CN104130316A

CN104130316A - Hepatitis B virus (HBV) surface L protein related peptide

Info

Publication number: CN104130316A
Application number: CN201410377176.9A
Authority: CN
Inventors: 刘宏利; 汪裕; 尹颖
Original assignee: SHANGHAI HEPU PHARMACEUTICAL Co Ltd
Current assignee: SHANGHAI HEPU PHARMACEUTICAL Co Ltd
Priority date: 2005-08-12
Filing date: 2005-08-12
Publication date: 2014-11-05
Anticipated expiration: 2025-08-12
Also published as: CN104130316B

Abstract

The invention relates to hepatitis B virus (HBV) surface L protein derived peptide which is used for preventing and curing HBV infection, a screening method which is used for removing polypeptide antigenicity and antigenicity-removing derived peptide which is screened and obtained through the screening method. An HBV surface L protein pre-S1 area has a key amino acid sequence of virus adherent cell surface receptors and an antigenicity amino acid sequence which is combined with antibody against L protein. Polypeptide in the HBV surface L protein pre-S1 area can inhibit the infection of the HBV on cells. The invention also provides the screening method which is used for removing polypeptide antigenicity. The antigenicity-removing derived peptide is screened, can inhibit HBV infection and does not combine with the antibody.; Therefore, the mutual interference between natural L protein derived peptide and the antibody against the L protein during HBV infection inhibition is avoided, and both the antigenicity-removing derived peptide and the antibody against the HBV surface L protein can inhibit the HBV infection.

Description

Hepatitis B virus surface L protein related peptide

Patent application of the present invention is that application number is dividing an application of 201210177978.6 Chinese invention patent application, and this application is the division of the application for a patent for invention that on August 12nd, 2005 submits to, denomination of invention is " hepatitis B virus surface L protein related peptide ".

Technical field

The present invention relates to the HBV surface L protein derived peptide infecting for prevention, treatment hepatitis B virus (human hepatitis B virus, HBV), the screening method of removing these polypeptide antigens and by the method screen obtain go 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 capable of blocking that obtains by the method infects and but do not go antigenicity derived peptide with the HBV surface L protein of antibodies.This goes antigenicity derived peptide to infect with the collaborative HBV of inhibition of anti-HBV surface L protein antibody.

Background technology

The epidemiology of 1.HBV

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 approximately has 2,000,000,000 people to infect HBV, and 75% world population is lived in high incidence of hepatitis b, and Patients with Chronic HBV Infection surpasses 3.5 hundred million.Every year infect relevant death toll up to 1,000,000 (1) to HBV, annual newly-increased infection estimates it is newly-increased 2.5～4 times of infecting of HIV.75% HBV chronic infection person concentrates on Asia (being about 2.87 hundred million) in the world, China is the high Endemic Area of hepatitis B, the sick data presentation of learning investigation of the end of the seventies and the beginning of the nineties twice national hepatitis epidemic, 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, HBsAg carrying rate 9.75%, existing chronic hepatitis B patient more than 2,000 ten thousand, and from nineteen ninety-five, Chinese population hepatitis B morbidity ratio is the trend that raises year by year, therefore in the < < of Ministry of Health sanitary work in 2005 will be put > >, hepatitis B having been listed in to China needs one of four large diseases of priority control.

The prevention that 2.HBV infects and treatment present situation

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

Since Krugman in 1970 obtains after hepatitis B vaccine the earliest, various countries utilize asymptomatic HBsAg carrier's blood plasma extraction haematogenous HBsAg to prepare 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, by gene recombination HBV vaccine, is replaced.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 after the omnidistance immunity of 3 pins, anti-HBsAg antibody male rotary rate is not less than 85%.But it is slower that its main drawback is antibody response, if infecting newborn infant's initial immunity of puerpera, HBV surpasses more than 7 days, lose HBV vaccine and newborn infant is blocked to the effect of vertical transmission.And 10%～15% 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 after hepatitis b vaccination Healthy People, immunoglobulin preparation prepared by the high-titer blood plasma of collection or serum separation and Extraction, and its antibody titer is more than 100IU/ml.The neonatal passive immunization prevention of inadvertent contamination crowd, immunologic hypofunction person and HBV infection puerpera is applicable to happen suddenly.The Patients with Chronic HBV Infection of China approximately has 30%-50% by mother-to-baby transmission; for blocking-up HBV mother and baby perinatal transmission; generally by HBIG and combined with hepatitis B vaccine application; although protection ratio can reach more than 80%; but the blocking-up that HBIG provides contribution is also not obvious; combined utilization is only compared with the blocking-up rate of the alone raising 5%～10% of Hepatitis B virus vaccine, and after combined utilization, HBV infects puerpera newborn infant HBV infection rate still more healthy puerpera newborn infant is high 4 times.In recent years find, intrauterine infection may be the important channel of mother-to-baby transmission, and the HBV that HBV infects pregnant woman induced labor fetus liver or blood infects the intrauterine infection rate approximately 16% that sign recall rate can reach the baby of 40%, HBsAg Positive Mothers.Therefore domestic some hospital last 3～4 middle of the month of gestation to HBV infect pregnant woman monthly a shot be greater than the HBIG of 200IU, to prevention fetus HBV intrauterine infection.But the method Intrauterine Transmission barrier effect is unsatisfactory, and may cause appearance and the immunoreactive generation of immunocomplex pathology of HBV S district variant.

HBIG is also widely used in the prevention of the relevant after liver transplantation HBV infection and recurrence of hepatitis B.In the situation that there is no preventive measures, in the relevant after liver transplantation of hepatitis B 6 months HBV reinfection rate up to approximately 40%, 2 year in reinfection rate up to approximately 60%.Majority again cases of infection is 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 approximately 60～70%, and easily there is HBV transgenation, YMDD resistance aberration rate is up to 21%.In addition patient transplants front many life-time service antiviral, and antiviral opposing phenomenon appears in majority, has increased the difficulty of transplanting rear virus infection recurrence rate and prevention.Therefore the heavy dose of HBIG of the additional use of state is with the relevant recurrence of Hepatitis B after Liver Transplantation of prevention hepatitis B.After liver transplantation do not accept, in the receptor of HBIG treatment, to occur the HBsAb of different titers in postoperative early stage body, and serum HBsAb fades away subsequently, and uses the patients serum HBsAb of HBIG can the higher titre of long term maintenance.High dosage unrestriction HBIG single therapy can stop 65%～80% Patients on Recurrence, but HBIG is expensive, and expense is every year up to approximately 1,300,000 yuans.Although domestic employing lamivudine and low dose of HBIG combined utilization stop the curative effect of transplanting rear HBV infection and recurrence to show well, confirm without large sample clinical trial.And the immune pressure that prolonged application HBIG brings can cause HBV genovariation and occur immunologic escape phenomenon, the prevention that HBIG is infected for the relevant after liver transplantation HBV of hepatitis B is again very limited.

In sum, for preventing and treating the means of HBV infection, be mainly limited at present the active passive immunity of nucleotide analog inhibition, antiviral cell factor and neutrality antibody, lack the inhibition means to other infection links of HBV virus.HBV surface L protein involved in the present invention goes antigenicity derived peptide 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 for HBV, infected 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, is only 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 generally infection of HBV and copying does not cause that the obvious pathologic of liver cell changes, and the virion of generation discharges in secretion mode.The relevant particle that HBV produces mainly contains 3 kinds of forms, comprises that the Dane particle, abundance of diameter 42-47nm are higher than Dane particle 10,000-1, the filamentary fibers that the 20nm spherical particle of 000,000 times and a small amount of diameter 20nm length do not wait.Wherein only having Dane particle to consist of genomic dna, viral dna polymerase, nucleocapsid and tunicle, is the unique viral particle with infection activity of HBV.

2) HBV genotype, serotype and Epidemiological distribution thereof

There are serotype and two kinds of different somatotype systems of genotype in HBV at present.Lebouvier in 1972 etc., according to the common antigenic determinant a of HBsAg HBsAg and the two couples of antigenic determinant d/y that mutually repel and w/r, are divided into adw by HBV, adr, tetra-serotypes of ayw and ayr.Courouce Pauty in 1978 etc. are divided into antigenic determinant w again w1～4, 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, according to the result of the full Phylogenetic analysis of HBV, take full gene nucleotide heterology >=8% as somatotype standard, HBV is divided into 8 genotype (A～H) (3,4).Combination between each genotype also can produce the different recombinant chous such as A/D, B/C, Ba and Bj.Corresponding relation between HBV serotype and genotype is in Table 1.

Although HBV infects, all over the world, all have in various degree popular, between country and domestic interzone there is very significantly difference (table 1).The genotype HBV such as China A, B, C, D infect and are all existence in various degree, northern area be take C genotype as main, by north to southern B genotype infection rate, increased gradually, Shenzhen B genotype is suitable with C genotype infection rate ratio, and the minority area D genotype such as Tibet have higher infection rate.The serotype HBV such as China adr, adw2, ayr, ayw1, ayw2 and ayw3 all have distribution, but take adr and adw2, are advantage serotype.In China B genotype and adw2 serotype, C genotype and adr serotype, there is obvious corresponding relation.

Relation and the Major Epidemic region of table 1.HBV genotype and serology hypotype, HBV albumen, PC/BCP sudden change

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

HBV peplos contain greatly (L), in (M), little (S) three kinds of surface antigen proteins, these albumen are encoded by the S district single open reading frame of 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 protein 26s of HBV S gene regions, 55 other amino acid of 165, Pre-S2 district alkali yl coding M albumen.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 Gai district lack respectively 33 and 3 bases, 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 is high, is the major protein of HBs in HBV infected patient circulating, and M and L albumen content are very micro-, only account for respectively 5～15% and 1～2% of whole HBs albumen.The envelope protein of 20nm spherical particle and filamentary fibers is mainly comprised of S albumen and quantity M albumen not etc., and hardly containing L albumen, the albumen of Dane particle coating is mainly L albumen.

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

According to competition principle, from the N-terminal myristoylation of L albumen 2-78 amino acids, to modify small peptide and can block the infection of HBV to liver cell and Hepa RG hepatic cell line, this small peptide can further foreshorten to the 2-48 amino acids of L albumen.To the infection of DHBV, also there is similar blocking effect in polypeptide corresponding to duck hepatitis B virus DHBV surface L protein preS the 2nd to the 41st amino acids.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, the infection of blocking virus to human liver cell, tree shrew (T.belangeri) liver cell and Hepa RG hepatic cell line.Its mechanism may be that antibody is combined with this section of sequence, has blocked the site that L albumen is combined with cell receptor, thereby has suppressed the infection of HBV to cell.Clinical data also confirms, can produce anti-preS1 antibody in patients with HBV infection 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 rare in chronic HBV infection patient, points out this antibody-like to play a significant role in removing HBV and controlling HBV infection chronicity process.

5. the prospect of meaning of the present invention and application

The chronically infected medicine for the treatment of HBV all acts on HBV time multiplexed cell link processed at present, and HBV surface L protein polypeptide can directly suppress the infection of HBV to cell, and the control of infecting for HBV provides new strategy.But such small peptide has strong antigenicity, there is the aminoacid sequence be combined with neutrality antibody, simultaneously this section of sequence be HBV must site in conjunction with infecting acceptor, be also the pass key sequence of L protein polypeptide inhibition HBV to the combination of cell infection acceptor.Have the neutrality antibody of being combined with this sequence in patient body, when applying such polypeptide HBV infected patient treat, polypeptide will combine with antibody, make polypeptide lose the effect with surface of hepatocytes HBV receptors bind, hinder the effect of polypeptide inhibition HBV infection.On the other hand, consume the neutrality antibody in body with the combination of polypeptide, be conducive on the contrary the infection of HBV.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 to infect HBV, the screening method of removing HBV surface L protein polypeptide antigen is provided on the other hand, but and by screening obtained HBV infection capable of blocking not with anti-HBV surface L protein antibodies go antigen derived peptide.The present invention also provides this coordinate repression that goes antigenicity derived peptide and anti-HBV surface L protein antibody to infect HBV.

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 this sequence N-terminal shortens certainly, or lacks completely, 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 sequence that this sequence C end shortens certainly, or lacks completely, 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, or 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 the sequence that this sequence C end shortens certainly or disappearance completely, 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 antigenicity derived peptide, retains the particular community of albumen for removing antigenicity, and the 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), under the condition existing at protein antibodies to be screened, screening still has the recombinant virus subgroup of infection ability, and obtains the aminoacid sequence of this albumen in this recombinant virus subgroup, obtains this albumen and removes antigenicity candidate peptide sequence;

(C) by going of obtaining, antigenicity candidate peptide sequence is synthetic removes antigenicity candidate peptide accordingly, screen adjustable virus infection ability but not with antigenic polypeptide that goes of this viral protein antibodies, obtain albumen and go antigenicity derived peptide.

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

α-β

In formula,

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

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

The invention still further relates to a kind of pharmaceutical composition, the polypeptide that it contains the HBV surface L protein described in first aspect present invention or second aspect or the HBV surface L protein described in fourth aspect go 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 antigenicity derived peptide to prevent and/or treat the purposes in the medicament of hepatitis B in preparation.

Accompanying drawing explanation

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 HBV liver cell.

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

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

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

Fig. 5 shows anti-HBV surface L protein antibody to HBV surface L protein derived peptide and goes antigenicity derived peptide to block the impact of HBV infection effect.

Fig. 6 shows HBV surface L protein derived peptide and goes the impact 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 with general formula X-Y-Z, and X represents the 1st to 12 amino acids sequences of HBV surface L protein here, or the sequence that the N-terminal of this sequence shortens certainly or disappearance completely, comprise the derived sequence that above sequence N-terminal is modified.Described " certainly the N-terminal of this sequence shorten " comprises from the 1st amino acids of this sequence N-terminal and starts 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, the integer that wherein a is 1-11, or X is only the 12nd amino acid.

Y represents the 13rd to the 59th amino acids sequence in B genotype adw serotype or 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 the sequence shortening from C-terminal or disappearance completely, comprises the derived sequence that above sequence C is end modified.Described " from C-terminal, shortening " comprises that the 1st amino acid (being the 89th amino acids of the present invention) from C-terminal starts 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, or Z is only the 60th amino acid.

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

Derived peptide provided by the invention, its sequence preference derives 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 sequences.

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

Above-described derived peptide is preferably with 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 other also preferred cholesterol and similar group thereof.

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-GTNLSVPNPLGFFPDHQLDPAFGANSN NPDWDFNPKKDHWPEANQVG (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 myristoyl group.In 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 HBV surface L protein sequence the 1st amino acids M or disappearance completely here, comprises the derived sequence that above sequence N-terminal is modified.Y represents the 2nd to the 32nd amino acids sequence in 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 the sequence shortening from C-terminal or disappearance completely, comprises the derived sequence that above sequence C is end modified.Described " from C-terminal, shortening " comprises that the 1st amino acid (being the 47th amino acids of the present invention) from C-terminal starts to shorten, can shorten to only surplus this sequence the 33rd amino acids,, z can be that the 33rd of described sequence is to c amino acids sequence, wherein, c represents the integer of 34-47, or z is only described the 33rd amino acids.

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

The preferred derived peptide of the present invention comprises the x sequence of disappearance completely, and the z sequence that preferred derived peptide contains is not affecting the condition of polypeptide inhibition 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 sequences.

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

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 myristoyl group.

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

The screening method that third aspect present invention provides a kind of albumen to go antigenicity derived peptide, retains the particular community of albumen for removing antigenicity.The method consists of A-B-C process.Here A procedural representation is introduced stochastic sequence in viral protein to be screened, sets up the recombinant virus storehouse of this albumen specific region or whole albumen hats.Under the condition that B procedural representation exists at protein antibodies to be screened, screening still has the recombinant virus subgroup of infection ability, and obtains the aminoacid sequence of this albumen in this recombinant virus subgroup, obtains this albumen and removes antigenicity candidate peptide sequence.By going of obtaining, antigenicity candidate peptide sequence is synthetic removes antigenicity candidate peptide accordingly to C procedural representation, screen adjustable virus infection ability but not with antigenic polypeptide that goes of this viral protein antibodies, obtain albumen and go antigenicity derived peptide.

The screening object of antigenicity derived peptide screening method that goes of the present invention comprises any type of proteins and peptides, and the source of its proteins and peptides comprises people, animal, plant, bacterium, virus etc. in interior all biologies and microorganism.In a preferred version of the present invention, applying screening method of the present invention screens the antigenicity derived peptide of going of the viral proteins such as HBV, people's acquired immunity defact virus, influenza A virus and Paramyxo virus (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 to 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 sequences is screened.More preferably the antigenicity derived peptide of going of B genotype adw serotype as described in SEQ ID 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 antigenicity derived peptide screening method, A process is included in the biomolecules level 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 and can introduce in above each level the method for stochastic sequence.In a preferred version of the present invention, the present invention introduces stochastic sequence in virogene picodna base level, further in preferred version, by gene recombination technology, is introducing stochastic sequence.

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

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 gene DNA order-checking to obtain recombinant virus subgroup protein sequence, 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 sequences (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 antigenicity derived peptide screening method, the producing method that C process comprises any polypeptide such as chemiluminescent polypeptide is synthetic, DNA recombinant expression.In preferred version of the present invention, adopt chemical synthesis to produce and remove antigenicity candidate peptide.In a preferred version of the present invention, screening obtains HBV surface L protein and goes antigenicity derived peptide (SEQ ID NO:17).

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

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

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

The present invention comprises through reducing or remove the viral protein derived sequence of antigenicity transformation, comprise especially through reducing or remove the HBV viral protein derived sequence of antigenicity transformation, more particularly comprise through reducing or remove the derived peptide sequence of the HBV surface L protein of antigenicity transformation.Here " transformation " refers to utilize and comprises that remove antigenicity derived peptide screening method, amino acid deletions, amino acid substitution etc. any provided by the invention take the technological method that reduction derived peptide antigenicity is object.

Above-described derived peptide is preferably with 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 other also preferred cholesterol and similar group thereof.

In a preferred version of the present invention, the N-terminal of polypeptide is modified through myristoylation.More preferably, to go antigenicity derived peptide be the polypeptide that the 1st glycine is connected with 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, the peptide sequence obtaining compared with the amino acid whose deletion of a good 1-8 amino acid, a better 1-5 amino acid, better 1-3, conservative/non-conservative amino acid substitution.The peptide modified product that further comprises the proteolysis resistant obtaining by one or more (as 1-10,1-8,1-5 etc.) L amino acid-D amino acid substitution etc.The application also comprises reducing modification and the transformation that the antigenicity of the application's polypeptide is object.Here " homologue " also comprises that relating to polypeptide with the application has and be greater than the polypeptide of 30% (as 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or larger) homology or contain glycine that myristoyl modifies and the binding sequence of anti-Pre-S1 antibody (or the anti-Pre-S1 antibodies sequence obtaining through past antigen selection corresponding go 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 produce the liver cell HBV infection inhibiting rate that is greater than 50% under the peptide concentration lower than 100mM.

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

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

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

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

The application provides HBV surface L protein to go the effect of antigenicity derived peptide and the collaborative anti HBV infecting of other anti-virus infection measures, these antiviral measures comprise that HBV vaccine immunity (comprises preventative vaccine and any actives relevant to HBV such as therapeutic vaccine, passive immunization measure), the anti-HBV antibody (monoclonal antibody that comprises any albumen of anti-HBV, polyclonal antibody, HBV immunoglobulin (Ig) etc. any with the HBV virus antibody of combination and the composition of antibody), cytokine (comprises interferon-alpha, cytokine and the composition thereof of all interventions such as interleukin-HBV), nucleoside analog (lamivudine, Adefovir, the compound of any inhibition virus replication such as Entecavir and Telbivudine) and other all intervention HBV infect, copy, medicine and the measure of any HBV processes such as release.In the application's a preferred version, 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 are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) described condition, 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 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 the B genotype adw serotype that N-terminal myristoyl is modified and C genotype adr serotype HBV virus surface L protein derived peptide, be expressed as Myr 13-59) with AB 431A type Peptide synthesizer by standard Fmoc scheme, take 0.25mM HMP as initial resin, and from carboxyl terminal, to aminoterminal, residue extension is synthetic one by one.For strengthening the stability of polypeptide, the further amidation of C-terminal of polypeptide.After peptide end of synthesis, through cutting liquid cutting, G6 glass sand hourglass filtering resin, filtrate vacuum is drained.Deionized water dissolves polypeptide cleaved products, explorer 100 type medium pressure liguid chromatograph C18 column purification, substep is collected main peak.Target peak is collected sample with the anti-phase high pressure liquid chromatography Symmetry C18 of Delta 600 type analytical column Purity, API2000LC/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 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 ℃ preservation of chronic HBV infection patients serum-70.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 precipitation, precipitation is dissolved in and in deionized water, is HBV DNA profiling.PCR reaction system 50 μ l, containing HBV DNA profiling 5 μ l, 1 * PCR Buffer, MgCl ₂1.5mmol/L, upstream primer (SEQ ID NO:19,5 ' AGTCTAGACTCGTGGTGGAC-3 ', position in HBV genome is 247-266, lower same) 25pmol/L, downstream primer (671-690,5 '-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 ℃ are extended 7 minutes.Pcr amplification product 443bp, direct Sequencing obtains its sequence.According to the aminoacid sequence of HBsAg DNA sequence dna conversion, by 122, 127, 134, 159 and 160 amino acids form 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 or integrated forms by amplification of the S-gene sequences by the polymerase chain reaction and 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 serotype variations within the four major subtypes ", J Gen Virol., in December, 1992, 73 (Pt 12): 3141-5), according to HBV S gene regions type specificity Nucleotide feature, determine HBV genotype (Norder H, Courouce AM, Magnius LO, " Molecular basis of hepatitis B virus serotype variations within the four major 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-S2region ", J Gen Virol., in June, 1994,75 (Pt 6): 1485-90, Erratum is at J Gen Virol, March nineteen ninety-five, 76 (Pt 3): 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, by method described in document, carry out separation (Guguen-Guillouzo, C., and A.Guillouzo, 1986.Methods for preparation of adult and fetal hepatocytes, the 1st 12 pages of –, .Les E ' ditions INSERM Paris in " Isolated and cultured hepatocytes " that A.Guillouzo and C.Guguen-Guillouzo edit, John Libbey and Co., Ltd., London), be incubated at and added 3.5 * 106M hydrocortisone monomester succinate (hydrocortisone hemisuccinate), the sub-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 to the human primary hepatocyte of cultivating 3 days, hatch for 37 ℃ and infect 24 hours.After infection completes, remove infection serum, washed cell 3 times, supplements 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, frozen in-80 ℃.

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

HBsAg in culture supernatant detects by sandwich sandwich assay (in-house sandwich) enzyme immunoassay (ELISA).37 ℃ of coated 96 orifice plates of 1ug/ml anti-hbs monoclonal antibodies 2 hours.Fully (0.1%Tween 20PBS washing 3 times, PBS washing 2 times) after washing, 10% 37 ℃ of foetal calf serums sealing 1 hour.Remove confining liquid, add 4 ℃ of liver cell culture supernatant 100 μ l that HBV infects to hatch 12 hours.Remove culture supernatant, fully washing, adds 37 ℃ of the anti-HBsAg antibody of peroxidase coupling to hatch 1 hour.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 supernatant.

6, the inhibition that HBV surface L protein derived peptide (Myr 13-59, SEQ ID NO:3) infects HBV liver cell

Human primary hepatocyte is cultivated 3 days in 12 well culture plates, and every hole approximately 106 cells and L protein derived peptide Myr13-59 pre-treatment 30 minutes add B genotype adw serotype or C genotype adr serotype HBV virus infection 24 hours.Remove infection supernatant, washed cell 3 times.Continue to cultivate after 10 days and detect HBsAg content in liver cell culture supernatant.As shown in Figure 1, take that not infected by the hepatocellular HBV of Myr 13-59 pre-treatment be contrast, HBV surface L protein derived peptide Myr 13-59 suppresses HBV to hepatocellular infection in dose-dependently mode.5,10,20,40nM can suppress respectively that HBV infects liver cell 28%, 74%, 87% and 95%, the Myr 13-59 that is greater than 80nM can block HBV completely to hepatocellular infection.Its 50% infection inhibition concentration (IC ₅₀) be 7.63nM.

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

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) that 1, N-terminal myristoyl is modified is with embodiment mono-.

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, frozen in-80 ℃.

3, HBV infects the detection (with embodiment mono-) of rear liver cell culture supernatant HBsAg

4, the inhibition that HBV surface L protein derived peptide Myr 2-47 infects HBV liver cell

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

Embodiment tri-: 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 structure of the 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/LTris-HCl (pH8.0), 200mmol/L NaCl, 10mmol/L EDTA, 2%SDS, 1 μ g/ml poly (A)].60 ℃ of cracking phenol/chloroform extractings after 1 hour, ethanol precipitation, precipitation is dissolved in H ₂in O, be HBV DNA solution.With upstream primer (Pst I) 5 ' ctgactgcagCACTGGATGGGGCTTGGCTATTGG (SEQ ID NO:21,1202-1225) and downstream primer (EcoR I) 5 ' ttatggaattcCGACGCGGCGATTGAGACCTTC (2201-2180, SEQ ID NO:22) the amplification C genotype adr serotype HBV genome (1202-2201nt) that contains complete EN II reproduction element.This amplification segment support virus completes and copies, and contains the HBV gene order (3996nt) that is greater than single-gene group.PCR reaction system 50 μ l, containing 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 ℃ are extended 10 minutes.Pcr amplification product 4017bp.The HBV DNA fragmentation of amplification is cloned into Pst I and the EcoR I restriction enzyme site of pUC18 plasmid vector, forms pUC-HBV recombinant plasmid.After this plasmid transfection cell, can produce the virion with infection ability.

3, the foundation in the restructuring 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 Bse59 I and Bsu36 I restriction enzyme site, wherein N represents any base in A, T, G, C, S represents any base in G, C.Two synthetic complementary DNA fragment annealing rear clones are entered between pUC-HBV restriction enzyme site Bse59 I and Bsu36 I, thereby introduce stochastic sequence in the corresponding coding region of HBV surface L protein Pre-S1 domain antibodies binding amino acid sequence HQLDPAFGANSNNPDWDFN.Again by the Transfected Recombinant Plasmid Hep G2 cell with hat district, cultured continuously 10 days.The virion that recombinant plasmid forms in Hep G2 cell is secreted in culture supernatant, has formed the restructuring HBV storehouse of the corresponding sequence hats 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 HBV surface L protein antibody existence condition, there is the screening of the restructuring 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 restructuring HBV storehouse is added in hepatocyte culture medium, infect 24 hours.Remove infection supernatant, add MA18/7 and 5a19 antibody to continue to cultivate 10 days.Culture supernatant is transferred to the human primary hepatocyte of new cultivation, continues screening and culturing 2 and takes turns, to screen the restructuring HBV subgroup still with liver cell infection ability in the presence of anti-HBV surface L protein antibody under MA18/7 and the existence of 5a19 antibody.

5, the restructuring random district of HBV surface L protein encoding sequence, the mensuration of removing antigenicity candidate peptide sequence.

50 μ l take turns through 3 the culture supernatant that screening contains restructuring HBV subgroup, add 310 μ l Proteinase K lysate [1mg/ml Proteinase K, 50mmol/LTris-HCl (pH8.0), 200mmol/L NaCl, 10mmol/L EDTA, 2%SDS, 1 μ g/ml poly (A)].60 ℃ of cracking phenol/chloroform extractings after 1 hour, ethanol precipitation, precipitation is dissolved in H ₂it in O, is 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, containing 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 ℃ are 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, transformed competence colibacillus intestinal bacteria, under penicillin selection condition, cultivate, picking list bacterium colony, the recombinant plasmid that extracting is entrained, to insertion sequence order-checking, obtain and under anti-HBV surface L protein antibody existence condition, still there is the restructuring HBV surface L protein gene coded sequence of liver cell infection ability.

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

According to above restructuring HBV surface L protein gene coded sequence, through translation, obtain corresponding aminoacid sequence, 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.Candidate's peptide (SEQ ID NO:15,16,17 and 18) of modifying by the synthetic N-terminal myristoyl of standard Fmoc scheme with AB 431A type Peptide synthesizer, concrete grammar is with embodiment mono-.

7, 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 (pH 9.6), concentration and is 10 μ g/ml goes the coated Immulon II elisa plate of antigenicity candidate peptide room temperature to spend the night.Phosphate buffered saline buffer (PBS) rinsing of elisa plate through containing 0.05%Tween 20 3 times, 10% 37 ℃ of foetal calf serums sealing 1 hour.Elisa plate PBS rinsing 2 times, adds monoclonal antibody MA 18/7 or 5a194 ℃ overnight incubation.Cleaning of enzyme yoke plate, adds the sheep anti mouse two of peroxidase coupling anti-, hatches 1 hour for 37 ℃.Rinsing unnecessary two is anti-, adds and contains phenylenediamine and H ₂o ₂citric acid phosphoric acid damping fluid (pH 5.0), room temperature reaction 5 minutes.2N H ₂sO4 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, HBV surface L protein go antigenicity candidate peptide SEQ ID NO:15 and 18 respectively separately and 5a19 and M18/7 have 57%, 34% combination, and antigenicity candidate peptide SEQ ID NO:16 and 17 is not all combined with two kinds of antibody.Therefore further detect the ability of these two candidate's peptide blocking-up HBV infected liver cells.

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

Human primary hepatocyte is cultivated 3 days in 12 well culture plates, every hole approximately 106 cells and HBV virus surface L protein go antigenicity candidate peptide SEQ ID NO:16 or SEQ ID NO:17 pre-treatment 30 minutes, add B genotype adw serotype or C genotype adr serotype HBV virus infection 24 hours.Remove infection supernatant, washed cell 3 times, continues to cultivate the concentration (measuring method is with embodiment mono-) of measuring afterwards HBsAg in culture supernatant for 10 days.As shown in Fig. 4 A, B, HBV surface L protein goes antigenicity candidate peptide SEQ ID NO:16 and SEQ ID NO:17 all in dose-dependently mode, to suppress the infection of two kinds of gene serotype HBV to primary hepatocyte.Two L albumen go antigenicity candidate peptide when 160nM, all can block HBV completely to hepatocellular infection, 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 is that HBV surface L protein goes antigenicity derived peptide.

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

1, HBV surface L protein goes antigenicity derived peptide to infect retarding effect to HBV and is not subject to anti-HBV surface L protein antibody interferes with.

Human liver cell is cultivated 3 days in 12 well culture plates, every hole approximately 10 ⁶cell, 1:1 adds anti-HBV surface L protein Pre-S1 monoclonal antibody M18/7 and 5a19, hatch after 30 minutes, add HBV surface L protein to remove the HBV surface L protein derived peptide Myr 13-59 (SEQ ID NO:3) of antigenicity derived peptide SEQ ID NO:17 or natural sequence, hatch 30 minutes, remove supernatant, washed cell 3 times, adds HBV virus infection 10 hours.Remove infection supernatant, washed cell 3 times, continues to cultivate the concentration (with embodiment mono-) of measuring afterwards HBsAg in culture supernatant for 10 days.As shown in Figure 5A, there is the HBV surface L protein derived peptide Myr 13-59 (SEQ ID NO:3) of natural sequence under antibody existence condition, the ability that its blocking-up HBV infects obviously declines, and the concentration Existence dependency relationship of decline degree and antibody, the antibody of 2 μ g/ml makes the viral barrier effect of 80nM Myr 13-59 decline 50%, the antibody of 8 μ g/ml is blocked the virus infection blocking effect of 160nM HBV surface L protein derived peptide Myr 13-59 completely, show that HBV surface L protein derived peptide infects to HBV the interference that retarding effect is obviously subject to anti-HBV surface L protein antibody.And HBV surface L protein goes under condition that antigenicity derived peptide SEQ ID NO:17 is pre-existing at 0,2,4,8 μ g/ml antibody, still in dose-dependently mode, suppress HBV to the infection of primary hepatocyte (Fig. 5 B), it suppresses curve does not have significant difference, under each condition, IC50 is close, and visible HBV surface L protein goes antigenicity derived peptide to eliminate the interference of anti-HBV surface L protein Pre-S1 antibody to virus infection blocking effect.

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

And the effect between antigenicity derived peptide SEQ ID NO:17 and HBV surface L protein antibody of going of HBV surface L protein is obviously different from the former.As shown in Figure 6B, 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 completely and reduces gradually.2 μ g/ml antibody+10nM go antigenicity derived peptide and 4 μ g/ml antibody+5nM to go antigenicity derived peptide all can block HBV virus completely to hepatocellular infection, antibody and go between antigenicity derived peptide to exist significantly synergy and complementary relationship.Its reason be antigenicity derived peptide not with antibodies, the effect that makes both suppress virus infection does not interfere with each other.In addition, the mechanism of going antigenicity derived peptide and antibody suppression HBV to infect is different, and the former is incorporated into liver cell virus and infects acceptor, the latter with viral combination, the Pre-S1 pass key sequence that sealing virus is combined with liver cell.Visible, 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.

Claims

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

X represents the sequence of the 1st to the 12nd Amino acid profile of B genotype adw serotype and C genotype adr serotype HBV virus surface L protein, or the sequence that this sequence N-terminal shortens certainly, or lacks completely, 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 the sequence that this sequence C end shortens certainly or disappearance completely, and wherein, the C-terminal of Z can be modified.

2. the polypeptide as described in right 1, 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. a polypeptide for 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, or 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 the sequence that this sequence C end shortens certainly or disappearance completely, and wherein, the C-terminal of z can be modified.

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

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

7. albumen removes a screening method for antigenicity derived peptide, retains the particular community of albumen for removing antigenicity, it is characterized in that, the method comprises:

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

α-β

In formula,

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

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

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