors bonded key amino acid sequence, is positioned the 2nd to the 77th amino acids of Pre-S1, and the Pre-S2 district does not bring into play keying action to virus infection.The myristoylation posttranslational modification is carried out at the 2nd glycine in the synthetic back of L albumen; if this position is not being substituted glycine by the L-Ala of myristoylation; its L albumen then can not be translated the back and modify; corresponding mutated viruses strain has also promptly lost hepatocellular infection ability, and the proteic translation of visible L back myristoylation is modified the importance to virus infection.L albumen is present in the Dane particle surface specifically, and be present in 20nm particle and filamentary fibers hardly, so both can avoid the 20nm particle liver cell to be infected the competition of acceptor, can utilize the antibody of anti-HBV surface protein in the 20nm particle attractor again, be convenient to the virus sweep effect that infectious Dane particle is escaped humoral immunization.

According to the competition principle, to modify small peptide from the N-terminal myristoylation of L albumen 2-78 amino acids and can block the infection of HBV liver cell and Hepa RG hepatic cell line, this small peptide can further foreshorten to the proteic 2-48 amino acids of L.Also there is similar blocking effect in the polypeptide of duck hepatitis B virus DHBV surface L protein preS the 2nd to the 41st amino acids correspondence to the infection of DHBV.The similar small peptide in marmoset monkey hepatitis B virus WMHBV source also can infect performance intersection blocking effect to HBV.This of Pre-S1 section sequence has very high antigenicity, contains the aminoacid sequence with antibodies such as MA18/7,5a19.These antibody can in and HBV virus, blocking virus is to the infection of human liver cell, tree shrew (T.belangeri) liver cell and Hepa RG hepatic cell line.Its mechanism may be that antibody combines with this section sequence, has blocked L albumen and cell receptor bonded site, thereby has suppressed the infection of HBV pair cell.Clinical data also confirms, can produce anti-preS1 antibody in hepatitis b virus infected patient's body, and it occurs with the clinical prognosis of hepatitis B closely related.PreS1 antibody often comes across acute self limiting HBV and infects, and rarely in the chronic HBV infection patient, points out this antibody-like to play a significant role in removing HBV and control HBV infection chronicity process.

5. meaning of the present invention and Application Prospect

The chronically infected medicine of treatment HBV all acts on HBV time multiplexed cell system link at present, and HBV surface L protein polypeptide can directly suppress the infection of HBV pair cell, and the control of infecting for HBV provides new strategy.But such small peptide has the intensive antigenicity, exist and neutrality antibody bonded aminoacid sequence, simultaneously this section sequence be HBV must the site in conjunction with infecting acceptor, also be that the L protein polypeptide suppresses the bonded that the HBV pair cell infects acceptor and closes key sequence.Exist and this sequence bonded neutrality antibody in patient's body, when using such polypeptide the HBV infected patient treat, polypeptide will combine with antibody, make polypeptide lose effect with surface of hepatocytes HBV receptors bind, hinder the effectiveness that polypeptide suppresses the HBV infection.On the other hand, consumed intravital neutrality antibody, helped the infection of HBV on the contrary with combining of polypeptide.May produce other untoward reactions such as immune complex immunopathogenesis in addition.

The restraining effect that one aspect of the present invention provides the pandemic B genotype adw serotype in South East Asia (particularly China), C genotype adr serotype HBV surface L protein 13-59 aminoacid sequence polypeptide and D genotype ayw serotype HBV surface L protein 2-47 aminoacid sequence polypeptide that HBV is infected, the screening method of removing HBV surface L protein polypeptide antigen is provided on the other hand, but and by screening obtained HBV capable of blocking infect not with anti-HBV surface L protein antibodies go the antigen derived peptide.The present invention also provides this coordinate repression that goes antigenicity derived peptide and anti-HBV surface L protein antibody that HBV is infected.

Summary of the invention

First aspect, the present invention relates to a kind of polypeptide of HBV surface L protein, this polypeptide has general formula X-Y-Z, wherein, X represents the 1st to the 12nd aminoacid sequence of B genotype adw serotype and C genotype adr serotype HBV virus surface L protein, or the sequence that shortens of this sequence N-terminal certainly, or disappearance fully, wherein, the N-terminal of X can be modified; Y represents the 13rd to the 59th aminoacid sequence of B genotype adw serotype and C genotype adr serotype HBV virus surface L protein, and wherein, the N-terminal of Y and/or C-terminal can be modified; Z represents B genotype adw serotype and C genotype adr serotype HBV virus surface L protein the 60th to the 89th aminoacid sequence, or the terminal sequence that shortens of this sequence C certainly, or disappearance fully, and wherein, the C-terminal of Z can be modified.

In one embodiment, the 1st amino acids glycine of described Y sequence is modified by myristoylation.In further embodiments, described polypeptide is selected from SEQ ID NO:2-7, and wherein the 1st amino acids glycine of SEQ ID NO:3-7 is modified by myristoylation.

Second aspect the present invention relates to a kind of polypeptide of HBV surface L protein, and this polypeptide has general formula x-y-z, and wherein, x represents methionine(Met), and it can be modified, and perhaps x does not exist; Y represents the 2nd to the 32nd aminoacid sequence of D genotype ayr serotype HBV surface L protein, and wherein, the N-terminal of y and/or C-terminal can be modified; Z represents D genotype ayr serotype HBV surface L protein the 33rd to the 47th aminoacid sequence, or terminal sequence that shortens of this sequence C or disappearance certainly fully, and wherein, the C-terminal of z can be modified.

In one embodiment, the 1st amino acids glycine of this y sequence is modified by myristoylation.In another embodiment, the sequence of this polypeptide is the SEQ ID NO:9 that the 1st amino acids glycine is modified by myristoylation.

The third aspect the present invention relates to the screening method that a kind of albumen goes the antigenicity derived peptide, is used to remove antigenicity and keeps proteic particular community, and this method comprises:

(A) in viral protein to be screened, introduce stochastic sequence, set up the recombinant virus storehouse of this albumen specific region or whole albumen hats;

(B) waiting to screen under the condition of protein antibodies existence, screening still has the recombinant virus subgroup of infection ability, and obtains this proteic aminoacid sequence in this recombinant virus subgroup, promptly obtains this albumen and removes antigenicity candidate peptide sequence;

(C) by obtain go antigenicity candidate peptide sequence synthetic go accordingly antigenicity candidate peptide, screening can regulate the virus infection ability but not with antigenic polypeptide that goes of this viral protein antibodies, promptly obtain albumen and go the antigenicity derived peptide.

Fourth aspect, the invention still further relates to a kind of HBV surface L protein and go the antigenicity derived peptide, it goes the antigenicity derived peptide by the described screening albumen of third aspect present invention method is screened B genotype adw serotype of the present invention and C genotype adr serotype HBV surface L protein polypeptide and is obtained, and it has general formula

α-β

In the formula,

α represents the 1st to 13 amino acids sequence of HBV surface L protein, or the sequence that shortens of this sequence N-terminal or only be the 13rd amino acids glycine certainly, and wherein, described N-terminal and/or the 13rd amino acids glycine can be modified;

β represents to use the described screening method of third aspect present invention B genotype adw serotype and C genotype adr serotype HBV virus surface L protein the 14th to the 58th aminoacid sequence is screened the aminoacid sequence of acquisition, wherein, the N-terminal of the aminoacid sequence that is obtained can be modified and/or its C-terminal can be shortened or modify.

The invention still further relates to a kind of pharmaceutical composition, polypeptide or the described HBV surface L protein of fourth aspect that it contains first aspect present invention or the described HBV surface L protein of second aspect remove antigenicity derived peptide and pharmaceutically acceptable carrier.This pharmaceutical composition also can contain other antiviral substances, as anti-HBV antibody, cytokine etc.

The invention still further relates to the polypeptide of HBV surface L protein of the present invention or HBV surface L protein goes the antigenicity derived peptide to prevent and/or treat purposes in the medicament of hepatitis B in preparation.

Description of drawings

Fig. 1 shows the inhibition that B genotype adw serotype and C genotype adr serotype HBV surface L protein derived peptide Myr 13-59 infect the HBV liver cell.

Fig. 2 shows the inhibition that D genotype ayw serotype HBV surface L protein derived peptide Myr 2-47 infects the HBV liver cell.

Fig. 3 shows that the HBV surface L protein goes the combination rate of antigenicity candidate peptide and antibody.

Fig. 4 shows that the HBV surface L protein goes antigenicity candidate peptide to suppress HBV to hepatocellular infection.

Fig. 5 shows that anti-HBV surface L protein antibody is to the HBV surface L protein derived peptide and the influence of removing antigenicity derived peptide blocking-up HBV infection effect.

Fig. 6 shows HBV surface L protein derived peptide and goes the influence of antigenicity derived peptide antagonism HBV surface L protein antibody blocking HBV infection effect.

Embodiment

As mentioned above, first aspect present invention provides the derived peptide that derives from B genotype adw serotype and C genotype adr serotype HBV surface L protein, and this derived peptide can suppress HBV to hepatocellular infection.These derived peptide sequences can represent that X represents the 1st to 12 amino acids sequence of HBV surface L protein with general formula X-Y-Z here, or terminal sequence that shortens of this sequence of N or disappearance certainly fully, comprise the derived sequence that above sequence N-terminal is modified.Described " certainly this sequence of N terminal shorten " comprises from the 1st amino acids of this sequence N-terminal and begins to shorten, can shorten to only surplus this sequence the 12nd amino acids, be that X can be a position to the 12 amino acids sequences of described sequence, wherein a is the integer of 1-11, and perhaps X only is the 12nd amino acid.

Y represents the 13rd to the 59th amino acids sequence in B genotype adw serotype or the C genotype adr serotype HBV virus surface L protein sequence, and comprises the derived sequence that this sequence N-terminal and/or C-terminal are modified.

Z represents B genotype adw serotype or C genotype adr serotype HBV virus surface L protein the 60th to the 89th amino acids sequence, or sequence that shortens from C-terminal or disappearance fully, comprises the derived sequence that above sequence C is end modified.Described " shortening from C-terminal " comprises that the 1st amino acid (being the 89th amino acids of the present invention) from C-terminal begins to shorten, can shorten to only surplus this sequence the 60th amino acids, be that Z can be that the 60th of described sequence is to b amino acids sequence, wherein, b is the integer of 61-89, and perhaps Z only is the 60th amino acid.

The preferred derived peptide of the present invention comprises the X sequence of N-terminal shortening and/or the Z sequence that C-terminal shortens, and preferred derived peptide only contains the Y sequence.

Derived peptide provided by the invention, its sequence preference derive from B genotype adw serotype and C genotype adr serotype HBV virus surface L protein the 1st to the 89th aminoacid sequence.More preferably come from B genotype adw serotype and C genotype adr serotype HBV virus surface L protein the 13rd to 59 aminoacid sequence.

In a preferred version of the present invention, its sequence of derived peptide provided by the invention derives from SEQ ID NO:1 described B genotype adw serotype and C genotype adr serotype HBV virus surface L protein the 1st to the 89th aminoacid sequence.More preferably come from SEQ ID NO:1 the 13rd to 59 amino acids sequence (SEQ ID NO:2).

Above-described derived peptide preferably has the hydrophobic grouping of chemically modified, these hydrophobic groupings preferably have the propylene residues of the saturated or unsaturated fatty acids of 4 above carbon atoms, more preferably myristic acid, palmitinic acid, stearic acid, oleic acid, linolic acid or arachidonic acid.Hydrophobic grouping is also preferred cholesterol and similar group thereof in addition.

In a preferred version of the present invention, the N-terminal of polypeptide is modified through myristoylation.More preferably; HBV surface L protein derived peptide provided by the invention is the polypeptide Myr-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPKKDHWPEANQVG (Myr 13-59, SEQ ID NO:3) that SEQ ID NO:1 the 13rd to the 59th aminoacid sequence and the 13rd glycine are connected with the myristoyl group.In the another one preferred version, polypeptide provided by the invention has SEQ ID NO:4,5,6 and 7 sequences, and their the 1st amino acid glycine modified through myristoylation.

Second aspect present invention provides the one group of derived peptide that derives from D genotype ayw serotype HBV surface L protein, and this group derived peptide can suppress HBV to hepatocellular infection.These derived peptide sequences can represent with general formula x-y-z, and x represents in the HBV surface L protein sequence the 1st amino acids M or disappearance fully here, comprises the derived sequence that above sequence N-terminal is modified.Y represents the 2nd to the 32nd amino acids sequence in the D genotype ayw serotype HBV surface L protein sequence, and the derived sequence of this sequence N-terminal and/or C-terminal modification.Z represents D genotype ayw serotype HBV surface L protein the 33rd to the 47th aminoacid sequence, or sequence that shortens from C-terminal or disappearance fully, comprises the derived sequence that above sequence C is end modified.Described " shortening from C-terminal " comprises that the 1st amino acid (being the 47th amino acids of the present invention) from C-terminal begins to shorten, can shorten to only surplus this sequence the 33rd amino acids, promptly, z can be that the 33rd of described sequence is to c amino acids sequence, wherein, c represents the integer of 34-47, and perhaps z only is described the 33rd amino acids.

The present invention comprises and derives from the corresponding sequence of other genotype serotype HBV surface L protein, meets the HBV surface L protein derived peptide of above-mentioned definition.

The preferred derived peptide of the present invention comprises the x sequence of disappearance fully, and the z sequence that preferred derived peptide contains is not influencing the condition that polypeptide suppresses the HBV infection activity, and its C-terminal shortens as far as possible.

Derived peptide sequence preference provided by the invention derives from D genotype ayw serotype HBV surface L protein the 2nd to 47 aminoacid sequence.

In a preferred version of the present invention, derived peptide sequence preference provided by the invention derives from the described D genotype of SEQ ID NO:8 ayw serotype HBV surface L protein the 2nd to 47 aminoacid sequence.

Above-described derived peptide preferably has the hydrophobic grouping of chemically modified, these hydrophobic groupings preferably have the propylene residues of the saturated or unsaturated fatty acids of 4 above carbon atoms, more preferably myristic acid, palmitinic acid, stearic acid, oleic acid, linolic acid or arachidonic acid.Hydrophobic grouping is also preferred cholesterol and similar group thereof in addition.

In a preferred version of the present invention, the N-terminal of polypeptide is modified through myristoylation.More preferably; HBV surface L protein derived peptide provided by the invention is the polypeptide Myr-GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKV (Myr 2-47, SEQ ID NO:9) that SEQ ID NO:8 the 2nd to the 47th aminoacid sequence and the 1st glycine are connected with the myristoyl group.

The present invention includes other genotype/serotype HBV that meet the present invention definition and the derived peptide in non-human HBV source, comprise the restraining effect that above derived peptide infects B genotype adw serotype and C genotype adr serotype HBV especially.

The screening method that third aspect present invention provides a kind of albumen to go the antigenicity derived peptide is used to remove antigenicity and keeps proteic particular community.This method is made of the A-B-C process.Here the A procedural representation is introduced stochastic sequence in viral protein to be screened, and sets up the recombinant virus storehouse of this albumen specific region or whole albumen hats.The B procedural representation is being waited to screen under the condition of protein antibodies existence, and screening still has the recombinant virus subgroup of infection ability, and obtains this proteic aminoacid sequence in this recombinant virus subgroup, promptly obtains this albumen and removes antigenicity candidate peptide sequence.The C procedural representation by obtain go antigenicity candidate peptide sequence synthetic go accordingly antigenicity candidate peptide, screening can regulate the virus infection ability but not with antigenic polypeptide that goes of this viral protein antibodies, promptly obtain albumen and go the antigenicity derived peptide.

The screening object of antigenicity derived peptide screening method that goes of the present invention comprises any type of albumen and polypeptide, and the source of its albumen and polypeptide comprises people, animal, plant, bacterium, virus etc. in interior all biologies and microorganism.In a preferred version of the present invention, using screening method of the present invention screens the antigenicity derived peptide of going of HBV, people's acquired immunodeficiency disease poison, influenza A virus and Paramyxo virus viral proteins such as (paramyxoviruses), further preferably the antigenicity derived peptide of going of the surface L protein of HBV is screened, more preferably HBV surface L protein Pre-S1 district is gone the screening of antigenicity derived peptide.

In a preferred version of the present invention, the antigenicity derived peptide of going of B genotype adw serotype and C genotype adr serotype HBV virus surface L protein the 14th to 58 aminoacid sequence is screened.More preferably the antigenicity derived peptide of going of B genotype adw serotype as described in SEQID NO:1 and C genotype adr serotype HBV virus surface L protein the 14th to 58 aminoacid sequence TNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPKKDHWPEANQV (SEQ ID NO:10) is screened.

Albumen of the present invention goes in the antigenicity derived peptide screening method, the A process is included in biomolecules levels such as gene picodna base level, messenger RNA(mRNA) nucleic acid base level, Argine Monohydrochloride residue level, polysaccharide sequence level and introduces stochastic sequence, and comprises the method that can introduce stochastic sequence in above each level.In a preferred version of the present invention, the present invention introduces stochastic sequence in virogene picodna base level, is further introducing stochastic sequence by gene recombination technology in the preferred version.

In a preferred version of the present invention, utilize gene recombination technology to introduce stochastic sequence (NNS) 19 in B genotype adw serotype and the pairing gene order of C genotype adr serotype HBV virus surface L protein the 28th to 46 aminoacid sequence, here N represents A, T, G, C base at random arbitrarily, and S represents G, C base at random arbitrarily.More preferably utilize gene recombination technology to introduce stochastic sequence (NNS) 19 in SEQ ID NO:1 described B genotype adw serotype and the pairing gene order of C genotype adr serotype HBV virus surface L protein the 28th to 46 aminoacid sequence HQLDPAFGANSNNPDWDFN (SEQ ID NO:11) (SEQ ID NO:12), here N represents A, T, G, C base at random arbitrarily, and S represents G, C base at random arbitrarily.

Albumen of the present invention goes in the B process of antigenicity derived peptide screening method, and the acquisition of this Argine Monohydrochloride sequence of recombinant virus subgroup comprises any order-checking means such as gene DNA order-checking, gal4 amino acid order-checking.In preferred version of the present invention, adopt the gene DNA order-checking to obtain recombinant virus subgroup protein sequence, promptly obtain viral protein and remove antigenicity candidate peptide sequence.In a preferred version of the present invention, what obtain four B genotype adw serotypes and C genotype adr serotype HBV surface L protein the 13rd to 58 aminoacid sequence (SEQ ID NO:10) removes antigenicity candidate peptide sequence (SEQ ID NO:15,16,17 and 18, its 1st amino acid glycine modified through myristoyl).

Albumen of the present invention goes in the antigenicity derived peptide screening method, and the C process comprises the producing method of any polypeptide such as chemiluminescent polypeptide is synthetic, dna recombinant expression.In preferred version of the present invention, adopt chemical synthesis production to remove antigenicity candidate peptide.In a preferred version of the present invention, screening obtains the HBV surface L protein and goes antigenicity derived peptide (SEQID NO:17).

Fourth aspect present invention provides and can suppress that HBV infects but do not go the antigenicity derived peptide with the HBV surface L protein of surface L protein antibodies, and provides this to go antigenicity derived peptide and HBV surface L protein antibody to suppress the synergistic effect of HBV liver cell infection.

These go antigenicity derived peptide sequence to derive to use The selection result of going B genotype adw serotype that antigenicity derived peptide screening method defines to the first aspect of the present invention and C genotype adr serotype HBV virus surface L protein derived peptide provided by the invention and with the combination of original B genotype adw serotype and C genotype adr serotype HBV virus surface L protein aminoacid sequence.

B genotype adw serotype provided by the invention and C genotype adr serotype HBV virus surface L protein go the antigenicity derived peptide to represent with the general formula alpha-beta, here α represents the 1st to 13 amino acids sequence of HBV virus surface L protein, or the sequence that shortens of this sequence N-terminal or only be the 13rd amino acids glycine certainly, comprise the derived sequence that above sequence N-terminal is modified, wherein, the definition of described " sequence of this sequence N-terminal shortening certainly " is with mentioned above identical.β represents to use the aminoacid sequence that goes antigenicity derived peptide screening method to B genotype adw serotype and C genotype adr serotype HBV virus surface L protein the 14th to the 58th aminoacid sequence screening acquisition provided by the invention, and comprises the derived sequence of this sequence N-terminal modification and/or the shortening sequence or the modification derived sequence of C-terminal.

Going in the antigenicity derived peptide that a preferred version of the present invention provides, α is the 1st to 13 an amino acids sequence in the SEQ ID NO:1 sequence, or the terminal sequence that shortens of this sequence of N or only be the 13rd amino acids glycine certainly, comprise the derived sequence that above sequence N-terminal is modified.β represents to use the aminoacid sequence that goes antigenicity derived peptide screening method to B genotype adw serotype and C genotype adr serotype HBV virus surface L protein (SEQ IDNO:1) the 14th to the 58th aminoacid sequence TNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPKKDHWPEANQV (SEQ ID NO:10) screening acquisition provided by the invention, and comprises the derived sequence of this sequence N-terminal modification and/or the shortening sequence or the modification derived sequence of C-terminal.

The present invention comprises through reducing or remove the viral protein derived sequence of antigenicity transformation, comprise HBV viral protein derived sequence especially, more particularly comprise through reducing or remove the derived peptide sequence of the HBV surface L protein of antigenicity transformation through reduction or the transformation of removal antigenicity.Here " transformation " is meant to utilize and comprises that provided by the invention to go any of antigenicity derived peptide screening method, amino acid deletion, amino acid replacement etc. be the technological method of purpose with reduction derived peptide antigenicity.

Above-described derived peptide preferably has the hydrophobic grouping of chemically modified, these hydrophobic groupings preferably refer to have the propylene residues of the saturated or unsaturated fatty acids of 4 above carbon atoms, more preferably myristic acid, palmitinic acid, stearic acid, oleic acid, linolic acid or arachidonic acid.Hydrophobic grouping is also preferred cholesterol and similar group thereof in addition.

In a preferred version of the present invention, the N-terminal of polypeptide is modified through myristoylation.More preferably, to go the antigenicity derived peptide be the polypeptide that the 1st glycine is connected with the myristoyl group to HBV surface L protein provided by the invention:

Myr-GTNLSVPNPLGFFPDCSLDWVWSWAPYFTPQWRTPKKDHWPEANQV(SEQ ID NO：17)。

Any homologue of the related polypeptide of the application is the application's a part, comprise through one or more amino acid mutations, as comprise 1-10 amino acid, a preferable 1-8 amino acid, a better 1-5 amino acid, a better 1-3 amino acid whose deletion, guard/non-conserved amino acid replacement and the peptide sequence of acquisition.The peptide modified product that further comprises the proteolysis resistant that obtains by one or more (as 1-10,1-8,1-5 etc.) L amino acid-D amino acid replacement etc.The application comprises that also the antigenicity to reduce the application's polypeptide is the modification and the transformation of purpose.Here " homologue " comprises that also relating to polypeptide with the application has greater than the polypeptide of 30% (as 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or bigger) homology or contain glycine that myristoyl modifies and the binding sequence of anti-Pre-S1 antibody (or the anti-Pre-S1 antibodies sequence that obtains through the past antigen selection corresponding go the antigenicity sequence).Here " homologue " also comprises B genotype adw serotype and C genotype adr serotype HBV surface L protein related polypeptide, and these polypeptide can be lower than the liver cell HBV infection inhibiting rate that produces under the peptide concentration of 100mM greater than 50%.

The application also comprises and utilizes contain the mixture that the application provide sequences polypeptide of genetic engineering technique by cell expressing.The related SEQ ID NO:3-7 of the application, 9 and first amino acid glycine of 15-18 all modify through myristoylation.

The application provides the purposes of polypeptide to comprise that the inside and outside sticks or internalization suppresses HBV and infects hepatocellular by blocking-up HBV particle.The application comprises that also the polypeptide that will provide is as blocking or prevent medicine that HBV infects and the pharmaceutical preparation of forming with suitable pharmaceutical carrier.The application also comprises the vaccine composition that contains described polypeptide.

The application is particularly including the blocking effect of the polypeptide that is provided to B genotype adw serotype and the infection of C genotype adr serotype HBV liver cell, the application also comprises the polypeptide of deriving in other genotype/serotype HBV and non-human HBV source, particularly the restraining effect of polypeptide to B genotype adw serotype and C genotype adr serotype HBV infection of deriving in Pre-S1 source.

The measure of infecting as treatment or prevention HBV with the polypeptide that provided also is provided the application, and relates to the application's polypeptide in the needed any prevention of interior patient, treatment measure.The application comprises especially and suppresses prevention and the treatment measure that HBV infects in the application's polypeptide body, comprising stoping HBV to propagate in the cell of organism uninfection.The preventive measures here are meant the possibility that reduces or avoid patient infection HBV, and the treatment measure is meant all measures of improvement or stable status of patient.The patient of indication is any people who is infected, may be infected by HBV and may soon be infected by HBV by HBV.

The effect that the application provides the HBV surface L protein to remove the collaborative anti HBV infecting of antigenicity derived peptide and other anti-virus infection measures, these antiviral measures comprise that the HBV vaccine immunity (comprises any actives relevant with HBV such as preventative vaccine and therapeutic vaccine, the passive immunization measure), anti-HBV antibody (comprises any proteic monoclonal antibody of anti-HBV, polyclonal antibody, any composition with HBV virus bonded antibody and antibody such as HBV immunoglobulin (Ig)), cytokine (comprises interferon-alpha, all intervene cytokine and the composition thereof of HBV interleukin-etc.), nucleoside analog (lamivudine, Adefovir, the compound of any inhibition virus replication such as Entecavir and Telbivudine) and other all intervene HBV and infect, duplicate, the medicine and the measure of any HBV processes such as release.In the application's a preferred version, the HBV surface L protein removes antigenicity derived peptide and the collaborative virus infection that suppresses of anti-HBV surface L protein antibody.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: the described condition of laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989), or the condition of advising according to manufacturer.

Embodiment 1: come from the inhibition that B genotype adw serotype and C genotype adr serotype HBV virus surface L protein derived peptide infect the HBV liver cell

1, the preparation of B genotype adw serotype and C genotype adr serotype HBV virus surface L protein derived peptide

(SEQ ID NO:3 the 1st amino acids glycine is modified through myristoyl for B genotype adw serotype that the N-terminal myristoyl is modified and C genotype adr serotype HBV virus surface L protein derived peptide, be expressed as Myr 13-59) press standard Fmoc scheme with AB 431A type Peptide synthesizer, with 0.25mM HMP is initial resin, and the residue extension is synthetic one by one to aminoterminal from carboxyl terminal.For strengthening the stability of polypeptide, the further amidation of the C-terminal of polypeptide.Behind the peptide end of synthesis, through the cutting of cutting liquid, G6 glass sand hourglass filtering resin, the filtrate vacuum is drained.Deionized water dissolving polypeptide cleaved products,  KTA explorer 100 type medium pressure liguid chromatograph C18 column purification, substep is collected main peak.Target peak is collected sample and is identified with the anti-phase high pressure liquid chromatography Symmetry C18 of Delta 600 types analytical column purity, API2000 LC/MS/MS type mass spectrograph molecular weight identification.The collection liquid freeze-drying of medium pressure liquid chromatography purifying gained is dissolved in 100 μ M DMSO and forms the polypeptide storage liquid, 0.20 μ M filtration sterilization, and-80 ℃ are frozen.

2, the mensuration of B genotype adw serotype and C genotype adr serotype HBV genotype serotype

Gather chronic HBV infection patients serum-70 ℃ preservation.Take out 50 μ l serum and add 310 μ l Proteinase K lysates [1mg/ml Proteinase K, 50mmol/LTris-HCl (pH8.0), 200mmol/L NaCl, 10mmol/LEDTA, 2%SDS, 1 μ g/ml poly (A)].60 ℃ of cracking phenol/chloroform extractings after 1 hour, ethanol sedimentation, precipitation is dissolved in and is the HBV dna profiling in the deionized water.PCR reaction system 50 μ l contain HBV dna profiling 5 μ l, 1 * PCR Buffer, MgCl ₂1.5mmol/L, upstream primer (SEQ ID NO:19,5 ' AGTCTAGACTCGTGGTGGAC-3 ', position in the HBV genome is 247-266,25pmol/L, downstream primer (671-690 down together), 5 '-T (G/T) GCACTAGTAAACTGAGCC-3 ', SEQ ID NO:20) 25pmol/L, 200 μ Mol/L dNTPs, 1.25U polysaccharase TaqDNA.Amplification condition: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 45s, 35 circulations, last 72 ℃ were extended 7 minutes.Pcr amplification product 443bp, directly order-checking obtains its sequence.Aminoacid sequence according to the conversion of HBsAg dna sequence dna, by 122,127,134,159 and 160 amino acids are formed the antigenic determinant sequence signature of determining HBsAg, and then the serotype of definite HBV (Yotsumoto S, Okamoto H, Tsuda F, Miyakawa Y, Mayumi M, " Subtyping hepatitis B virus DNA in free orintegrated forms by amplification of the S-gene sequences by the polymerase chain reactionand single-track sequencing for adenine ", J Virol Methods, May nineteen ninety, 28 (2): 107-16; Norder H, Courouce AM, Magnius LO, " Molecular basis of hepatitis B virus serotypevariations within the four major subtypes ", J Gen Virol., in December, 1992,73 (Pt 12): 3141-5), determine HBV genotype (Norder H, Courouce AM, Magnius LO according to HBV S gene regions type specificity Nucleotide feature, " Molecular basis of hepatitis B virus serotype variations within the fourmajor subtypes ", J Gen Virol., in December, 1992,73 (Pt 12): 3141-5; Norder H, Hammas B.J Gen Virol, 1993,74:1341-1348; Kidd-Ljunggren K, Courouce AM, Oberg M, Kidd AH; " Genetic conservation within subtypes in the hepatitis B virus pre-S2 region ", J GenVirol., in June, 1994,75 (Pt 6): 1485-90, Erratum is at J Gen Virol, March nineteen ninety-five, 76 (Pt3): 727).

3, the cultivation of human primary hepatocyte

The adjacent healthy tissues of liver cancer patient surgical operation tumor resection thing that non-HBV is relevant, separate (Guguen-Guillouzo by the described method of document, C., and A.Guillouzo, 1986.Methods for preparation of adultand fetal hepatocytes, the 1-12 page or leaf, .Les E ' ditions INSERM Paris in " Isolated and cultured hepatocytes " that A.Guillouzo and C.Guguen-Guillouzo edit, John Libbey andCo., Ltd., the London), be incubated at and added 3.5 * 106M hydrocortisone monomester succinate (hydrocortisone hemisuccinate), the inferior sulfoxide of 2% dimethyl, in the H substratum of 5% human serum and 5% foetal calf serum.

4, the cultivation of B genotype adw serotype and C genotype adr serotype HBV virus.

The HBV serum virus 2ml that is defined as B genotype adw serotype and C genotype adr serotype is added the human primary hepatocyte of cultivating 3 days, hatch for 37 ℃ and infected 24 hours.Remove infection serum after infection is finished, washed cell 3 times replenished the fresh culture cultured continuously 10 days.Collect culture supernatant, 6% polyoxyethylene glycol (PEG8000) centrifugation virion, precipitation is resuspended in the phosphate buffered saline buffer that contains 25% foetal calf serum, and is frozen in-80 ℃.

5, HBV infects the detection of back liver cell culture supernatant HBsAg

HBsAg in the culture supernatant detects by sandwich sandwich assay (in-house sandwich) enzyme immunoassay (ELISA).37 ℃ of bags of 1ug/ml anti-hbs monoclonal antibodies were by 96 orifice plates 2 hours.Behind the thorough washing (0.1%Tween 20 PBS washing 3 times, PBS washing 2 times), 37 ℃ of sealings of 10% foetal calf serum 1 hour.Remove confining liquid, the liver cell culture supernatant 100 μ l that add the HBV infection were hatched 12 hours for 4 ℃.Remove culture supernatant, thorough washing adds peroxidase link coupled anti-HBsAg antibody and hatched 1 hour for 37 ℃.Remove unnecessary antibody, add phenylenediamine-H ₂O ₂Reaction substrate room temperature reaction 15 minutes, 2N H ₂SO ₄Stopped reaction, the OD492 of assaying reaction product calculates and infects HBsAg content in the supernatant.

6, HBV surface L protein derived peptide (Myr 13-59, SEQ ID NO:3) the inhibition human primary hepatocyte that the HBV liver cell is infected was cultivated 3 days in 12 well culture plates, about 106 cells in every hole and L protein derived peptide Myr13-59 pre-treatment 30 minutes added B genotype adw serotype or C genotype adr serotype HBV virus infection 24 hours.Remove the infection supernatant, washed cell 3 times.Continue to cultivate and detect HBsAg content in the liver cell culture supernatant after 10 days.As shown in Figure 1, be contrast not infected by the hepatocellular HBV of Myr 13-59 pre-treatment, HBV surface L protein derived peptide Myr 13-59 suppresses HBV to hepatocellular infection in the dose-dependently mode.5,10,20,40nM can suppress respectively HBV to liver cell infect 28%, 74%, 87% and 95%, can block HBV fully to hepatocellular infection greater than the Myr 13-59 of 80nM.Its 50% infection inhibition concentration (IC ₅₀) be 7.63nM.

Embodiment two: come from the inhibition that D genotype ayw serotype HBV virus surface L protein derived peptide infects the HBV liver cell

1, the cultivation of the synthetic and human primary hepatocyte of the D genotype ayw serotype HBV surface L protein derived peptide Myr 2-47 (SEQ ID NO:9) of N-terminal myristoyl modification is with embodiment one.

2, the cultivation of D genotype ayw serotype HBV virus

Can secrete the 2.2.15 clone cultured continuously 10 days of complete D genotype ayw serotype HBV infecting virus particle.Collect culture supernatant, 6% polyoxyethylene glycol (PEG8000) centrifugation virion, precipitation is resuspended in the phosphate buffered saline buffer that contains 25% foetal calf serum, and is frozen in-80 ℃.

3, HBV infects the detection (with embodiment one) of back liver cell culture supernatant HBsAg

4, HBV surface L protein derived peptide Myr 2-47 is to the inhibition of HBV liver cell infection

Human primary hepatocyte was cultivated 3 days in 12 well culture plates, and about 106 cells in every hole and L protein derived peptide Myr2-47 pre-treatment 30 minutes added D genotype ayw serotype HBV virus infection 24 hours.Remove the infection supernatant, washed cell 3 times.Continue to cultivate and detect HBsAg content in the liver cell culture supernatant after 10 days.As shown in Figure 2, be contrast not infected by the hepatocellular HBV of Myr 2-47 pre-treatment, HBV surface L protein derived peptide Myr2-47 suppresses HBV to hepatocellular infection in the dose-dependently mode.5,10,20,40nM can suppress HBV respectively to hepatocellular infection 23%, 69%, 82% and 97%, can block HBV fully to hepatocellular infection greater than the Myr 2-47 of 80nM.Its IC50 is 8.33nM.

Embodiment three: the HBV surface L protein goes the screening of antigenicity derived peptide

1, the cultivation (with example one) of B genotype adw serotype and C genotype adr serotype HBV virogene type serotype mensuration, virus culture, human primary hepatocyte.

2, carry the complete full genome of HBV and possess the construction of recombinant plasmid of HBV complete copy ability.

50 μ l HBV virus culture suspensions add 310 μ l Proteinase K lysates [1mg/ml Proteinase K, 50mmol/L Tris-HCl (pH8.0), 200mmol/L NaCl, 10mmol/L EDTA, 2%SDS, 1 μ g/mlpoly (A)].60 ℃ of cracking phenol/chloroform extractings after 1 hour, ethanol sedimentation, precipitation is dissolved in H ₂It among the O HBV dna solution.With upstream primer (Pst I) 5 ' ctgactgcagCACTGGATGGGGCTTGGCTATTGG (SEQ IDNO:21,1202-1225) and downstream primer (EcoR I) 5 ' ttatggaattcCGACGCGGCGATTGAGACCTTC (2201-2180, SEQ ID NO:22) amplification contain the C genotype adr serotype HBV genome (1202-2201nt) of complete EN II reproduction element.This amplification segment support virus is finished and is duplicated, and contains the HBV gene order (3996nt) greater than the single-gene group.PCR reaction system 50 μ l contain HBV dna profiling 5 μ l, 1 * PCR Buffer, MgCl ₂1.5mmol/L, upstream and downstream primer 2 5pmol/L, 200 μ Mol/L dNTPs, high-fidelity TaqDNA polysaccharase 1.25U.Amplification condition: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s, 35 circulations, last 72 ℃ were extended 10 minutes.Pcr amplification product 4017bp.The HBV dna fragmentation of amplification is cloned into the Pst I and the EcoR I restriction enzyme site of pUC18 plasmid vector, forms the pUC-HBV recombinant plasmid.Can produce virion behind this plasmid transfection cell with infection ability.

3, the foundation in the reorganization HBV storehouse of surface L protein Pre-S1 district hat.

Chemical synthesising DNA fragment (SEQ ID NO:13 and 14), corresponding to fragment between HBV DNA genome Bse59I and the Bsu36 I restriction enzyme site, wherein N represents any base among A, T, G, the C, and S represents any base among G, the C.Two synthetic complementary DNA fragment annealing rear clones are gone between pUC-HBV restriction enzyme site Bse59I and the Bsu36I, thereby in the corresponding coding region of HBV surface L protein Pre-S1 domain antibodies binding amino acid sequence HQLDPAFGANSNNPDWDFN, introduce stochastic sequence.The recombinant plasmid transfection Hep G2 cell that will have the hat district again, cultured continuously 10 days.The virion that recombinant plasmid forms in Hep G2 cell is secreted in the culture supernatant, has promptly formed the reorganization HBV storehouse of the corresponding sequence hat of surface L protein (SEQ ID NO:1) the 28th to 46 amino acid HQLDPAFGANSNNPDWDFN.Wherein DPAF and NSNNPDWDFN are respectively the binding sequences of anti-HBV surface L protein neutralizing monoclonal antibody MA18/7 and 5a19.

4, under the HBV surface L protein antibody existence condition, has the screening of the reorganization HBV subgroup of liver cell infection ability.

The human primary hepatocyte of cultivating 3 days adds 10 μ g/ml anti-HBV monoclonal antibody MA18/7 and 5a19, hatches 30 minutes.The above-mentioned Hep G2 culture supernatant that contains hat sequence reorganization HBV storehouse is added in the hepatocyte culture medium, infected 24 hours.Remove the infection supernatant, add MA18/7 and 5a19 antibody and continue to cultivate 10 days.Culture supernatant is transferred to the human primary hepatocyte of new cultivation, continues screening and culturing 2 and take turns in the presence of MA18/7 and 5a19 antibody, still has the reorganization HBV subgroup of liver cell infection ability with screening in the presence of anti-HBV surface L protein antibody.

5, reorganization HBV surface L protein is distinguished encoding sequence at random, promptly goes the mensuration of antigenicity candidate peptide sequence.

50 μ l take turns the culture supernatant that screening contains reorganization HBV subgroup through 3, add 310 μ l Proteinase K lysates [1mg/ml Proteinase K, 50mmol/LTris-HCl (pH8.0), 200mmol/L NaCl, 10mmol/LEDTA, 2%SDS, 1 μ g/ml poly (A)].60 ℃ of cracking phenol/chloroform extractings after 1 hour, ethanol sedimentation, precipitation is dissolved in H ₂It among the O HBV dna solution.With upstream primer (Pst I) 5 ' ctgactgcagTTTCCGCACTCATTTACAAGA (1539-1561, SEQ ID NO:23) and downstream primer (EcoR I) 5 ' ttatggaattcATGCTCCCGCTC CTACCTGATTT (2032-2010, SEQ ID NO:24) amplification HBV surface L protein Pre-S1 encode fragment (494bp).PCR reaction system 50 μ l contain HBV dna profiling 5 μ l, 1 * PCRBuffer, MgCl ₂1.5mmol/L, upstream and downstream primer 2 5pmol/L, 200 μ Mol/L dNTPs, high-fidelity TaqDNA polysaccharase 1.25U.Amplification condition: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 45s, 35 circulations, last 72 ℃ were extended 7 minutes.Pcr amplification product 515bp.The HBV dna fragmentation of amplification is cloned between the Pst I and EcoR I restriction enzyme site of pUC 18 plasmid vectors, the transformed competence colibacillus intestinal bacteria, the penicillin selection condition is cultivated down, picking list bacterium colony, the recombinant plasmid that extracting is entrained, to the insertion sequence order-checking, obtain under anti-HBV surface L protein antibody existence condition, still to have the reorganization HBV surface L protein gene coded sequence of liver cell infection ability.

6, the HBV virus surface L protein goes the preparation of antigenicity screening peptide

According to above reorganization HBV surface L protein gene coded sequence, obtain corresponding amino acid sequence through translation, intercept its 12nd to 58 aminoacid sequence for going antigenicity candidate peptide ammino acid sequence, this example obtains 4 HBV surface L proteins through screening and removes antigenicity candidate peptide-coding sequence.With candidate's peptide (SEQ ID NO:15,16,17 and 18) that AB 431A type Peptide synthesizer is modified by the synthetic N-terminal myristoyl of standard Fmoc scheme, concrete grammar is with embodiment one.

7, the HBV virus surface L protein goes the antigenic detection of antigenicity candidate peptide

The HBV surface L protein that is dissolved in 0.1M carbonic acid buffer (pH9.6), concentration and is 10 μ g/ml goes antigenicity candidate peptide room temperature bag to be spent the night by Immulon II elisa plate.Phosphate buffered saline buffer (PBS) rinsing of elisa plate through containing 0.05% Tween 20 3 times, 37 ℃ of sealings of 10% foetal calf serum 1 hour.Elisa plate PBS rinsing 2 times adds monoclonal antibody MA 18/7 or 4 ℃ of overnight incubation of 5a19.The cleaning of enzyme yoke plate adds peroxidase link coupled sheep anti mouse two and resists, and hatches 1 hour for 37 ℃.Rinsing unnecessary two is anti-, adds and contains phenylenediamine and H ₂O ₂Citric acid phosphoric acid damping fluid (pH5.0), room temperature reaction 5 minutes.2N H ₂The SO4 stopped reaction reads reaction solution OD492 value, calculates the combination rate that L albumen removes antigenicity candidate peptide and anti-L protein antibodies.As shown in Figure 3, the HBV surface L protein goes antigenicity candidate peptide SEQ ID NO:15 and 18 separately with 5a19 and M18/7 57%, 34% combine to be arranged respectively, and antigenicity candidate peptide SEQ ID NO:16 and 17 and two kind of equal debond of antibody.Therefore further detect the ability of these two candidate's peptide blocking-up HBV infected liver cells.

8, the HBV virus surface L protein goes the retarding effect that antigenicity candidate peptide infects HBV

Human primary hepatocyte was cultivated 3 days in 12 well culture plates, about 106 cells in every hole and HBV virus surface L protein went antigenicity candidate peptide SEQ ID NO:16 or SEQ ID NO:17 pre-treatment 30 minutes, added B genotype adw serotype or C genotype adr serotype HBV virus infection 24 hours.Remove the infection supernatant, washed cell 3 times continues to cultivate the concentration (measuring method is with embodiment one) of measuring HBsAg in the culture supernatant after 10 days.Shown in Fig. 4 A, B, the HBV surface L protein goes antigenicity candidate peptide SEQ ID NO:16 and SEQ ID NO:17 all to suppress the infection of two kinds of gene serotype HBV to primary hepatocyte in the dose-dependently mode.Two L albumen go antigenicity candidate peptide all can block HBV fully to hepatocellular infection when 160nM, and wherein HBV surface L protein candidate peptide SEQ ID NO:16 is respectively 12.06nM and 10.79nM to the IC50 of B genotype adw serotype and C genotype adr serotype HBV virus.And HBV surface L protein candidate peptide SEQ ID NO:17 has the activity of higher inhibition HBV, IC50 to B genotype adw serotype and C genotype adr serotype HBV virus is respectively 8.34nM and 7.89nM, and therefore obtaining SEQ ID NO:17 goes the antigenicity derived peptide for the HBV surface L protein.

Embodiment four: the HBV surface L protein goes antigenicity derived peptide and the collaborative HBV of inhibition of antibody to hepatocellular infection

1, the HBV surface L protein goes the antigenicity derived peptide that HBV is infected retarding effect and is not subjected to anti-HBV surface L protein antibody interferes with.

Human liver cell was cultivated 3 days in 12 well culture plates, every hole about 10 ⁶Cell, add anti-HBV surface L protein Pre-S1 monoclonal antibody M18/7 and 5a19 at 1: 1, after hatching 30 minutes, adding HBV surface L protein removes the HBV surface L protein derived peptide Myr 13-59 (SEQ IDNO:3) of antigenicity derived peptide SEQ ID NO:17 or natural sequence, hatched 30 minutes, remove supernatant, washed cell 3 times added the HBV virus infection 10 hours.Remove the infection supernatant, washed cell 3 times continues to cultivate the concentration (with embodiment one) of measuring HBsAg in the culture supernatant after 10 days.Shown in Fig. 5 A, HBV surface L protein derived peptide Myr 13-59 (SEQ IDNO:3) with natural sequence is under the antibody existence condition, the ability that its blocking-up HBV infects obviously descends, and there is dependence in the concentration of decline degree and antibody, the antibody of 2 μ g/ml makes the viral barrier effect of 80nM Myr 13-59 descend 50%, the antibody of 8 μ g/ml is then blocked the virus infection blocking effect of 160nM HBV surface L protein derived peptide Myr 13-59 fully, shows that HBV surface L protein derived peptide infects the interference that retarding effect obviously is subjected to anti-HBV surface L protein antibody to HBV.And the HBV surface L protein goes antigenicity derived peptide SEQ ID NO:17 under the condition that 0,2,4,8 μ g/ml antibody are pre-existing in, still suppress the infection (Fig. 5 B) of HBV in the dose-dependently mode to primary hepatocyte, it suppresses curve does not have significant difference, IC50 is close under each condition, and visible HBV surface L protein goes the antigenicity derived peptide to eliminate the interference of anti-HBV surface L protein Pre-S1 antibody to the virus infection blocking effect.

2, the HBV surface L protein goes the effect that antigenicity derived peptide and the collaborative blocking-up of HBV surface L protein antibody performance HBV infect.

Human primary hepatocyte was cultivated 3 days in 12 well culture plates, about 106 cells in every hole, add anti-HBV surface L protein Pre-S1 monoclonal antibody M18/7 and 5a19 at 1: 1, after hatching 30 minutes, adding HBV surface L protein removes the HBV surface L protein derived peptide Myr13-59 (SEQ ID NO:3) of antigenicity derived peptide SEQ ID NO:17 or natural sequence, hatched 30 minutes, added the HBV virus infection 24 hours.Remove culture supernatant, washed cell 3 times continues to cultivate the concentration (with embodiment one) of measuring HBsAg in the culture supernatant after 10 days.As shown in Figure 6A, do not having under the Myr 13-59 derived peptide condition, anti-HBV surface L protein Pre-S1 antibody infects HBV and has neutralizing effect, and the inhibition of virus infection is had dose-dependently.But along with the increase of peptide concentration, the virus neutralization that Myr 13-59 dose-dependently ground has reduced antibody almost completely offsets the virus neutralizing capacity of antibody at the 20nM place.The counteracting of this virus neutralization is because HBV surface L protein derived peptide and antibodies, makes antibody consumption and can not combine with viral.The virus infection blocking effect that occurs when the antibody lower concentration is to be provided by excessive relatively Myr 13-59, and the Myr 13-59 that add this moment has surpassed the binding ability of antibody, the surplus of Myr 13-59 occurred.Along with the increase of antibody concentration, when reaching Myr 13-59 and antibody binding capacity when suitable, the mixture of polypeptide and antibody almost disappears to the restraining effect that HBV infects.Therefore as seen, there is the phenomenon of obviously cancelling out each other between HBV surface L protein derived peptide Myr 13-59 and the HBV surface L protein antibody.

And the effect between antigenicity derived peptide SEQ ID NO:17 and the HBV surface L protein antibody of going of HBV surface L protein obviously is different from the former.Shown in Fig. 6 B, SEQ ID NO:17 dose-dependently ground strengthens the effect that HBV surface L protein antibody blocking HBV infects, and along with the increase gradually of SEQ ID NO:17 concentration, blocks the dosage that HBV infects required HBV surface L protein antibody fully and reduces gradually.2 μ g/ml antibody+10nM go antigenicity derived peptide and 4 μ g/ml antibody+5nM to go the antigenicity derived peptide all can block HBV virus fully to hepatocellular infection, antibody and go to exist between the antigenicity derived peptide obvious synergistic effect and complementary relationship.Its reason be the antigenicity derived peptide not with antibodies, the effect that makes both suppress virus infection is not disturbed mutually.In addition, the mechanism of going antigenicity derived peptide and antibody inhibition HBV to infect is different, and the former is incorporated into liver cell virus and infects acceptor, and the latter then combines with viral, and sealing is viral closes key sequence with liver cell bonded Pre-S1.As seen, the HBV surface L protein goes antigenicity derived peptide and HBV surface L protein antibody can bring into play the effect that collaborative blocking-up HBV infects.

Sequence table

＜110〉Shanghai Xiqun Biotech Co., Ltd

＜120〉b hepatitis virus surface L protein related peptide

<130>055004

<160>24

<170>PatentIn version 3.1

<210>1

<211>119

<212>PRT

＜213〉HBV surface L protein

<400>1

Met Gly Gly Trp Ser Ser Lys Pro Arg Gln Gly Met Gly Thr Asn Leu

1 5 10 15

Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro

20 25 30

Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe Asn Pro Lys

35 40 45

Lys Asp His Trp Pro Glu Ala Asn Gln Val Gly Ala Gly Ala Phe Gly

50 55 60

Pro Gly Phe Thr Pro Pro His Gly Gly Leu Leu Gly Trp Ser Pro Gln

65 70 75 80

Ala Gln Gly Ile Leu Thr Thr Val Pro Val Ala Pro Pro Pro Ala Ser

85 90 95

Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro Ile Ser Pro Pro Leu

100 105 110

Arg Asp Ser His Pro Gln Ala

115

<210>2

<211>47

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＜213〉HBV surface L protein

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Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His

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Gln Leu Asp Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp

20 25 30

Phe Asn Pro Lys Lys Asp His Trp Pro Glu Ala Asn Gln Val Gly

35 40 45

<210>3

<211>47

<212>PRT

＜213〉HBV surface L protein derived peptide

<400>3

Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His

1 5 10 15

Gln Leu Asp Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp

20 25 30

Phe Asn Pro Lys Lys Asp His Trp Pro Glu Ala Asn Gln Val Gly

35 40 45

<210>4

<211>47

<212>PRT

＜213〉HBV surface L protein derived peptide

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Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His

1 5 10 15

Gln Leu Asp Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp

20 25 30

Phe Asn Pro Asn Lys Asp His Trp Pro Glu Ala Asn Gln Val Gly

35 40 45

<210>5

<211>47

<212>PRT

＜213〉HBV surface L protein derived peptide

<400>5

Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His

1 5 10 15

Gln Leu Asp Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp

20 25 30

Phe Asn Pro Asn Lys Ala His Trp Pro Glu Ala Asn Gln Val Gly

35 40 45

<210>6

<211>47

<212>PRT

＜213〉HBV surface L protein derived peptide

<400>6

Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His

1 5 10 15

Gln Leu Asp Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp

20 25 30

Phe Asn Pro Asn Lys Asp His Trp Pro Lys Ala Asn Gln Val Gly

35 40 45

<210>7

<211>47

<212>PRT

＜213〉HBV surface L protein derived peptide

<400>7

Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His

1 5 10 15

Gln Leu Asp Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp

20 25 30

Phe Asn Pro Asn Lys Asp His Trp Pro Lys Ala Lys Gln Val Gly

35 40 45

<210>8

<211>108

<212>PRT

＜213〉HBV surface L protein

<400>8

Met Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp

1 5 10 15

His Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp

20 25 30

Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala His Lys Val Gly

35 40 45

Ala Gly Ala Phe Gly Leu Gly Phe Thr Pro Pro His Gly Gly Leu Leu

50 55 60

Gly Trp Ser Pro Gln Ala Gln Gly Ile Leu Gln Thr Leu Pro Ala Asn

65 70 75 80

Pro Pro Pro Ala Ser Thr Asn Arg Gln Ser Gly Arg Gln Pro Thr Pro

85 90 95

Leu Ser Pro Pro Leu Arg Asn Thr His Pro Gln Ala

100 105

<210>9

<211>46

<212>PRT

＜213〉HBV surface L protein derived peptide

<400>9

Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His

1 5 10 15

Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp

20 25 30

Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val

35 40 45

<210>10

<211>45

<212>PRT

＜213〉HBV virus surface L protein

<400>10

Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln

1 5 10 15

Leu Asp Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp Phe

20 25 30

Asn Pro Lys Lys Asp His Trp Pro Glu Ala Asn Gln Val

35 40 45

<210>11

<211>19

<212>PRT

＜213〉HBV virus surface L protein

<400>11

His Gln Leu Asp Pro Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp

1 5 10 15

Asp Phe Asn

<210>12

<211>57

<212>DNA

＜213〉HBV virus surface L protein encoding sequence

<400>12

caccagttgg accctgcgtt cggagccaac tcaaacaatc cagattggga cttcaac 57

<210>13

<211>268

<212>DNA

＜213〉artificial sequence

<220>

<221>misc feature

<222>(1)..(268)

＜223〉n=a, t, c, or g

<400>13

gtcaccatat tcttgggaac aagagctaca gcatgggagg ttggtcttcc aaacctcgac 60

aaggcatggg gacgaatctt tctgttccca atcctctggg attctttccc gatnnsnnsn 120

nsnnsnnsnn snnsnnsnns nnsnnsnnsn nsnnsnnsnn snnsnnsnns cccaaaaagg 180

atcactggcc agaggcaaat caggtaggag cgggagcatt cgggccaggg ttcaccccac 240

cacacggcgg tcttttgggg tggagccc 268

<210>14

<211>266

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(266)

＜223〉n=a, t, c, or g

<400>14

tgagggctcc accccaaaag accgccgtgt ggtggggtga accctggccc gaatgctccc 60

gctcctacct gatttgcctc tggccagtga tcctttttgg gsnnsnnsnn snnsnnsnns 120

nnsnnsnnsn nsnnsnnsnn snnsnnsnns nnsnnsnnat cgggaaagaa tcccagagga 180

ttgggaacag aaagattcgt ccccatgcct tgtcgaggtt tggaagacca acctcccatg 240

ctgtagctct tgttcccaag aatatg 266

<210>15

<211>46

<212>PRT

＜213〉the HBV surface L protein goes the antigenicity derived peptide

<400>15

Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp Thr

1 5 10 15

Arg Gly Asp Arg Gly Gln Asn Leu Ala Ala Gly Val Ala Leu Lys Asp

20 25 30

Phe Gly Pro Lys Lys Asp His Trp Pro Glu Ala Asn Gln Val

35 40 45

<210>16

<211>46

<212>PRT

＜213〉the HBV surface L protein goes the antigenicity derived peptide

<400>16

Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp Ala

1 5 10 15

Leu Gly Asp Leu Ser Phe Ser Arg Phe Gln Leu Tyr Thr Thr Asp Leu

20 25 30

Ser Gly Pro Lys Lys Asp His Trp Pro Glu Ala Asn Gln Val

35 40 45

<210>17

<211>46

<212>PRT

＜213〉the HBV surface L protein goes the antigenicity derived peptide

<400>17

Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp Cys

1 5 10 15

Ser Leu Asp Trp Val Trp Ser Trp Ala Pro Tyr Phe Thr Pro Gln Trp

20 25 30

Arg Thr Pro Lys Lys Asp His Trp Pro Glu Ala Asn Gln Val

35 40 45

<210>18

<211>46

<212>PRT

＜213〉the HBV surface L protein goes the antigenicity derived peptide

<400>18

Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp Phe

1 5 10 15

Ser Ser Asp Pro Lys Ser Arg Phe Ala Arg Arg Val Pro Val Ala Ala

20 25 30

Pro Val Pro Lys Lys Asp His Trp Pro Glu Ala Asn Gln Val

35 40 45

<210>19

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>19

agtctagact cgtggtggac 20

<210>20

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>20

tkgcactagt aaactgagcc 20

<210>21

<211>34

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>21

ctgactgcag cactggatgg ggcttggcta ttgg 34

<210>22

<211>33

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>22

ttatggaatt ccgacgcggc gattgagacc ttc 33

<210>23

<211>31

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>23

ctgactgcag tttccgcact catttacaag a 31

<210>24

<211>34

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>24

ttatggaatt catgctcccg ctcctacctg attt 34

Claims (10)

1. the polypeptide of a HBV surface L protein is characterized in that, it has general formula X-Y-Z, wherein,

X represents the sequence that the 1st to the 12nd amino acid of B genotype adw serotype and C genotype adr serotype HBV virus surface L protein constitutes, or the sequence that shortens of this sequence N-terminal certainly, or disappearance fully, and wherein, the N-terminal of X can be modified;

Y represents the 13rd to the 59th aminoacid sequence of B genotype adw serotype and C genotype adr serotype HBV virus surface L protein, and wherein, the N-terminal of Y and/or C-terminal can be modified;

Z represents B genotype adw serotype and C genotype adr serotype HBV virus surface L protein the 60th to the 89th aminoacid sequence, or terminal sequence that shortens of this sequence C or disappearance certainly fully, and wherein, the C-terminal of Z can be modified.

2. as right 1 described polypeptide, it is characterized in that the 1st amino acids glycine of described Y sequence is modified by myristoylation.

3. polypeptide as claimed in claim 1 or 2 is characterized in that, described sequence is selected from SEQ ID NO:2-7, and wherein the 1st of SEQ ID NO:3-7 the amino acid glycine modified by myristoylation.

4. the polypeptide of a HBV surface L protein is characterized in that, this polypeptide has general formula x-y-z, wherein,

X represents methionine(Met), and it can be modified, and perhaps x does not exist;

Y represents the 2nd to the 32nd aminoacid sequence of D genotype ayr serotype HBV surface L protein, and wherein, the N-terminal of y and/or C-terminal can be modified;

Z represents D genotype ayr serotype HBV surface L protein the 33rd to the 47th aminoacid sequence, or terminal sequence that shortens of this sequence C or disappearance certainly fully, and wherein, the C-terminal of z can be modified.

5. as right 4 described polypeptide, it is characterized in that the 1st amino acids glycine of its y sequence is modified by myristoylation.

6. as right 4 or 5 described polypeptide, it is characterized in that the sequence of described polypeptide is SEQ ID NO:9, its 1st amino acids glycine is modified by myristoylation.

7. an albumen removes the screening method of antigenicity derived peptide, is used to remove antigenicity and keeps proteic particular community, it is characterized in that this method comprises:

(A) in viral protein to be screened, introduce stochastic sequence, set up the recombinant virus storehouse of this albumen specific region or whole albumen hats;

(B) waiting to screen under the condition of protein antibodies existence, screening still has the recombinant virus subgroup of infection ability, and obtains this proteic aminoacid sequence in this recombinant virus subgroup, promptly obtains this albumen and removes antigenicity candidate peptide sequence;

(C) by obtain go antigenicity candidate peptide sequence synthetic go accordingly antigenicity candidate peptide, screening can regulate the virus infection ability but not with antigenic polypeptide that goes of this viral protein antibodies, promptly obtain albumen and go the antigenicity derived peptide.

8. a HBV surface L protein goes the antigenicity derived peptide, and it screens right 1 described B genotype adw serotype and C genotype adr serotype HBV surface L protein polypeptide by right 7 described methods and obtains, and it is characterized in that it has general formula

α-β

In the formula,

α represents the 1st to 13 amino acids sequence of HBV surface L protein, or the sequence that shortens of this sequence N-terminal or only be the 13rd amino acids glycine certainly, and wherein, described N-terminal and/or the 13rd amino acids glycine can be modified;

β represents that application rights requires 7 described screening methods that B genotype adw serotype and C genotype adr serotype HBV virus surface L protein the 14th to the 58th aminoacid sequence are screened the aminoacid sequence of acquisition, wherein, the N-terminal of the aminoacid sequence that is obtained can be modified and/or its C-terminal can be shortened or modify.

9. a pharmaceutical composition is characterized in that, polypeptide or the described HBV surface L protein of claim 8 that it contains claim 1 or 4 described HBV surface L proteins remove antigenicity derived peptide and pharmaceutically acceptable carrier.

10. the described HBV surface L protein of the polypeptide of claim 1 or 4 described HBV surface L proteins or claim 8 goes the antigenicity derived peptide to prevent and/or treat purposes in the medicament of hepatitis B in preparation.

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