CN104211784B

CN104211784B - It is used to prepare the protein and method of Hepatitis E virus sample particle

Info

Publication number: CN104211784B
Application number: CN201310218823.7A
Authority: CN
Inventors: 李少伟; 王楠; 张晓�; 王凯航; 郑明华; 夏宁邵
Original assignee: Xiamen University; Xiamen Innovax Biotech Co Ltd
Current assignee: Xiamen University; Xiamen Innovax Biotech Co Ltd
Priority date: 2013-06-04
Filing date: 2013-06-04
Publication date: 2018-05-22
Anticipated expiration: 2033-06-04
Also published as: HK1205148A1; CN104211784A

Abstract

The present invention relates to molecular biology and field of virology.Particularly, the present invention relates to one kind can be assembled into Hepatitis E virus in vitro（Hepatitis E Virus, HEV）Virus-like particle（Virus like particle, VLP）Protein, coded sequence and preparation method and include its virus-like particle, the albumen and virus-like particle can be used for preventing or treating HEV infection and the disease caused by HEV infects, such as Hepatitis E etc..Pharmaceutical composition or the purposes of vaccine are used to prepare the invention further relates to above-mentioned albumen and virus-like particle, and described pharmaceutical composition or vaccine are used to preventing or treat HEV infection and disease caused by HEV infects such as Hepatitis E.In addition, the present invention also provides the methods for preparing HEV virus-like particles.

Description

It is used to prepare the protein and method of Hepatitis E virus sample particle

Technical field

The present invention relates to molecular biology and field of virology.Particularly, the present invention relates to one kind to assemble in vitro Into Hepatitis E virus（Hepatitis E Virus, HEV）Virus-like particle（Virus-like particle, VLP）Egg White matter, coded sequence and preparation method and the virus-like particle for including it, the albumen and virus-like particle can be used for pre- Anti- or treatment HEV infection and the disease caused by HEV infects, such as Hepatitis E etc..The invention further relates to above-mentioned albumen and Virus-like particle is used to prepare pharmaceutical composition or the purposes of vaccine, and described pharmaceutical composition or vaccine are used to prevent or treat HEV infects and the disease caused by HEV infects is such as Hepatitis E.In addition, the present invention also provides prepare HEV virus-likes The method of particle.

Background technology

Viral hepatitis type E is by Hepatitis E virus（Hepatitis E Virus, HEV）Caused by infection, it is One of main virus hepatitis, and most is in self limiting.The symptom of viral hepatitis type E is similar with hepatitis A, but disease Dead rate higher, symptom are heavier：Pregnant woman's case fatality rate may be up to 20%, and patients with chronic liver merges HEV infection and easily liver triggered to decline It exhausts, the death rate is up to 70%.

HEV can also be propagated mainly by intestinal transmitted by blood transfusion or vertical approach, frequently result in large-scale outburst It is popular.Since nearly half a century, document describes the Hepatitis E great outburst nearly ten of ten thousand or more, wherein largest one The secondary Xinjiang, China for betiding 1986-1988 is fallen ill 119,280 altogether, dead 707 people, including 414 pregnant woman.By It is very limited to the research of HEV in people, therefore Hepatitis E is in a kind of ignored state for a long time.Into 21 century with Come, increased substantially with the sensitivity and specificity of Hepatitis E diagnostic reagent, more and more Hepatitis E case quilts It makes a definite diagnosis.According to statistics, the population in the whole world about 1/3rd once infected HEV.

More and more evidences show that Hepatitis E is a kind of zoonosis.Pig is the main animal place of HEV It is main, and be the important infection sources of mankind's Hepatitis E.Chinese scholar examines national 20 provinces and cities, 120 pig farms and import The 9 of epidemic disease, the HEV infection conditions of 055 pig have carried out serosurvey, as a result confirm that the HEV in Chinese commodity pig infects extremely Common, total infection rate is up to 83%, and is isolated to HEV Strain from the pig in domestic multiple areas.

Hepatitis E virus (HEV) is a kind of RNA virus of single-stranded positive, and capsid is assembled by single structure albumen T=3 icosahedral symmetry structure, wrap up about 7.2kb genome.A diameter of about 27~34nm of HEV virions, nothing Coating.HEV genomes include 5 ' end cap-like structures and poly A tract, and include three overlapped open reading frames （ORF：ORF1, ORF2 and ORF3）, both sides are respectively a 5 ' short end noncoding regions（5′UTR）With one by polyadenosine 3 ' short end the noncoding regions that acid terminates（3′UTR）.ORF1 is about 5kb, and positioned at 5 ' ends of code area, being that HEV is maximum opens Reading frame is put, main function is：In the presence of RNA synzyme, the coding non-structural protein related with viral RNA duplication （pORF1）.ORF2 is about 2kb, positioned at 3 ' ends of code area, the primary structure gene coding region for being HEV, main function For：A kind of encoding virus coat proteins pORF2 --- glycosylation albumen for containing 660 amino acid.Recently also directed to ORF2 albumen It is studied with the special RNA combinations activity of 5 ' end of HEV genomes, the results show that the interaction of the two may help In the formation of viral capsid.ORF3 is a small opening code-reading frame, and function is not yet elucidated with.There is research to prompt at present, HEV ORF3 albumen in cell signalling, the assembling of HEV particles, release and immune etc. may play an important role.

According to the homology of ORF2 structural protein genes, HEV can at least be divided into 4 main genotype.Molecular Epidemic It learns research and shows 1 type of HEV genes and the 2 type main infection mankind, often result in eruption and prevalence；3 type of gene and 4 types can infect simultaneously Animals and humans, in sporadic prevalence.The HEV representative strains of four kinds of types are respectively Burma's strain, Mexico's strain, U.S.'s strain and China Variant.At present, chicken HEV is classified as 5 type of gene.Various HEV has higher homology on ORF2 amino acid sequences, this leads It is same serotype to cause the HEV in HEV serology all over the world.

At present, the cell culture of HEV and tissue cultures fail to succeed.However, the virus-like particle due to HEV （Virus-like particle, VLP）Natural HEV particles can be preferably simulated, basic research for activity and functionally And the development of Hepatitis E vaccine.Therefore, the virus-like particle research of domestic and international HEV is main as follows：It is by various expression System obtains structural proteins, and structural proteins then are assembled into virus-like particle in vivo or in vitro（VLP）, so as to obtain HEV Virus-like particle.At present, include on the report for preparing HEV VLP researchs as follows：（1）By using baculoviral/ Insect cell expression HEV ORF2 segment aa112-aa607 can obtain the HEV VLP of internal self assembly in cell lysates （Robinson etc., Protein Expr Purif.1998,12 (1):75-84）；（2）By using baculoviral/insect cell HEV ORF2 segment aa14-aa608 are expressed, can be self-assembled into vitro as HEV VLP（Li et al., JBC.2010, August18）；（3）Using Bacillus coli expression HEV ORF2 segment aa368-aa606, can be self-assembled into vitro as HEV VLP（Li et al., Vaccine.2005,23:2893-2901;Li et al., JBC.2005,28 (5):3400-3406）.

So far, in the world into clinical test Hepatitis E vaccine there are two types of：It is a kind of to be developed by GSK companies The VLP vaccines of HEV ORF2 segments aa112-606 comprising baculoviral/insect cell expression compel you in Buddhist nun and enter II Clinical trial phase, it is shown that good immunogenicity, yet with a variety of causes, which is interrupted；Another kind is comprising big The VLP vaccines of the HEV ORF2 segments aa368-aa606 of enterobacteria expression, group in vitro after which is expressed with inclusion bodies Dress（Li et al., JBC, 2005；Yang etc., Protein Science, 2013）, completed III clinical trial phases, it is shown that Good security and protectiveness（Zhu etc., Lancet, 2010）, and listed in October, 2012.It can be seen that HEV VLP have There are good immunoreactivity and immunogenicity, be the ideal form of Hepatitis E vaccine available for Hepatitis E vaccine is developed.

As described above, HEV virus-like particles can be prepared and obtain by 2 kinds of methods at present：1）Utilize insect cell The protein fragments of solubility expression HEV ORF2（aa112-606）, HEV virus-like particles are assembled into insect cell；With 2）The protein fragments of HEV ORF2 are expressed in inclusion body using Escherichia coli（aa368-606）, dissolve in vitro, renaturation simultaneously It is assembled into HEV virus-like particles.This 2 kinds of methods respectively have advantage and disadvantage.The advantages of first method, is, can express length more Big albumen so as to which the virus-like particle obtained is closer to wild-type virus, is conducive to by the immune system knowledge of body Not and induce efficient immune response；Itself the disadvantage is that, HEV virus-like particles are assembled in the cell, so as to there is The risk of the impurity such as host protein or nucleic acid is introduced in HEV virus-like particles.In addition, first method uses eukaryotic cell expression System, of high cost, the cycle is long, and less efficient.Second method is then just the opposite.

However, never reporting before, HEV ORF2 segments aa112-606 can be reassembled into virus-like in vitro Grain.In fact, the present inventor has attempted to express wild type HEV ORF2 segments aa112-606 and external group using Escherichia coli HEV virus-like particles are filled, the shortcomings that eliminate first method（That is, avoid introducing host protein in HEV virus-like particles Or the impurity such as nucleic acid, and reduce cost by using efficient, cycle short prokaryotic expression system）.It however, the results show that should Protein fragments can express in inclusion body, be but difficult to carry out renaturation and assembling in vitro, can not obtain HEV virus-like particles.

Therefore, there is a need in the field to provide a kind of new method for preparing HEV virus-like particles, asked with solving above-mentioned technology Topic.

The content of the invention

In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The normally understood meaning of personnel institute.Also, cell culture used herein, molecular genetics, nucleic acid chemistry, immunological experiment Room operating procedure is widely used conventional steps in corresponding field.Meanwhile for a better understanding of the present invention, it is provided below The definition and explanation of relational language.

According to the present invention, term " escherichia expression system " refers to the expression being made of Escherichia coli (bacterial strain) and carrier System, wherein Escherichia coli（Bacterial strain）From bacterial strain available on the market, it is such as, but not limited to：GI698,ER2566, BL21(DE3),B834(DE3),BLR(DE3)。

According to the present invention, term " carrier（vector）" refer to, a kind of nucleic acid fortune that polynucleotide can be inserted Load instrument.When carrier can make the albumen of the polynucleotide encoding of insertion obtain expression, carrier is known as expression vector.Carrier can be with By conversion, transduction or transfection import host cell, its inhereditary material element carried is made to be expressed in host cell. Carrier is well known to those skilled in the art, and is included but not limited to：Plasmid；Bacteriophage；Coemid etc..

According to the present invention, term " HEV ORF2 " refers to the ORF2 of Hepatitis E virus (HEV) genome, and sequence is this Well known to field（See, e.g. DDBJ data entries number：D11092）, and encode the capsid protein of HEV.In the present invention, When being related to the sequence of HEV ORF2, DDBJ data entries number are used：Sequence shown in D11092 is described.For example, Statement " the 112-606 amino acids residue of the polypeptide of HEV ORF2 codings "（It is also abbreviated as herein, HEV ORF2 segments Aa112-aa606 or HEV ORF2aa112-aa606）In 112-606 amino acids residues refer to, D11092 coding it is more The 112-606 amino acids residues of peptide.However, it will be appreciated by those skilled in the art that HEV ORF2 or its coding polypeptide in, Can be naturally-produced or it be artificially introduced mutation or variation（Including but not limited to, replace, missing and/or addition）, without influencing its life Object function（In the present invention i.e., coding can form the capsid protein of HEV VLP）.Therefore, in the present invention, term " HEV ORF2 " should include all such sequences, including the sequence for example shown in D11092 and its natural or artificial variant.Also, As description HEV ORF2（Or the polypeptide of its coding）Sequence fragment when, not only include D11092（Or the polypeptide of its coding）'s Sequence fragment further includes D11092（Or the polypeptide of its coding）Natural or artificial variants in corresponding sequence segment.For example, table Stating " the 112-606 amino acids residue of the polypeptide of HEV ORF2 codings " includes, the 112-606 of the polypeptide of D11092 codings The variant of the polypeptide of amino acids residue and D11092 codings（It is natural or artificial）In respective segments.According to the present invention, table It states " corresponding sequence segment " or " respective segments " refers to, when carrying out optimal comparison to sequence, i.e., when sequence is compared to obtain When obtaining highest percentage homogeneity, the segment of equivalent site is located in the sequence being compared.

According to the present invention, term " HEV capsid protein " refers to, the albumen encoded by HEV ORF2 can be assembled into HEV Virus-like particle.

According to the present invention, when in the background in protein/polypeptide in use, term " variant " refers to such albumen, ammonia Base acid sequence with reference to protein/polypeptide（For example, the HEV capsid protein of the present invention）Amino acid sequence have one or more （Such as 1-10 or 1-5 or 1-3）Amino acid of differences（For example, conservative amino acid replacement）Or have at least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity, and it remains necessity spy with reference to protein/polypeptide Property.In the present invention, protein/polypeptide（For example, the HEV capsid protein of the present invention）Necessary characteristic can refer to, have be assembled into The ability of HEV virus-like particles.

According to the present invention, term " homogeneity " for refer to two polypeptides between or two nucleic acid between sequence matching feelings Condition.When some position in the sequence that two are compared all is occupied by identical base or amino acid monomer subunit（Example Such as, some position in each of two DNA moleculars by adenine occupy or two polypeptides each in some position It puts and is all occupied by lysine）, then each molecule is same on the position." percentage homogeneity " between two sequences is The matched position number shared by the two sequences divided by the function for position number × 100 being compared.If for example, two There are 6 matchings in 10 positions of sequence, then the two sequences have 60% homogeneity.For example, DNA sequence dna CTGACT and CAGGTT shares 50% homogeneity (having 3 location matches in 6 positions in total).In general, by two sequence alignments to generate It is compared during maximum homogeneity.Such comparison can be by using for example, can pass through computer program such as Align programs （DNAstar,Inc.）Needleman easily carried out et al.（1970）J.Mol.Biol.48：The method of 443-453 is come real It is existing.E.Meyers and the W.Miller (Comput.Appl for being integrated into ALIGN programs (version 2 .0) also can be used Biosci., 4:11-17 (1988)) algorithm, use PAM120 weight residue tables（weight residue table）, 12 Gap Length Penalty and 4 Gap Penalty measure the percentage homogeneity between two amino acid sequences.In addition, it can be used The Needleman and Wunsch (J MoI being integrated into the GAP programs of GCG software packages (can be obtained on www.gcg.com) Biol.48:444-453 (1970)) algorithm, using Blossum62 matrixes or PAM250 matrixes and 16,14,12,10,8,6 or 4 Gap Weight（gap weight）And 1,2,3,4,5 or 6 Length Weight measures the percentage between two amino acid sequences Number homogeneity.

As used in this article, term " conservative substitution " means to influence or change comprising amino acid sequence The amino acid replacement of the biological function of protein/polypeptide.It is lured for example, can for example be pinpointed by standard technique known in the art Become and the mutagenesis of PCR mediations introduces conservative substitution.Conservative amino acid replacement includes being replaced with the amino acid residue with similar side chain For the displacement of amino acid residue, it is used for example in physically or functionally similar with corresponding amino acid residue（Such as with phase Like size, shape, charge, chemical property, the ability including forming covalent bond or hydrogen bond etc.）Residue carry out displacement.Exist The family in the art for defining the amino acid residue with similar side chain.These families include having basic side chain（For example, rely Propylhomoserin, arginine and histidine）, acid side-chain（Such as aspartic acid, glutamic acid）, uncharged polar side chain（It is such as sweet Propylhomoserin, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan）, non-polar sidechain（Such as Alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine）, β branched building blocks（For example, Soviet Union's ammonia Acid, valine, isoleucine）And beta-branched side（For example, tyrosine, phenylalanine, tryptophan, histidine）Amino acid. It is therefore preferable that substitute corresponding amino acid residue with another amino acid residue from same side chain family.Identify amino acid The method of conservative substitution is well known in the art（See, e.g., Brummell et al., Biochem.32:1180-1187 (1993);Kobayashi et al. Protein Eng.12 (10):879-884(1999);With Burks et al. Proc.Natl Acad.Set USA 94:412-417 (1997), is incorporated herein by reference).

As used in this article, term " p239 ", " 239 albumen " or " p239 albumen " refer to HEV ORF2 segments aa368- aa606（It also includes the methionine encoded by initiation codon before aa368）, have and be assembled into HEV VLP's Ability, available for preparing Hepatitis E vaccine, and available for the composition mechanism of research HEV VLP.

As used in this article, term " p495 ", " p495 albumen " or " 495 albumen " refer to HEV ORF2 segments aa112- aa606（It also includes the methionine encoded by initiation codon before aa112）, have and be assembled into HEV VLP's Ability available for composition mechanism, the infection mechanism of research HEV VLP, and can be used for grinding for the structure biology of HEV VLP Study carefully.

According to the present invention, term " virus-like particle（VLP）" refer to without viral nucleic acid, in structure with natural disease The similar hollow shell structure of malicious particle is usually made of viral capsid proteins or its variant or segment.Due to VLP in structure with Natural virion is quite similar, therefore, has very strong immunogenicity and immunoreactivity.Simultaneously as VLP is free of Therefore the inhereditary material of virus, does not have infectivity.Due to above-mentioned advantage, VLP is had been developed that as vaccine, some of VLP vaccines have been successfully applied to clinic, illustrate but are not limited to：The tetravalence VLP Gardasil vaccines of Merck, p239VLP epidemic diseases Seedling etc..VLP allows the insertion of foreign gene or genetic fragment in structure and forms mosaic type VLP, and can be by external source Property antigen is illustrated in its surface.In addition, major part VLP also has package nucleic acid or the ability of other small molecules, therefore, may be used also As gene or the delivery vehicle of drug.Virus-like particle is also referred to as particle in this application.As used in the present invention, when Protein there is no become sexual factor under conditions of can assembling assembly virus-like particle when, the protein be referred to as " have assembling Into the ability of virus-like particle ".In the present invention, " change sexual factor " refers to, the factor that can be denatured protein, bag It includes but is not limited to, high temperature（For example, heating）, denaturant（Such as urea）Etc..

According to the present invention, term " 239 particle " and " 495 particle " refer respectively to, the HEV virus-likes formed by 239 albumen Particle and the HEV virus-like particles formed by 495 albumen or its mutant.

According to the present invention, term " assembling " refers to the structural proteins of virus（Such as capsid protein）Between or structural proteins with By various interactions between nucleic acid, the process of well-regulated nutty structure is formed, includes the group of natural viral particle The assembling of dress and virus-like particle.

According to the present invention, the broken of host cell can be realized by various methods well known to those skilled in the art, be wrapped Include but be not limited to homogenizer crush, homogeneous crusher machine, ultrasonication, grinding, high-pressure extrusion, bacteriolyze enzymatic treatment etc..

According to the present invention, various buffer solutions are it is known in the art that including but not limited to, phosphate buffer etc..

According to the present invention, term " redissolution buffer solution " refers to, the solution that can dissolve protein precipitation, is this field Well known to technical staff, include but not limited to Tris-HCl buffer solutions.

According to the present invention, term " assembling solution " refers to, can make the capsid protein assembling assembly virus-like particle of virus Solution.It typically includes the mild buffer solution of salt, such as the phosphate buffer comprising salt.It is particularly preferred that comprising The phosphate buffer of NaCl.

Salt in method for use in the present invention includes but not limited to acid salt, basic salt, neutral salt, such as alkali metal Salt, alkali salt, ammonium salt, hydrochloride, sulfate, bicarbonate, phosphate or hydrophosphate, particularly NaCl, NH₄Cl、 (NH₄)₂SO₄、Na₂SO₄One or more of.According to the present invention, particularly preferred salt is NaCl.

According to the present invention, term " polypeptide " and " protein " have identical meaning, are used interchangeably.According to the present invention, Amino acid is usually represented with single-letter well known in the art and trigram abbreviation.For example, alanine can be represented with A or Ala. In the application, can clearly it determine unless explicitly stated otherwise or based on context, otherwise when describing the position of amino acid residue, Usually using the sequence of HEV ORF2 as reference.For example, aa244 refers to, the 244th amino acids residue of HEV ORF2；H244 is Refer to, the 244th amino acids residue of HEV ORF2, histidine.For another example, H244L refers to, the 244th amino acids of HEV ORF2 Residue is leucine by Histidine mutagenesis.Similarly, aa188, aa284, aa335, V188A, L284P, A335T etc. have similar Meaning.

According to the present invention, term " pharmaceutically acceptable carrier and/or excipient " refers in pharmacology and/or physiologically The carrier and/or excipient compatible with subject and active ingredient, is well known in the art（See, for example, Remington ' s Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995）, and include but not limited to：PH adjusting agent, surfactant, adjuvant, ionic strength increase Strong agent.For example, pH adjusting agent includes but not limited to phosphate buffer；Surfactant includes but not limited to cation, it is cloudy from Son or nonionic surface active agent, such as Tween-80；Adjuvant includes but not limited to aluminium adjuvant（Such as aluminium hydroxide）, not Family name's adjuvant（Such as complete Freund's adjuvant）；Ionic strength reinforcing agent includes but not limited to sodium chloride.

The present invention is at least partially based on having now surprisingly been found that for the present inventor：It is mutated comprising H244L（That is, HEV ORF2 The 244th amino acids residue histidine sported leucine）P495 protein mutants can be in the culture of Escherichia coli Solubility expression in supernatant, and HEV virus-like particles can be assembled into vitro, and thus obtained HEV virus-likes Grain has excellent immunogenicity and immunoreactivity, available for preparing Hepatitis E vaccine.

Therefore, in one aspect, the present invention provides a kind of mutant of p495 albumen, it includes HEV ORF2's Aa112-aa606, and with including leucine on the corresponding sites of the aa244 of HEV ORF2.

In a preferred embodiment, compared with the aa112-aa606 of wild type HEV ORF2, the mutant is also Include one or more other mutation, such as 1,2 or 3 other mutation.In a preferred embodiment, institute One or more other mutation are stated to be located at selected from following sites：It is corresponding with aa188, aa284 and aa335 of HEV ORF2 Site.In a preferred embodiment, the mutant includes, with the corresponding sites of the aa188 of HEV ORF2 On alanine, with the proline on the corresponding sites of the aa284 of HEV ORF2 and/or in the aa335 with HEV ORF2 Threonine on corresponding site.

In a preferred embodiment, the mutant has SEQ ID NO：2, SEQ ID NO：4, SEQ ID NO：6、SEQ ID NO：8 or SEQ ID NO：Amino acid sequence shown in 10.In another preferred embodiment, it is described Mutant has such as SEQ ID NO：Amino acid sequence shown in 2.

On the other hand, the present invention relates to coding the present invention mutant polynucleotides and contain the polynucleotides Carrier.

Carrier available for insertion polynucleotide of interest is it is known in the art that including but not limited to cloning vector and expression Carrier.In one embodiment, carrier is such as plasmid, clay, bacteriophage, coemid etc..

On the other hand, the invention further relates to the host cells for including above-mentioned polynucleotides or carrier.Such host is thin Born of the same parents include but not limited to, prokaryotic cell such as Bacillus coli cells and eukaryocyte such as yeast cells, insect cell, plant Object cell and zooblast（Such as mammalian cell, such as mouse cell, people's cell etc.）.The host cell of the present invention can be with It is cell line, such as 293T cells.However, it is particularly preferred to host cell of the invention is Bacillus coli cells.

On the other hand, the present invention relates to a kind of HEV virus-like particles, the wherein virus-like particle contains the present invention's The mutant of p495 albumen is formed or formed by the mutant of the p495 albumen of the present invention.

In an especially preferred embodiment, HEV virus-like particles of the invention, which include, has SEQ ID NO：2, The mutant of the p495 albumen of sequence shown in 4,6,8 or 10 is formed or formed by the mutant.

On the other hand, the invention further relates to the mutant comprising above-mentioned p495 albumen or above-mentioned polynucleotides or loads The composition of body or host cell or HEV virus-like particles.In a preferred embodiment, the composition includes this hair The mutant of bright p495 albumen.In another preferred embodiment, the composition includes the HEV virus-likes of the present invention Particle.

On the other hand, the invention further relates to a kind of pharmaceutical composition or vaccine, it includes the HEV virus-likes of the present invention Particle optionally also includes pharmaceutically acceptable carrier and/or excipient.The pharmaceutical composition or vaccine of the present invention can be used for Prevention or treatment HEV infect or the disease caused by HEV infects is such as Hepatitis E.

In a preferred embodiment, the HEV virus-like particles with prevent or treat HEV infection or Hepatitis E Effective quantity exists.

The pharmaceutical composition or vaccine of the present invention can be administered by means commonly known in the art, such as, but not limited to logical Oral or injection is crossed to be administered.In the present invention, particularly preferred method of application is injection.

In a preferred embodiment, pharmaceutical composition of the invention or vaccine are applied in a unit With.Such as but be not intended to limit the present invention, the amount of the HEV virus-like particles included in per unit dose is 5 μ g-80 μ g, preferably 20 μ g-40 μ g.

On the other hand, the present invention relates to it is a kind of prepare the present invention p495 albumen mutant method, including Using the mutant of the escherichia expression system solubility expression present invention, then the cracking supernatant containing the mutant is carried out Purification process.

In a preferred embodiment, the condition for the solubility expression of escherichia expression system for example can be with Be, when 15 DEG C of isopropylthiogalactosides (IPTG) induction 10 is small, 20 DEG C of isopropylthiogalactosides (IPTG) inductions 8 it is small When or 25 DEG C of isopropylthiogalactosides (IPTG) induction 8 it is small when.In a further preferred embodiment, the expression item When part is that 25 DEG C of isopropylthiogalactoside (IPTG) inductions 8 are small.

In a preferred embodiment, the purification process includes, and ammonium sulfate is added into cracking supernatant（It is it is preferred that full And ammonium sulfate）Protein precipitation to be come out, then albumen precipitation is redissolved using redissolving buffer solution, then using it is cloudy from Sub- displacement chromatography purifies the protein of redissolution, so as to obtain the mutant of purified p495 albumen.

In a preferred embodiment, the final concentration of the ammonium sulfate added for example can be 10%, 20%, 30%, 40% or 50%, particularly preferably be 30%.In a preferred embodiment, the redissolution buffer solution is Tris-HCl bufferings Liquid.In a preferred embodiment, the anion-exchange chromatography can be DEAE FF anion-exchange chromatographies.

The invention further relates to a kind of methods for the HEV virus-like particles for preparing the present invention, are being obtained as described above the present invention's On the basis of mutant, including step：Purified mutant is dialysed into assembling solution, and it is made to be self-assembled into VLP.

In a preferred embodiment, the temperature for assembling for example can be 4 DEG C, 15 DEG C, 25 DEG C or 37 DEG C, special It You Xuandishi not be 37 DEG C.In a preferred embodiment, assembling solution is well known to those skilled in the art, such as comprising The phosphate buffer of salt, the particularly preferably phosphate buffer comprising NaCl.In a preferred embodiment, institute It for example can be 10mM, 20mM, 50mM or 100mM to state phosphate buffering liquid concentration, particularly preferably be 50mM.At one preferably Embodiment in, the phosphate buffer include 0.4M NaCl, 0.5M NaCl or 0.6M NaCl, be particularly preferred ground Include 0.5M NaCl.In a preferred embodiment, the phosphate buffer pH for example can be 5.5,6.0, 6.5th, 7.0 or 7.5, particularly preferably be 6.5.

The invention further relates to a kind of method for preparing vaccine, including can with pharmacy by the HEV virus-like particles of the present invention Carrier and/or the excipient mixing of receiving.As discussed above, the vaccine obtained can be used for preventing or treat HEV infection Or the disease caused by HEV infects is such as Hepatitis E.

On the other hand, prevent or treatment HEV infection or the disease caused by HEV infects the present invention relates to a kind of Method, including that will prevent or the HEV virus-like particles according to the present invention or pharmaceutical composition or vaccine of therapeutically effective amount is applied With to subject.In a preferred embodiment, the disease caused by HEV infects includes but not limited to, penta type Hepatitis.In another preferred embodiment, the subject is mammal, such as people.

On the other hand, further relate to mutant or HEV virus-like particle according to the present invention and prepare pharmaceutical composition Or the purposes in vaccine, described pharmaceutical composition or vaccine are used to preventing or treating HEV infection or the disease caused by HEV infects Disease.In a preferred embodiment, the disease caused by HEV infects includes but not limited to, Hepatitis E.

Advantageous effect of the invention

Expression system currently used for preparing HEV virus-like particles can be divided into eukaryotic expression system and prokaryotic expression system System.

The HEV capsid protein native conformation expressed in eukaryotic expression system destroys less, can spontaneously form VLP, often table It is the VLP with correct conformation up to after coming out.But current eukaryotic expression system such as baculovirus expression system and yeast table It is low there are expression quantity up to system, the defects of toxigenic capacity is high, extreme difficulties are brought to large-scale industrial production, and it is unfavorable In the composition mechanism for studying VLP in the cell.For vaccine development, such as impurity that need to be eliminated as much as in particle, host protein Or nucleic acid, this method can not provide assembled in vitro method, and big difficulty is caused to removal impurity.

In prokaryotic expression system, escherichia expression system has many advantages, such as that toxigenic capacity is low, and expression quantity is high.However, Correct native conformation is often lost in the albumen of expression in escherichia coli, is expressed in inclusion bodies in precipitation.To forgive Body form is expressed in the protein in precipitation（For example, p495 albumen）Renaturation be a global problem.In many cases, It is difficult in vitro effectively to the protein of inclusion bodies（For example, p495 albumen）Renaturation is carried out, recovers its native conformation.

Present inventors have surprisingly discovered that when the aa244 of the HEV ORE2 in p495 albumen is mutated into leucine, The mutant obtained can solublely express in Escherichia coli, and being capable of assembling assembly virus-like particle in vitro.With The prior art is compared, and method of the invention employs prokaryotic expression system efficient, that the cycle is short, and is obtained by assembled in vitro HEV virus-like particles not only avoid the use of of high cost, cycle long and less efficient eukaryotic cell expression system, The risk for introducing the impurity such as host protein or nucleic acid when being assembled in the cell in HEV virus-like particles is eliminated, and The albumen of length bigger can be expressed（aa112-606）, so as to which the virus-like particle obtained is closer to wild-type virus, Be conducive to be identified and induced by the immune system of body efficient immune response.The HEV virus-like particles obtained can be used for penta The development of type hepatitis vaccine, the structure determination of HEV, the preparation of HEV pseudovirus, protein body composition mechanism and viral infection mechanism Deng research.By with the parallel comparison of the p239 vaccines listed, the application confirms, HEV that the method for the present invention is obtained disease Malicious sample particle has excellent immunoreactivity and immunogenicity, can be used for preparing Hepatitis E vaccine.

From the above analysis, the present invention at least tool has the advantage that：First, the present invention uses Bacillus coli expression system It unites to express HEV capsid protein（P495 albumen）, it is ensured that high expression quantity and low cost；Secondly, method of the invention provides nothing External particle assemble method need to be denatured, controllable, and confirm HEV VLP that assembling obtains have good immunoreactivity and Immunogenicity, it is similar to natural viral particle shape, more suitable for natural viral particle infection mechanism and its structure biology Etc. research；3rd, the present invention can express the albumen identical with the p495 length of insect cell expression, closer to natural Hepatitis E virus particle, and operation is simple for VLP assemble methods.

Embodiment of the present invention is described in detail below in conjunction with drawings and examples, but people in the art Member will be understood that drawings below and embodiment are merely to illustrate the present invention rather than the restriction to the scope of the present invention.With reference to the accompanying drawings With the following detailed description of preferred embodiment, various purposes of the invention and favourable aspect are to those skilled in the art It will be apparent.

Description of the drawings

Fig. 1 shows the expression of 495 albumen and its mutant.

Figure 1A shows expression of 495 albumen of HEV Xinjiang Strains in Escherichia coli（Pass through coomassie brilliant blue staining Detection）, wherein, the expression in the cracking supernatant of culture of Escherichia coli of 95 albumen of swimming lane Isosorbide-5-Nitrae under 15 DEG C of inductions；Swimming lane 2, Expression in the inclusion body precipitation of culture of Escherichia coli of 495 albumen under 15 DEG C of inductions；3,495 albumen of swimming lane is lured at 25 DEG C Expression in the cracking supernatant of culture of Escherichia coli under leading；Escherichia coli training of 4,495 albumen of swimming lane under 25 DEG C of inductions Support the expression in the inclusion body precipitation of object.Figure 1A's the result shows that, 495 albumen are in Escherichia coli mainly in the form of inclusion body Expression, and can not be substantially expressed in soluble form in the cracking supernatant of culture of Escherichia coli.

Figure 1B shows expression of the mutant 1 of 495 albumen of HEV Xinjiang Strains in Escherichia coli（Pass through Western blot analysis detects）, wherein, swimming lane 1, protein markers；2,495 albumen of swimming lane is in culture of Escherichia coli Inclusion body precipitation in expression；Swimming lane 3, culture of Escherichia coli of the mutant 1 under 15 DEG C of inductions are cracked in supernatant Expression；Swimming lane 4, mutant 1 15 DEG C induction under culture of Escherichia coli inclusion body supernatant in expression；Swimming lane 5, mutation Expression in the cracking supernatant of culture of Escherichia coli of the body 1 under 20 DEG C of inductions；Swimming lane 6, mutant 1 is under 20 DEG C of inductions Expression in the inclusion body supernatant of culture of Escherichia coli；Swimming lane 7, culture of Escherichia coli of the mutant 1 under 25 DEG C of inductions Cracking supernatant in expression；Swimming lane 8, mutant 1 25 DEG C induction under culture of Escherichia coli inclusion body supernatant in Expression.Figure 1B's the result shows that, the mutant 1 of 495 albumen is mainly expressed in cracking supernatant in Escherichia coli with soluble form In, and do not expressed with inclusion bodies substantially.

Fig. 1 C show expressions of the mutant 2-5 of 495 albumen of HEV Xinjiang Strains in Escherichia coli（Pass through Western blot analysis detects）, wherein, swimming lane 1：Expression of the mutant 2 in the cracking supernatant of culture of Escherichia coli；Swimming Road 2：Expression of the mutant 2 in the inclusion body precipitation of culture of Escherichia coli；Swimming lane 3：Mutant 3 is in culture of Escherichia coli Cracking supernatant in expression；Swimming lane 4：Expression of the mutant 3 in the inclusion body precipitation of culture of Escherichia coli；Swimming lane 5：It is prominent Expression of the variant 4 in the cracking supernatant of culture of Escherichia coli；Swimming lane 6：Mutant 4 is in the inclusion body of culture of Escherichia coli Expression in precipitation；Swimming lane 7：Expression of the mutant 5 in the cracking supernatant of culture of Escherichia coli；Swimming lane 8：Mutant 5 exists Expression in the inclusion body precipitation of culture of Escherichia coli.Fig. 1 C's the result shows that, the mutant 2-5 of 495 albumen is in large intestine bar It is mainly expressed in cracking supernatant with soluble form in bacterium, and is not expressed with inclusion bodies substantially.

Fig. 1 D show expression of the mutant 6 of 495 albumen of HEV Xinjiang Strains in Escherichia coli（Pass through Western blot analysis detects）, wherein, 95 protein control of swimming lane Isosorbide-5-Nitrae；Swimming lane 2, large intestine bar of the mutant 6 under 37 DEG C of inductions Expression in the cracking supernatant of bacterium culture；Swimming lane 3, the inclusion body of culture of Escherichia coli of the mutant 6 under 37 DEG C of inductions Expression in precipitation；Swimming lane 4, the expression cracked in supernatant of culture of Escherichia coli of the mutant 6 under 25 DEG C of inductions；Swimming lane 5, mutant 6 25 DEG C induction under culture of Escherichia coli inclusion body precipitation in expression；Swimming lane 6, mutant 6 is at 20 DEG C Expression in the cracking supernatant of culture of Escherichia coli under induction；Swimming lane 7, Escherichia coli of the mutant 6 under 20 DEG C of inductions Expression in the inclusion body precipitation of culture；Swimming lane 8, mutant 6 15 DEG C induction under culture of Escherichia coli cracking on Expression in clear；Swimming lane 9, mutant 6 15 DEG C induction under culture of Escherichia coli inclusion body precipitation in expression.Fig. 1 D The results show that the mutant 6 of 495 albumen is mainly expressed in Escherichia coli in the form of inclusion body, and substantially can not It is expressed in soluble form in the cracking supernatant of culture of Escherichia coli.

Fig. 2 shows the saturated ammonium sulphate of the cracking supernatant containing 495 protein mutants 1.Swimming lane 1, protein molecular Amount mark；The control of the inclusion bodies of 2,495 albumen of swimming lane；Swimming lane 3, using 10% saturated ammonium sulfate from the cracking of mutant 1 The precipitation that supernatant obtains；Swimming lane 4, the precipitation obtained using 20% saturated ammonium sulfate from the cracking supernatant of mutant 1；Swimming lane 5, makes The precipitation obtained with 30% saturated ammonium sulfate from the cracking supernatant of mutant 1；Swimming lane 6, using 40% saturated ammonium sulfate from mutant 1 Cracking supernatant obtain precipitation；Swimming lane 7, the precipitation obtained using 50% saturated ammonium sulfate from the cracking supernatant of mutant 1.Knot Fruit shows that the saturated ammonium sulfate solution of 10-50% can precipitate the albumen in supernatant, and 30% saturated ammonium sulfate solution Sedimentation effect is preferable.

Fig. 3 shows the redissolution that the saturated ammonium sulphate of 495 protein mutants 1 is carried out with different buffer solutions.Swimming lane 1, Protein markers；Swimming lane 2, the precipitation after being redissolved with 20mM TB pH9.0；Swimming lane 3, after being redissolved with 20mM TB pH9.0 Supernatant；Swimming lane 4, the precipitation after being redissolved with 20mMTB pH8.5；Swimming lane 5, the supernatant after being redissolved with 20mM TB pH8.5；Swimming Road 6, the precipitation after being redissolved with 20mM TB pH8.0；Swimming lane 7, the supernatant after being redissolved with 20mM TB pH8.0；8,495 egg of swimming lane The control of white inclusion bodies.The results show that used various buffer solutions can redissolve the saturated ammonium sulfate of mutant 1 Precipitation, and the redissolution effect of 20mM TB pH8.5 is preferable.

Fig. 4 A show the DEAE column chromatographies purifying collection of illustrative plates of 495 protein mutants 1.Column balance buffering liquid：20mM TB8.5； Elution buffer：20mM TB8.5+0.1M/0.3M/0.5M/1M NaCl；Chromatography media is DEAE Fast Flow.

Fig. 4 B：The SDS-PAGE analyses for each fraction collected in the DEAE column chromatography purification process of 495 protein mutants 1. Swimming lane 1, protein markers；Swimming lane 2, the supernatant after being redissolved with 20mM TB8.5；Swimming lane 3, penetrates sample；Swimming lane 4, The 100mM NaCl elutriated fractions of DEAE-FF chromatographies；Swimming lane 5, the 300mM NaCl elutriated fractions of DEAE-FF chromatographies；Swimming lane 6, The 500mM NaCl elutriated fractions of DEAE-FF chromatographies；Swimming lane 7, the 1M NaCl elutriated fractions of DEAE-FF chromatographies；Swimming lane 8, The 100mM NaOH elutriated fractions of DEAE-FF chromatographies.It is washed the results show that 495 protein mutants 1 focus primarily upon 100mM NaCl In de- fraction.

Fig. 5 A show that the SDS-PAGE of 495 purified protein mutants 1 is analyzed.Swimming lane 1, protein markers；Swimming Road 2, the 495 purified protein mutants 1 without boiling water bath processing；Swimming lane 3 handles 10 minutes purified through boiling water bath 495 protein mutants 1.

Fig. 5 B show the sieve chromatography of 495 purified protein mutants 1.min：Minute, similarly hereinafter；Reference protein is E2 albumen.

Fig. 5's the results show that 495 protein mutants 1 after purification mainly exist in the solution with dimeric forms, and There is no grain fractions.

Fig. 6 A show, the influence that salinity assembles the particle of 495 protein mutants 1.The salinity of selection for 0.1M, 0.3M、0.4M、0.5M、0.6M、1M。

Fig. 6 B show, the influence that the pH value of buffer solution assembles the particle of 495 protein mutants 1.The pH of selection for 4.0, 5.0、6.0、6.5、7.0、7.5、8.0、8.5。

Fig. 6 C show, the influence that temperature assembles the particle of 495 protein mutants 1.The temperature of selection for 4 DEG C, 15 DEG C, 25 ℃、37℃。

Fig. 6 D show, the influence that the concentration of phosphate solution assembles the particle of 495 protein mutants 1.The phosphoric acid of selection Salt buffer concentration is 10mM, 20mM, 50mM, 100mM.

Fig. 7 A show the electron microscopic observation result of 495 particles.Bar（Bar）, 100nm.

Fig. 7 B show the sieve chromatography result of 495 particles.It compares as 239 particles.

Fig. 7 C show the dynamic scattering analysis result of 495 particles.

Fig. 7 D show the sedimentation velocity analysis result of 495 particles.

Fig. 7 E-7G are shown by the transmission electron microscope observing result of the mutant 2-4 VLP formed.

Fig. 8 shows 495 particles and various HEV（I）The WB reactivities of specific antibody.Used antibody 1-10 （Corresponding to swimming lane 1-10）Respectively：8C11、8G12、8H3、9F7、6F8、12A10、16D7、4A6、1B7、3G4.

Fig. 9 shows 495 particles and HEV（I）The Elisa reactivities of specific antibody.Used antibody is respectively： 8C11、8G12、8H3、9F7、6F8、12A10、16D7、4A6、1B7、3G4；It compares as 239 particles.

Figure 10 shows 495 particles and HEV（I）The EC50 analyses of specific antibody 8C11.8C11 initial concentrations are 4000ng/ml, is serially diluted 24 gradients continuous twice；It compares as 239 particles.

Figure 11 shows 495 particles and HEV（I）The WB reactivities of specific antibody 8C11.8C11 dilution ratios are 1: 2K；It compares as 239 particles.

Fig. 8's -11 by the HEV VLP that 495 protein mutants 1 assemble the results show that remains good immunoreactivity, It can be identified by a variety of HEV specific antibodies.

Figure 12 shows the ability of 495 granular absorption cells.Blocking antibody is 8C11, detection antibody be 16D7, thinner ratio Example is 1:2K；It compares as 239 particles.The results show that 495 particles and 239 particles can absorb into cell, and the process Antibody can be blocked（Such as monoclonal antibody 8C11）It is blocked.

Figure 13 shows the electron microscopic observation result of 495 pseudovirus.Bar（Bar）, 100nm.

Figure 14 shows the sieve chromatography analysis of 495 pseudovirus.A:The double UV check result of 495 particles；B:N31- The double UV check result of GFP plasmids；C:495 particles wrap up N31-GFP plasmids（Form pseudovirus）Double UV check knot afterwards Fruit；Detection wavelength is 260nm and 280nm.The results show that 495 particles can wrap up N31-GFP plasmids, 495 pseudovirus are formed, And the retention time of 495 pseudovirus is 10.8min.

Figure 15 shows the inverted fluorescence microscope observation result after 495 pseudovirus transfection HepG2.Figure 15 A:Individually GFP plasmids can Successful transfection HepG2 cells；Figure 15 B:Inverted fluorescence microscope observation after 495 pseudovirus adherent cells three days As a result；Figure 15 C:Inverted fluorescence microscope observation result after 495 pseudovirus adherent cells seven days.The results show that 495 pseudovirus HepG2 cells can be infected, and reporter GFP is expressed in cell.

Figure 16 shows the immunogenicity of 495 particles.It compares as 239 particles.The dosage used is respectively 0.5ug（Figure 16A）And 5ug（Figure 16 B）.The results show that 495 particles have the immunogenicity similar to 239 particles, can body be triggered to produce Raw neutralizing antibody.In addition, result is also shown, in low dosage（0.5ug）Under, 495 particles are even exempted from more higher than 239 particles Epidemic focus.

Sequence information

In the table 1 that the information of sequence of the present invention is provided below.

Table 1：The description of sequence

SEQ ID NO:	Sequence description	Sequence information
			1	Encode the nucleotide sequence of p495 protein mutants 1	It sees below
2	The amino acid sequence of p495 protein mutants 1	It sees below
			3	Encode the nucleotide sequence of p495 protein mutants 2	It sees below
4	The amino acid sequence of p495 protein mutants 2	It sees below
			5	Encode the nucleotide sequence of p495 protein mutants 3	It sees below
6	The amino acid sequence of p495 protein mutants 3	It sees below
			7	Encode the nucleotide sequence of p495 protein mutants 4	It sees below
8	The amino acid sequence of p495 protein mutants 4	It sees below
			9	Encode the nucleotide sequence of p495 protein mutants 5	It sees below
10	The amino acid sequence of p495 protein mutants 5	It sees below
			11	Encode the nucleotide sequence of p495 protein mutants 6	It sees below
12	The amino acid sequence of p495 protein mutants 6	It sees below
			13	495F	5’-CATATGGCGGTCGCTCCGGCTC-3’
14	495R	5’-GAATTCTTACGCAGAGTGGGGGGCT-3’

(the SEQ ID NO of sequence 1:1)：

ATGGCGGTCGCTCCGGCTCATGACACCCCGCCAGTGCCTGATGTTGACTCCCGCGGCGCTATCCTGCGC CGGCAGTATAACCTA

TCAACATCTCCCCTTACCTCTTCCGTGGCCACCGGTACAAACTTGGTTCTTTACGCCGCTCCTCTTAGC CCGCTTCTACCCCTC

CAGGACGGCACCAATACTCATATAATGGCTACAGAAGCTTCTAATTATGCCCAGTACCGGGTTGCTCGT GCTACAATTCGCTAC

CGCCCGCTGGTCCCCAACGCTGTTGGTGGCTACGCCATCTCCATCTCGTTCTGGCCACAGACCACCACC ACCCCGACGTCCGTC

GACATGAATTCAATAACCTCGACGGATGTCCGTATTTTAGTCCAGCCCGGCATAGCCTCCGAGCTTGTT ATCCCAAGTGAGCGC

CTACACTATCGTAACCAAGGTTGGCGCTCTGTTGAGACCTCCGGGGTGGCGGAGGAGGAGGCCACCTCT GGTCTTGTTATGCTC

TGCATACATGGCTCACTTGTAAATTCTTATACTAACACACCTTATACCGGTGCCCTTGGGCTGTTGGAT TTTGCCCTCGAACTT

GAGTTCCGCAACCTCACCCCCGGTAATACCAATACGCGGGTCTCCCGTTACTCCAGCACTGCCCGTCAC CGCCTTCGTCGCGGT

ACAGATGGGACTGCCGAGCTCACCACCACGGCTGCTACCCGCTTCATGAAGGACCTCTATTTTACTAGT ACTAATGGTGTCGGT

GAGATCGGCCGCGGGATAGCGCTTACCCTGTTTAACCTTGCTGACACCCTGCTTGGCGGTCTACCGACA GAATTGATTTCGTCG

GCTGGTGGCCAGCTGTTCTACTCTCGCCCTGTCGTCTCAGCCAATGGCGAGCCGACTGTTAAGCTGTAT ACATCTGTAGAGAAT

GCTCAGCAGGATAAGGGTATTGCAATCCCGCATGACATCGACCTCGGGGAATCTCGAGTTGTTATTCAG GATTATGACAACCAA

CATGAGCAGGACCGACCGACACCTTCCCCAGCCCCATCGCGCCCTTTTTCTGTCCTCCGAGCTAATGAT GTGCTTTGGCTTTCT

CTCACCGCTGCCGAGTATGACCAGTCCACTTACGGCTCTTCGACCGGCCCAGTCTATGTCTCCGACTCT GTGACCTTGGTTAAT

GTTGCGACCGGCGCGCAGGCCGTTGCCCGGTCGCTCGACTGGACCAAGGTCACACTTGATGGTCGCCCC CTTTCCACCATCCAG

CAGCATTCAAAGACCTTCTTTGTCCTGCCGCTCCGCGGTAAGCTCTCCTTTTGGGAGGCGGGTACTACT AAAGCCGGGTACCCT

TATAATTATAATACCACTGCTAGcGACCAACTGCTCGTTGAGAATGCCGCCGGGCATCGGGTTGCTATTTCCACTTA CACCACT

AGCCTGGGTGCTGGCCCCGTCTCTATTTCTGCGGTTGCTGTTTTAGCCCCCCACTCTGCGTAA

(the SEQ ID NO of sequence 2:2)：

MAVAPAHDTPPVPDVDSRGAILRRQYNLSTSPLTSSVATGTNLVLYAAPLSPLLPLQDGTNTHIMATEA SNYAQYRVARATIRY

RPLVPNAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASELVIPSERLHYRNQGWRSVET SGVAEEEATSGLVML

CIHGSLVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYSSTARHRLRRGTDGTAELTTTAAT RFMKDLYFTSTNGVG

EIGRGIALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGIAIPHDI DLGESRVVIQDYDNQ

HEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLD WTKVTLDGRPLSTIQ

QHSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVA VLAPHSA

(the SEQ ID NO of sequence 3:3)：

CAGGACGGCACCAATACTCATATAATGGCTACAGAAGCTTCTAATTATGCCCAGTACCGGGTTGTTCGT GCTACAATTCGCTAC

TGCATACATGGCTCACCTGTAAATTCTTATACTAACACACCTTATACCGGTGCCCTTGGGCTGTTGGAT TTTGCCCTCGAACTT

TATAATTATAATACCACTGCTAGCGACCAACTGCTCGTTGAGAATGCCGCCGGGCATCGGGTTGCTATT TCCACTTACACCACT

AGCCTGGGTGCTGGCCCCGTCTCTATTTCTGCGGTTGCTGTTTTAGCCCCCCACTCTGCGTAA

(the SEQ ID NO of sequence 4:4)：

MAVAPAHDTPPVPDVDSRGAILRRQYNLSTSPLTSSVATGTNLVLYAAPLSPLLPLQDGTNTHIMATEA SNYAQYRVVRATIRY

CIHGSPVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYSSTARHRLRRGTDGTAELTTTAAT RFMKDLYFTSTNGVG

QHSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVA VLAPHSA

(the SEQ ID NO of sequence 5:5)：

AGCCTGGGTGCTGGCCCCGTCTCTATTTCTGCGGTTGCTGTTTTAGCCCCCCACTCTGCGTAA

(the SEQ ID NO of sequence 6:6)：

QHSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVA VLAPHSA

(the SEQ ID NO of sequence 7:7)：

GAGTTCCGCAACCTCACCCCCGGTAATACCAATACGCGGGTCTCCCGCTACTCCAGCACTGCCCGTCAC CGCCTTCGTCGCGGT

GCAGATGGGACTGCCGAGCTCACCACCACGGCTGCTACCCGCTTCATGAAGGACCTCTATTTTACTAGT ACTAATGGTGTCGGT

GCTGGTGGCCAGCTGTTCTACTCTCGCCCCGTCGTCTCAGCCAATGGCGAGCCGACTGTTAAGCTGTAT ACATCTGTAGAGAAT

AGCCTGGGTGCTGGCCCCGTCTCTATTTCTGCGGTTGCTGTTTTAGCCCCCCACTCTGCGTAA

(the SEQ ID NO of sequence 8:8)：

CIHGSLVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYSSTARHRLRRGADGTAELTTTAAT RFMKDLYFTSTNGVG

QHSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVA VLAPHSA

(the SEQ ID NO of sequence 9:9)：

TCAACATCTCCCCTTACCTCTTCCGTGGCCACCGGTACAAACTTGGTTCTTTACGCCGCTCCTCTTAgCCCGCTTCT ACCCCTC

CATGAGCAGGACCGACCGACACCTTCCCCAGCCCCATCGCGCCCTTTTTcTGTCCTCCGAGCTAATGATGTGCTTTG GCTTTCT

AGCCTGGGTGCTGGCCCCGTCTCTATTTCTGCGGTTGCTGTTTTAGCCCCCCACTCTGCGTAA

(the SEQ ID NO of sequence 10:10)：

CIHGSPVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYSSTARHRLRRGADGTAELTTTAAT RFMKDLYFTSTNGVG

QHSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVA VLAPHSA

(the SEQ ID NO of sequence 11:11)：

GACATGAATTCAATAACCTCGACGGATGTCCGTATTTTAGTCCAGCCCGGCATAGCCTCCGAGCATGTT ATCCCAAGTGAGCGC

AGCCTGGGTGCTGGCCCCGTCTCTATTTCTGCGGTTGCTGTTTTAGCCCCCCACTCTGCGTAA

(the SEQ ID NO of sequence 12:12)：

RPLVPNAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASEHVIPSERLHYRNQGWRSVET SGVAEEEATSGLVML

QHSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLVENAAGHRVAISTYTTSLGAGPVSISAVA VLAPHSA

Specific embodiment

It is intended to illustrate the present invention referring now to following（Rather than limiting the invention）Embodiment the present invention described.

Unless specifically stated otherwise, the experimental methods of molecular biology and immunodetection used in the present invention, substantially joins According to J.Sambrook et al., molecular cloning：Laboratory manual, second edition, CSH Press, 1989 and F.M.Ausubel et al., fine works molecular biology experiment guide, the 3rd edition, described in John Wiley＆Sons, Inc., 1995 Method carry out；The condition that the use of restriction enzyme is recommended according to goods producer.Those skilled in the art know, implement The example description present invention, and be not intended to limit scope of the present invention by way of example.

The structure of 1.495 albumen of embodiment and its mutant

With HEV Xinjiang Strain sequences（NC_001434.1）For template, ORF2 gene orders are synthesized（Invitrogen）And it connects Onto pMD-18T carriers, plasmid pMD-18T-ORF2 is obtained.Using pMD-18T-ORF2 as template, using 495F as forward primer（Its The end of sequence 5 ' introduces restriction enzyme Nde I sites（CATATG）, ATG is the initiation codon in escherichia expression system Son）, using 495R as reverse primer（Its sequence 3 ' end introduces restricted type restriction endonuclease BamHI sites）, in PCR thermal cyclers （Biometra T3）According to following condition carry out PCR reactions：

Amplified production is the DNA fragmentation of 1500bp or so.By the PCR product and pMD18-T carriers commercially（TAKARA is public Department's production）Connection is identified through NdeI/BamHI digestions, obtains the positive colony plasmid pMD 18-T-495 of 495 genes of insertion. Using M13 (+) primer, sequence verification is carried out to pMD 18-T-495 plasmids for Shanghai Bo Ya bio-engineering corporations.

By well known to a person skilled in the art method, carrying out point mutation to pMD 18-T-495 plasmids, preparing following prominent The cloned plasmids of variant, and it is respectively designated as pMD 18-T-495-Mut1, pMD 18-T-495-Mut2, pMD 18-T-495- Mut3, pMD 18-T-495-Mut4, pMD 18-T-495-Mut5 and pMD 18-T-495-Mut6.

Table 2：The site mutation explanation of mutant 1-6

Hereafter by taking mutant 1 as an example, schematically illustrate the structure of the expression plasmid of above-mentioned mutant.By mutant 1 （495-Mut1）Cloned plasmids pMD 18-T-495-Mut1 NdeI/BamHI digestions, and through NdeI/BamHI digestions PTO-T7 prokaryotic expression carriers（Luo Wenxin etc., bioengineering journal, 2000,16:53-57）It is connected, and is transferred to Escherichia coli； Plasmid is extracted, identifies to obtain the positive expression plasmid pTO-T7-495-Mut1 of insertion target fragment through NdeI/BamHI digestions.

Take 1 μ L plasmids（0.15mg/ml）Convert the competence Escherichia coli ER2566 that 40 μ L are prepared with Calcium Chloride Method（It is purchased from Invitrogen companies）, it is coated on containing kanamycins（Final concentration 100mg/ml, similarly hereinafter）Solid LB media（Ingredient： 10g/L peptones, 5g/L dusty yeasts, 10g/L sodium chloride, similarly hereinafter）, and 37 DEG C of quiescent cultures 10-12 hours are clear to single bacterium colony It is distinguishable.Picking single bacterium drops down onto LB liquid medium containing 4mL（Containing kanamycins）Test tube, under 37 DEG C 180 revs/min vibrate training Support 10 it is small when, therefrom take 1mL bacterium solutions in -70 DEG C preservation.

By method similar to the above, the expression plasmid of the mutant 2-6 of 495 albumen is prepared, is respectively designated as pTO- T7-495-Mut2, pTO-T7-495-Mut3, pTO-T7-495-Mut4, pTO-T7-495-Mut5 and pTO-T7-495-Mut6. In addition, also prepare HEV ORF2（Xinjiang Strain）495 albumen expression plasmid, be named as pTO-T7-495.

The expression of 2.495 albumen of embodiment and its mutant

5 μ L bacterium solutions are taken out from -70 DEG C of ultra low temperature freezers, are inoculated in LB liquid mediums of the 5mL containing kanamycins, Shaken cultivation under 37 DEG C of 250rpm until OD600 up to 0.5 or so；Then, it is transferred to LB culture mediums of the 500mL containing kanamycins In, when shaken cultivation 4-5 is small under 37 DEG C of 250rpm.When OD600 is up to 1.5 or so, IPTG to final concentration 0.4mM is added in, Under 250rpm, vibration induces 10,8 or 8 hours at 15 DEG C, 20 DEG C or 25 DEG C respectively.By culture with 8000rpm from Heart 5min collects thalline, and carries out ultrasonication to thalline.It after bacterial cell disruption, is centrifuged, and supernatant and precipitation is taken to be examined It surveys（Using coomassie brilliant blue staining or Western blot analysis, Western blot analysis uses anti-HEVORF2 monoclonal antibodies 16D7 （Dilution ratio 1：2000, the self-control of this laboratory））, to identify the expression of 495 albumen and its mutant.Testing result is as schemed Shown in 1.

The result of analysis chart 1A-1D understands that the amino acid residue of the aa244 of HEV ORF2 is for 495 albumen and its mutation The solubility expression of body is particularly significant.When aa244 is H, the albumen is mainly with inclusion bodies in culture of Escherichia coli Middle expression（Referring to Figure 1A and 1D）；However, when aa244 is L, the albumen is mainly with soluble form in culture of Escherichia coli Cracking supernatant in express（Referring to Figure 1B and 1C）.At the same time, the result of Fig. 1 is also shown, the mutation in other sites（For example, Aa188, aa284 and aa335）The inclusion body expression of solubility expression and 495 albumen in itself to 495 protein mutants is basic On do not influence（Referring to Figure 1B -1D）.

The purifying of 3.495 protein mutant of embodiment

The present embodiment by taking 495 protein mutants 1 as an example, illustratively illustrate can in Escherichia coli solubility expression 495 protein mutants purifying.

After IPTG induced expressions, 5min is centrifuged to collect thalline by 8000rpm, then corresponding to 10mL by 1g thalline splits Solve liquid（20mM Tris pH of buffer 7.2,300mM NaCl）Ratio thalline is resuspended with lysate, ice bath uses Ultrasonic Cell Disruptor （Sonics VCX750 type Ultrasonic Cell Disruptors）Handle thalline（Treatment conditions：Working time 15min, pulse 2s suspend 4s, output Power 55%）.Cellular lysate liquid is with 12000rpm, 4 DEG C of centrifugation 5min（Similarly hereinafter）, abandon precipitation and retain supernatant（That is, soluble table It reaches）.

It is pure at the beginning of saturated ammonium sulfate

With final concentration of 10%, 20%, 30%, 40%, 50%（Percent by volume）Saturated ammonium sulfate solution previous step is obtained The centrifugation supernatant obtained is precipitated, and is centrifuged after ice bath stirring 30min, and SDS-PAGE analyses are carried out to precipitation.Analysis result such as Fig. 2 It is shown.

The redissolution of Tris-HCl buffer solutions (TB) and phosphate buffer to precipitation

Precipitation is redissolved with 20mM TB pH8.0,20mM TB pH8.5,20mM TB pH9.0 buffer solutions respectively, SDS-PAGE analyses are carried out to redissolving supernatant and precipitation.Analysis result is as shown in Figure 3.

Fig. 3 shows the redissolution that the saturated ammonium sulphate of 495 protein mutants 1 is carried out with different buffer solutions.Swimming lane 1, Protein markers；Swimming lane 2, the precipitation after being redissolved with 20mM TB pH9.0；Swimming lane 3, after being redissolved with 20mM TB pH9.0 Supernatant；Swimming lane 4, the precipitation after being redissolved with 20mM TB pH8.5；Swimming lane 5, the supernatant after being redissolved with 20mM TB pH8.5；Swimming Road 6, the precipitation after being redissolved with 20mM TB pH8.0；Swimming lane 7, the supernatant after being redissolved with 20mM TB pH8.0；8,495 egg of swimming lane The control of white inclusion bodies.The results show that used various buffer solutions can redissolve the saturated ammonium sulfate of mutant 1 Precipitation, and the redissolution effect of 20mM TB pH8.5 is preferable.

DEAE anion-exchange chromatographies purify

Chromatographic purifying is carried out with DEAE anion-exchange columns.In short, loading is balanced with 20mM TB8.5 buffer solutions, then It is eluted with 20mM TB8.5+0.1M/0.3M/0.5M/1M NaCl, is finally eluted with 0.1M NaOH successively.Purifying Chromatogram and the SDS-PAGE analysis charts of each elutriated fraction are respectively as shown in Fig. 4 A, 4B.

Similarly, the mutant 2-5 of 495 albumen is purified using the above method.

Embodiment 4:The Property Verification of 495 protein mutants after purification

It according to the method described before, is analyzed using SDS-PAGE and sieve chromatography, during by the reservation of sample to be tested Between（Schuck P etc., Biophys J, 2000,78:1606-1619）Measure purified 495 protein mutants in the solution State.Measurement result is as shown in Fig. 5 A, 5B.

The results show that 495 protein mutants 1 after purification mainly exist in the solution with dimeric forms, and do not deposit In grain fraction.

Similar analysis is carried out to the mutant 2-5 of 495 albumen using the above method, and obtains similar result.

Embodiment 5：The particle assembling of 495 protein mutants

In the present embodiment, have studied using above-mentioned purified mutant 1-5 to assemble the optimal of HEV virus-like particles Condition.

Embodiment is confirmed as before, present inventors have surprisingly found that, 495 of the H244L mutation comprising HEV ORF2 Protein mutant can be expressed in the cracking supernatant of culture of Escherichia coli with soluble form.495 albumen obtained as a result, Mutant as inclusion body with denaturant without being handled, always close to its native state, so as to easily carry out Particle assembles.It for example, generally can be by dialysing 495 protein mutants to the phosphate containing certain salinity of certain temperature In buffer solution, 495 protein mutants to be made to be assembled into particle.

Below by taking 495 protein mutants 1 as an example, analyze 495 protein mutants 1 particle assembling ability and for The optimum condition of grain assembling.

The influence that salinity assembles the particle of 495 protein mutants 1

Purified 495 protein mutant, 1 solution is diluted to 0.7mg/ml, and is dialyzed to containing final concentration It is assembled for the 50mM PB 6.5 of 0.1M, 0.3M, 0.4M, 0.5M, 0.6M, 1M NaCl in buffer solution.It in addition, will be by 239 albumen The particle of assembling（Li et al., JBC, 2005）As control.Each 495 protein mutant 1 is analyzed by sieve chromatography after dialysis State of the sample in each assembling solution.

As a result as shown in Figure 6A.The results show that used buffer solution can make 495 protein mutants 1 that particle occur Assembling, and when NaCl concentration is 0.5M, assembling effect is preferable.Particularly, it is main in sample when NaCl concentration is 0.5M There are two types of components, one of which for 495 protein mutants 1 of the foregoing description dimeric forms, during the reservation of another component Between it is roughly the same with the VLP that p239 is assembled into, this shows in solution there are grain fraction, and particle shape and size and p239 The VLP being assembled into is similar.

The influence that the pH value of buffer solution assembles the particle of 495 protein mutants 1

Purified 1 solution of p495 protein mutants is diluted to 0.7mg/ml, and be dialyzed to pH4.0, 5.0th, 6.0,6.5,7.0,7.5,8.0,8.5 and the assembling solution containing 0.5M NaCl in.Pass through sieve chromatography point after dialysis Analyse state of the mutant 1 in each assembling buffer solution.

As a result as shown in Figure 6B.The results show that the influence unobvious that pH value assembles the particle of p495 protein mutants 1. However, when pH is 6.5, the efficiency of 495 protein mutant, 1 assembling assembly virus-like particle is higher.

The influence that temperature assembles the particle of 495 protein mutants 1

Purified 1 solution of p495 protein mutants is diluted to 0.7mg/ml, and is dialyzed to temperature as 4 DEG C, 15 DEG C, 25 DEG C, in the phosphate assembling solution of the 50mM pH6.5 of 37 DEG C of the NaCl containing 0.5M final concentrations.After dialysis State of the mutant 1 in each assembling solution is analyzed by sieve chromatography.

As a result as shown in Figure 6 C.The results show that at various temperatures, particle assembling can occur for p495 protein mutants 1. However, when it is 37 DEG C to assemble temperature, the packaging efficiency of p495 protein mutants 1 significantly improves.

The influence that the concentration of phosphate solution assembles the particle of 495 protein mutants 1

Purified 495 protein mutant, 1 solution is diluted to 0.7mg/ml, and is dialyzed to final concentration of In the NaCl containing 0.5M of 10mM, 20mM, 50mM, 100mM, the phosphate assembling solution of pH6.5.Pass through molecular sieve layer after dialysis State of the analysis analysis mutant 1 in each assembling solution.

As a result as shown in Figure 6 D.The results show that the concentration of phosphate solution influences unobvious to the assembling of 495 albumen.10- The phosphate solution of 100mM can make 495 protein mutant, 1 assembling assembly virus-like particle.

Embodiment 6：By the end-state of the virus-like particle of 495 protein mutants assembling

In the present embodiment, by taking 495 protein mutants 1 as an example, the optimal particle assembling condition that is described using embodiment 5, 495 purified protein mutants are dialysed at 37 DEG C to temperature be 37 DEG C, the phosphate that is 6.5 containing 0.5M NaCl, PH In solution, so as to obtain HEV virus-like particles（Hereinafter referred to as, 495 virus-like particles or 495 particles）.Use transmission electron microscope The methods of observation, sieve chromatography analysis, dynamic scattering analysis, sedimentation velocity analysis the property and final shape to 495 particles State is studied.

Transmission electron microscope observing

The 200kV transmission electron microscopes that the instrument used produces for Japan Electronics Corporation, 25,000 times of amplification factor.It will obtain 495 particles with 2% phosphotungstic acid pH7.0 negative staining, be fixed on the copper mesh of spray charcoal and observed.The result is shown in Fig. 7 A for Electronic Speculum.As a result show Show, a diameter of 30nm or so, virus-like particle of uniform size is largely presented in sample.

Molecule mesh analysis

Using the 1120Compact LC high performance liquid chromatography tomographic systems and TSK Gel of German Agilent PW5000xl7.8x300mm pillars carry out sieve chromatography analysis.With the buffer solution 20mM PB6.5+0.5M of 2 times of column volumes NaCl pre-balance chromatographic columns, until 280nm wavelength absorption values are without significant change.The absorption value of detector is zeroed, Ran Houyou Autosampler sample introduction is simultaneously analyzed.As a result as shown in Figure 7 B.The results show that the retention time of 495 particles is 13.7min.

Dynamic light scattering measures

The instrument used is the DynaPro MS/X type dynamic light scatterings of Protein Solutions companies of U.S. production （Containing temperature controller）, the algorithm used is Regulation algorithms.495 particulate samples obtained are through 0.22 μm of membrane filtration After measure.Measurement result is shown in Fig. 7 C.The results show that 495 particles are in the solution for component is single, hydrated molecule dynamics Radius is the particle of 15nm.

Sedimentation velocity analysis

The instrument used is U.S.'s Beckman XL-A analytic type ultracentrifuges, is furnished with Systems for optical inspection and An- 50Ti and An-60Ti rotary heads.The sedimentation coefficient of 495 particles is analyzed using sedimentation rate method.As a result as illustrated in fig. 7d.As a result show Show, the sedimentation coefficient of 495 particles is about 47.56S.

Similar analysis is carried out to the mutant 2-5 of 495 albumen using the above method, and obtains similar result. Particularly, Fig. 7 E-7G are shown by the transmission electron microscope observing result of the mutant 2-4 VLP formed.The results show that the big portion of sample Divide and a diameter of 30nm or so, virus-like particle of uniform size is presented.

Embodiment 7：The research of the immune response activity of 495 particles

The present embodiment is specifically single using existing HEV-I genotype by taking the HEV VLP assembled by 495 protein mutants 1 as an example It is anti-, pass through Western immunoblot experiments and enzyme-linked immunosorbent assay（ELISA）, the reactivity of 495 particles of analysis.

Pass through the reactivity of 495 particle of western blot experimental analysis and the special monoclonal antibody of HEV-I genotype

495 particulate samples after assembling are transferred to after 12% SDS-PAGE separation on nitrocellulose filter, with 5% degreasing Milk room temperature closes 2h；Add in diluted monoclonal antibody（1:1000）, it is placed in room temperature reaction 1h（Used monoclonal antibody 8C11,8G12, 8H3,9F7,6F8,12A10,16D7,4A6,1B7,3G4 are HEV-I idiotype specific antibodies prepared by this laboratory）；With TNT solution（8.765g NaCl, 1.21g Tris Base and 0.5ml Tween-20, adding deionized water, it is 8.0 to adjust pH to 1L） Wash film 3 times, each 10min；Then sheep anti mouse alkaline phosphatase is added in（1:5000）（KPL products）, it is placed in room temperature reaction 1h；With TNT washes film 3 times, each 10min；With NBT (Nitro blue tetrazolium) and BCIP (5-Bromo-4-chloro-3- Indolyl phosphate) substrate develops the color.As shown in Figure 8.

Pass through the reactivity of 495 particle of ELISA experimental analyses and the special monoclonal antibody of HEV-I genotype

With buffer solution 50mM PB6.5+0.5M NaCl diluted proteins to final concentration 1ng/ μ l, then carried out on 96 orifice plates Coating（It is coated with 100 μ l of volume）, 37 DEG C of incubation 2h.It after 37 DEG C are closed 2h, drains, it is 1 to add in dilution ratio:100 monoclonal antibody （Selected monoclonal antibody 8C11,8G12,8H3,9F7,6F8,12A10,16D7,4A6,1B7,3G4 are HEV-I prepared by this laboratory Idiotype specific antibody）, three times are dilute again, dilute 12 gradients；37 DEG C of reaction 30min；Then board-washing adds in HRP marks Anti- mouse IgG secondary antibodies（KPL products）, 37 DEG C of reaction 30min；Board-washing, 37 DEG C of colour developing 15min, then reads light absorption value at 450nm （Reference wavelength is 620nm）.Positive control is p239.The readings mapping in the range of linearity is chosen, as shown in Figure 9.

The EC50 of 495 particles monoclonal antibody 8C11 special to HEV-I genotype

It is detected using direct method.In short, 495 particles after assembling are coated with according to 100ng/ holes, in 37 DEG C When incubation 2 is small in incubator, confining liquid is added in after washed once, when incubation 2 is small in 37 DEG C of incubators, taking-up pats dry.Add in 8C11 （Initial concentration 4000ng/ml）, 2 times dilute, and dilute 24 gradients, and 60min is incubated in 37 DEG C of incubators, wash 5 times, add in two Anti- GAM-HRP（Dilution gradient 1:5000）It in 37 DEG C of incubation 30min, washs 5 times, adds in developing solution A, B each 50ul, 37 DEG C Develop the color 15min, readings immediately after termination.Reference protein is p239.The results are shown in Figure 10.

The reactivity of 495 particles monoclonal antibody 8C11 special to HEV-I genotype is analyzed by WB

495 particulate samples after assembling are transferred to after 12% SDS-PAGE separation on nitrocellulose filter, with 5% degreasing Milk room temperature closes 2h；Add in diluted monoclonal antibody 8C11（Prepared by laboratory, dilution factor 1:1000）, it is placed in room temperature reaction 1h；With TNT solution（8.765g NaCl, 1.21g Tris Base and 0.5ml Tween-20, adding deionized water, it is 8.0 to adjust pH to 1L） Wash film 3 times, each 10min；Then sheep anti mouse alkaline phosphatase is added in（1:5000）（KPL products）, it is placed in room temperature reaction 1h；With TNT washes film 3 times, each 10min；With NBT (Nitro blue tetrazolium) and BCIP (5-Bromo-4-chloro-3- Indolyl phosphate) substrate develops the color.Reference protein is p239.As shown in figure 11.

Above-mentioned experiment the results show that remaining good immune response by the HEV VLP that 495 protein mutants 1 assemble Property, it can be identified by a variety of HEV specific antibodies.Using the above method to the HEV of the mutant 2-5 assemblings by 495 albumen VLP carries out similar analysis, and obtains similar result.

Embodiment 8：495 granular absorptions enter the research of cell

Those skilled in the art are known, 239 particles can simulate natural viral granular absorption to cell surface mucous membrane and oneself Main intrusion cell interior.Whether the present embodiment analyzes 495 particles by taking the HEV VLP assembled by 495 protein mutants 1 as an example With the property similar to 239 particles.

Respectively by 495 particles（20ug）, 239 particles（20ug）（239 granule proteins are positive control）, 495 particles+mono- After anti-8C11 or 239 particles+monoclonal antibody 8C11 and HepG2 cell incubations 30min, cell is cleaned to remove residual particles sample. After cell cracking, obtain supernatant and pass through the presence of 495 albumen of Western immunoblotting assays and 239 albumen.For examining The sample concentration of survey is strictly set to 1mg/ml, and the used primary antibody that detects is 16D7, extension rate 1：2000, secondary antibody GAM- HRP, extension rate 1：5000, detecting system is 400 mini of GE ImageQuant LAS.Western immunoblotting assays Result it is as shown in figure 12.

The results show that 495 particles and 239 particles can absorb into cell, and the process can be blocked antibody （Such as monoclonal antibody 8C11）It is blocked.

Similar analysis is carried out to the HEV VLP of the mutant 2-5 assemblings by 495 albumen using the above method, and is obtained Obtained similar result.

Embodiment 9：The research of HEV pseudovirus is prepared using 495 particles

If embodiment 5 and 6 is described, p495 albumen can be self-assembled into VLP in vitro.The present embodiment is with by 495 eggs The HEV VLP and reporter gene green fluorescence protein gene that white mutant 1 assembles（GFP）Exemplified by, it analyzes 495 particles and whether can It is enough to prepare HEV pseudovirus（That is, whether 495 particles can wrap up foreign gene in its assembling process, form HEV cape horn fevers Poison）.

In short, by 495 protein mutants 1 and GFP plasmids in molar ratio 1：1 mixing adsorbs at least 2h, then in 37 DEG C Dialysis is assembled into assembling buffer solution.After being completed, nuclease Benzonase Nuclease are utilized（It is purchased from Merck KgaA companies）By GFP plasmid removings remaining in mixed system（Action condition：37ug DNA/1u, 37 DEG C, 30min）.Then, Mixture through nucleic acid enzymatic treatment is used for infection cell.Control group GFP plasmids for individual 495VLP and individually.

Using transmission electron microscope, sieve chromatography analyze, inverted fluorescence microscope the methods of verify pseudovirus prepare whether into Work(.

Use transmission electron microscope observing pseudovirus form

The 200kV transmission electron microscopes that the instrument used produces for Japan Electronics Corporation, 25,000 times of amplification factor.It will obtain 495 particles with 2% phosphotungstic acid pH7.0 negative staining, be fixed on the copper mesh of spray charcoal and observed.As a result as shown in figure 13.As a result show Show, the form of pseudovirus and the form of 495 particles are basically identical.

Sieve chromatography analyzes pseudovirus retention time

Using 5000 PWxl 7.8x300mm pillars of U.S. Waters high performance liquid chromatography tomographic system and TSK Gel Carry out sieve chromatography analysis.With the buffer solution 50mM PB6.5+0.5M NaCl pre-balance chromatographic columns of 2 times of column volumes, until Absorption value at 280nm is without significant change.The absorption value of detector is zeroed, then by autosampler sample introduction and is divided Analysis.As a result as shown in figure 14, Figure 14 A, B, C be respectively individual 495 particle, individual N31-GFP plasmids and 495 particles+ The analysis result of N31-GFP plasmids.The results show that 495 particles in mixture can wrap up N31-GFP plasmids, it is false to form 495 Virus, and the retention time of 495 pseudovirus is 10.8min.

Use infection of the inverted fluorescence microscope observation pseudovirus to cell

By pseudovirus with the 3rd day after cell incubation, observed using inverted fluorescence microscope within the 7th day（NIKON is public Department, model：TE2000-U）.Observe result such as Figure 15 B（3rd day）, Figure 15 C（7th day）It is shown；Figure 15 A, which are shown, passes through PEI Method is with N31-GFP plasmid profit transfection HepG 2 cells as a result, it is used as positive control.The results show that pseudovirus can infect HepG2 cells, and reporter GFP is expressed in cell.

The above results show that 495 particles have the ability of infection cell, and can wrap up foreign gene and form cape horn fever Poison, for foreign gene to be imported aim cell.Using the above method to the HEV VLP of the mutant 2-5 assemblings by 495 albumen Similar analysis is carried out, and obtains similar result.

Embodiment 10：The research of the immunogenicity of 495 particles

If embodiment 5 and 6 is described, p495 albumen can be self-assembled into VLP in vitro.The present embodiment is with by 495 eggs Exemplified by the HEV VLP that white mutant 1 assembles, the immunogenicity of 495 particles is further studied.

The ED50 of 495 particles

Using aluminium adjuvant, single intraperitoneal injection mode is female to 6 week old BalB/c, male each 3 of mouse is immunized.Control group For 239 particles, immunizing dose is 1.6 μ g, 0.4 μ g, 0.1 μ g, 0.025 μ g.495 particle of experimental group separately have 0.6 μ g of immunizing dose, 0.4 μ g, 0.1 μ g, 0.025 μ g, 0.00625 μ g, 0.0015625 μ g, it is 1mL that volume, which is immunized,.Eye socket is taken a blood sample during 4th week, to blood HEV antibody in liquid is detected, and calculates ED50 by Reed-Muench methods.The results are shown in Table 3.

Table 3

Table 3A.239 particles are in the ED of mouse Immune inducing in vivo HEV antibody male rotaries₅₀

Table 3B.495 particles are in the ED of mouse Immune inducing in vivo HEV antibody male rotaries₅₀

The results show that when mouse 4 weeks is immunized with 495 particles, ED50 is 0.068 μ g, and it is good that this shows that 495 particles have Good, even better than 239 particles immunogenicities.

The investigation of serum titer after BalB/c mouse are immunized with 495 granule proteins

Using aluminium adjuvant, injected s.c. is female to 6 week old BalB/c, male each 3 of mouse is immunized.Control group is 239 particles, immunizing dose are 5 μ g or 0.5 μ g, and it is 200 μ l that volume, which is immunized,.Just exempt from after the blood sampling of zero circle eye socket, then respectively at the 2nd Booster immunization was carried out with 4 weeks.To mouse, blood was collected weekly, analyzes the titre of the Anti-HEV antibody in serum.As a result such as Figure 16 A （0.5μg）With Figure 16 B（5μg）It is shown.The results show that 495 particles have the immunogenicity similar to 239 particles, can draw It sends out body and generates neutralizing antibody.In addition, result is also shown, in low dosage（0.5μg）Under, 495 particles are even with than 239 particles Higher immunogenicity.

Embodiments herein confirms there is 495 protein mutation physical efficiencys of leucine on the aa244 of HEV ORF2 for the first time It is enough to be expressed in solublely in the cracking supernatant of culture of Escherichia coli, and such mutant can be assembled into and has in vitro Good immunogenicity and immunoreactive HEV virus-like particles.Therefore, such mutant can be used for efficient, low cost to prepare HEV vaccines have significant advantageous effects.

Although the specific embodiment of the present invention has obtained detailed description, it will be appreciated by those skilled in the art that：Root According to all introductions having disclosed, details can be carry out various modifications and changed, and these change in the guarantor of the present invention Within the scope of shield.The four corner of the present invention is provided by appended claims and its any equivalent.

Claims

1. a kind of mutant of p495 albumen, is made of the aa112-aa606 of HEV ORF2, and with HEV ORF2's Leucine is included on the corresponding sites of aa244；Optionally, it is described prominent compared with the aa112-aa606 of wild type HEV ORF2 Variant also includes, on the corresponding sites of the aa188 of HEV ORF2 alanine, opposite with the aa284 of HEV ORF2 The proline on site answered and with 1 in the threonine on the corresponding sites of the aa335 of HEV ORF2 or 2；Institute State mutant can in the culture supernatant of Escherichia coli solubility expression, and HEV virus-likes can be assembled into vitro Grain.

2. the mutant of claim 1, the amino acid sequence such as SEQ ID NO of the mutant：2, SEQ ID NO：4, SEQ ID NO：6, SEQ ID NO：8 or SEQ ID NO：Shown in 10.

3. the mutant of claim 1, the amino acid sequence such as SEQ ID NO of the mutant：Shown in 2.

4. a kind of mutant of any one of separated nucleic acid, coding claim 1-3.

5. the carrier of the separated nucleic acid comprising claim 4.

6. the host cell of the carrier of separated nucleic acid and/or claim 5 comprising claim 4.

7. a kind of HEV virus-like particles, the mutant containing any one of claim 1-3 or any by claim 1-3 The mutant composition of item.

8. a kind of composition, it includes the mutant of any one of claim 1-3 or the separated nucleic acid of claim 4 or power Profit requires 5 carrier or the host cell of claim 6 or the HEV virus-like particles of claim 7.

9. a kind of pharmaceutical composition or vaccine, optionally can also comprising pharmacy it includes the HEV virus-like particles of claim 7 The carrier and/or excipient of receiving.

10. the pharmaceutical composition or vaccine of claim 9, the HEV virus-like particles are to prevent or treat HEV infection or penta type Hepatitis effective quantity exists.

11. the method for the mutant of the p495 albumen of any one of claim 1-3 is prepared, including utilizing Bacillus coli expression Then cracking supernatant containing the mutant is carried out purification process by mutant described in system solubility expression.

12. the method for claim 11, the condition for the solubility expression of escherichia expression system are, 15 DEG C of isopropyl sulphur For galactoside (IPTG) induction 10 it is small when, 20 DEG C of isopropylthiogalactosides (IPTG) induction 8 it is small when or 25 DEG C of isopropyls When thiogalactoside (IPTG) induction 8 is small.

13. the method for claim 11 or 12, the purification process include, ammonium sulfate is added into cracking supernatant with by protein It is precipitated out, is then redissolved albumen precipitation using redissolution buffer solution, then using anion-exchange chromatography to redissolution Protein is purified, so as to obtain the mutant of purified p495 albumen.

14. the method for claim 13, the ammonium sulfate is saturated ammonium sulfate solution.

15. a kind of method for preparing HEV virus-like particles, including dialysing the mutant of any one of claim 1-3 to group It fills in solution, and it is made to be self-assembled into VLP.

16. the method for claim 15, the mutant be prepared by the method for claim 11 it is purified prominent Variant.

17. a kind of method for preparing vaccine, including by the HEV virus-like particles of claim 7 or passing through claim 15 The HEV virus-like particles that method obtains are mixed with pharmaceutically acceptable carrier and/or excipient.

18. the mutant of any one of claim 1-3 or the HEV virus-like particles of claim 7 pass through claim 11 The mutant that obtains of method or pharmaceutical composition is being prepared by the HEV virus-like particles that the method for claim 15 obtains Or the purposes in vaccine, described pharmaceutical composition or vaccine are used to preventing or treating HEV infection or the disease caused by HEV infects Disease.

19. the purposes of claim 18, the disease caused by HEV infects are Hepatitis Es.