CN104857510A - Hepatitis E virus-like particle vaccine preparation method and application - Google Patents

Hepatitis E virus-like particle vaccine preparation method and application Download PDF

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CN104857510A
CN104857510A CN201510197903.8A CN201510197903A CN104857510A CN 104857510 A CN104857510 A CN 104857510A CN 201510197903 A CN201510197903 A CN 201510197903A CN 104857510 A CN104857510 A CN 104857510A
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hepatitis
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chicken
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CN104857510B (en
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吕宏亮
张澍
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Abstract

The invention discloses a hepatitis E virus-like particle vaccine preparation method, and belongs to the biological medicine technical field. According to the method, hepatitis E virus truncated capsid protein gene codon optimization is performed, prokaryotic expression system escherichia coli strain B834-pRARE2 is used, by fusion of purification tag MBP expression, different cracking agent screening and combination, bacterial membrane extraction, maltose amylose affinity chromatography, molecular sieve chromatography for removal of MBP and non-virus structural protein, and promotion of virus-like particle assembly, the hepatitis E virus-like particle purity is more than 99.0%, and is free of protein label and bioactive (in the form of non-inclusion body, and soluble). The hepatitis E virus-like particle vaccine produces complete protection to pig, dog and chicken HEV infection, cross protective antigens are between different genotype HEV virus, and the hepatitis E virus-like particle vaccine can be used as an animal vaccine to prevent the diseases and infection caused by pig, dog and poultry HEV.

Description

A kind of preparation method of hepatitis E virus sample particle vaccines and application thereof
Technical field
The present invention relates to a kind of preparation method and application thereof of hydrophobicity viral capsid proteins, particularly a kind of preparation method of hepatitis E virus sample particle vaccines and application thereof, belong to biomedicine technical field.
Background technology
Hepatitis E virus (Hepatitis E virus, HEV) is one of hepatitis virus section hepatovirus member, and containing a forward single stranded RNA, genome has 3 overlapping open reading frame framves (ORFs), comprises ORF1, ORF2, ORF3.Virus is divided into 4 kinds of genotype, and Genotype I is mainly separated from human body with gene II type virus, and gene type III, Genotype IV virus are mainly derived from the animals such as pig, chicken, rabbit, dog, even has gene V-type virus to find.Genotype I virus mainly China and Southeast Asia popular, HEV is the main pathogen of acute hepatitis, and cause moderate, Severe Hepatitis, the order of severity increased with the age.
HEV is popular in many developing countries, particularly South Asia, Southeast Asia, north African, Middle East, and the Epidemiological study whole world has 1/3 people to infect.HEV is propagated by the fecal-oral route drinking polluted source.The mortality rate 1-15% that ordinary people infects, but lethal actute infection is caused to pregnant woman, mortality rate is up to 30%, and high probability miscarriage, premature labor occur the pregnant woman infecting survival.To chronic hepatitis patient, HEV superinfection causes serious consequence.So the HEV sanitarian disease that is serious threat, diagnose fast and prevent HEV to be necessary.
Although the hepatitis E of people sickness rate in general population is less than 1%, in developing country or the bad area of sanitary condition, because the extensive acute hepatitis E in water source polluted often occurs, it is popular that developed country also has food pollution to distribute.So far, identify and determined 4 genotype HEV viruses, Genotype I, gene II type virus is only propagated crowd, and gene type III, Genotype IV virus is propagated between animal, has even found popular gene V-type, genotype VI hepatitis E virus in wild boar and rat.All pig hepatitis E virus distinguished belong to gene type III or Genotype IV virus, can propagate, the HEV Ye Neng infected pigs of other animal dogs, rabbit across the barrier between kind.The animal derived hepatitis e virus infection of people is often multiple is born in the meat products or water of having consumed the Hepar Sus domestica, Carnis Sus domestica, Carnis Cervi or other pollutions infectiousness HEV that pollute.
Chicken hepatitis E virus starts an inflammation of the liver splenomegaly syndrome (hepatitis splenomegaly syndrome (HSsyndrome)), causes the laying hen ovarian atrophy in 30-72 week, hepatosplenomegaly, a large amount of red hydrops in abdominal cavity.The genomic gene order-checking display fowl HEV genome structure of fowl HEV is similar with mammal, and nucleotides sequence shows the homology of 50%, with people HEV and pig HEV gene structure and functional similarity.The capsid protein of fowl HEV and mammal have common antigenicity and epitope.The nucleotide homology of fowl HEV that the fowl HEV in U.S. state, European fowl HEV, Southeast Asia are separated reaches 80%, should belong to the genus that hepatitis virus is different, and other people, pig, dog HEV are that the different hepatitis virus of hepatitis virus section belongs to.
The SPF chicken intravenous injection 5 × 10 in 60 week age 4.550% fowl infective dose, cause HS syndrome, lymphocyte lymphocytic periphlebitis and phlebitis is had to be the hepatic injury of feature, about have the chicken of 25% infection to have the loose phenomenon of the hemorrhage or liver right middle lobe of subcapsular, slurry liver enzyme lactase dehydrogenase slightly rises (the plasmaliverenzyme lactate dehydrogenase).The pathogenic discovery of fowl HEV is caused a disease to HEV and the research of immunity provides little individual animal model, and be also that assessment and the research of HEV vaccine provides platform, particularly vaccine is to the control effect of HS syndrome simultaneously.
Usually, healthy chicken infects HEV has antibody to produce, and is shown as inferior clinical symptom.Be 91% from the chicken of HS with the homology of the viral nucleotide be separated of healthy chicken, its pathogenic difference, therefore vaccine is different to the protectiveness of the strain of different HEV, the protected effect of example highlights to the different pathogenic strain of fowl.
Fowl hepatitis E virus is popular in chicken group, the type that starts an inflammation of the liver splenomegaly syndrome, Australia, Spain, the U.S., China find authenticated at least 3 genotype HEV, and people, pig source HEV nucleotide homology 50-60%.In addition, rat, Mongoose, deer, rabbit, fish all find that there is HEV, and mammals HEV only has 1 serotype.
The vaccine of attenuation or deactivation is because fail to develop without the cell system be applicable to, China's exploitation is only had to have registered the HEV virus sample particle vaccines of an escherichia coli expression, but the virus-like particle manufacturing process expressed is complicated, the albumen of expressing is inclusion body, needs complicated refolding strategy process ability assembling assembly virus-like particle.In HEV albumen, ORF1 encodes nonstructural proteins, does not form neutralizing antibody in immunity, and ORF3 encodes the unclear non-structural protein of a kind of function, and short-term forms antibody, but without neutralization.ORF2 encodes 72kD capsid protein, is made up of 660 aminoacid, and composition fragment wherein has immunogenicity and at each genotype hepatitis E virus high conservative.In the antibody in vitro of capsid protein and HEV, protection non-human primate ape and monkey are from infection, and digital preservation.ORF2 total length capsid protein (72kDa) is not suitable for as vaccine antigen, because it covers immunodominant epitope, has certain hydrophobic nature.The capsid protein 55kDa blocked is that one is comparatively stable, safety and have immunogenic vaccine antigen.Because HEV lacks effective tissue culturing system, the development for HE deactivation and attenuated live vaccine causes difficulty, and current HE vaccine research mainly concentrates on the development of HE recombinant vaccine.ORF2 contains important Neutralization and crystallization, and the existing many fragments of ORF2 are successfully expressed in different expression system at present, and as expression systems such as protokaryon, insecticide, yeast, plant cells, and expression product all has certain immunogenicity.
The capsid protein of HEV restructuring can induce HEV neutralizing antibody on people, non-human primate, chicken; the capsid protein wherein blocked; containing HEV immune epitope and form virus-like particle, strengthen immune effect of vaccine, and to allos HEV virus attack produce cross protection.
Summary of the invention
Hepatitis E virus is owing to lacking suitable vaccine bebcell substrate, the requirement of biological product antigen or vaccine can not be reached by the method for cell culture, now prepared by the gene engineering method that adopts more, and the general eukaryotic system that adopts is expressed, as insecticide, mammalian cell, yeast.With eukaryotic system express, extract, the step of purification is many, yield is low, and the cost of instrument and Factory Building is expensive and require high.Total length capsid protein (72kDa) is not suitable for, as HEV virus sample particle vaccines antigen, because it covers immunodominant epitope, having certain hydrophobic nature.The capsid protein 55kDa blocked is that one is comparatively stable, safety and have immunogenic vaccine antigen.
For above problem, the invention discloses a kind of method preparing hepatitis E virus sample particle vaccines, it is characterized in that, comprise the following steps:
(1) synthesize wild type hepatitis E virus total length ORF2 gene, after gene chemical synthesis, insert cloned plasmids pGEM-T;
(2) the Δ ORF2 gene that hepatitis E virus blocks is synthesized
Use the codon of escherichia coli hobby, the Δ ORF2 gene blocked of design coding hepatitis E virus 112-660 amino acids, then with wild type hepatitis E virus total length ORF2 gene for template, design primer synthesis Δ ORF2 gene, the Δ ORF2 gene outcome of synthesis connects with the pMAL-p4x expression plasmid fragment of same enzyme action;
(3) expression of Δ ORF2 gene in escherichia coli blocked of hepatitis E virus
Connect product conversion escherichia coli, select positive colony, order-checking, obtains pMAL-p4X MBP-Δ ORF2 recombiant plasmid; Recombiant plasmid pMAL-p4xMBP-Δ ORF2 inoculates LB culture medium, be transferred in the fermentation tank of the 20L containing 18L LB culture medium by 1: 200 ~ 1: 1000 (V/V), 4 ~ 6h is cultivated with 250rpm rotating speed 37 DEG C, when bacterium liquid OD600 reaches 0.6 ~ 0.8, add IPTG by concentration 0.5 ~ 0.7mmol/L and induce 4h, the centrifugal 25min of 4000rpm, obtains the yeast culture thing that weight in wet base is 110 ~ 120g/L;
(4) Isolation and purification of capsid protein Δ ORF2 that blocks of hepatitis E virus
The screening of a decomposition agent and bacterial membrane protein extract
Yeast culture thing step (3) obtained melts, be resuspended in chromatography buffer, with Ultrasound Instrument fracturing cell walls in ice bath, ultrasonic liquid 75, 000g, 4-8 DEG C is separated upper cleer and peaceful precipitation for centrifugal 1 hour, precipitation lysate dissolves, add decomposition agent and stir 2h extraction escherichia coli memebrane protein at 4 DEG C, measure the activity of extract, to determine the ratio of total protein and decomposition agent, extract is with 100, 000g ultracentrifugation 1 hour, obtain soluble upper, be that 0.5-1.0% (w/v) is so that subsequent purification by the concentration that chromatography buffer is diluted to final lysate again,
Wherein, described chromatography buffer contains 20mM NaH 2pO 4, 100mM NaCl, 1 μM of protease inhibitor E-64,0.3mM tricarboxylic methyl acid phosphate, pH 7.5;
Described lysate contains 20mM Tris-HCl, 300mMNaCl, 1mM 2 mercapto ethanol, pH 8.0;
Described decomposition agent is the mixture of Triton X-100 and DDM;
B maltose-binding protein-Δ ORF2 purification
Under 4 DEG C of conditions, 20mL amylose medium dress post, balance with the balance solution containing 1.0% (w/v) Triton-X100 and 1.0% (w/v) DDM of 3-5 times of column volume, supernatant containing maltose-binding protein-Δ ORF2 carries out siphon loading with the speed of 2mL/min, wash with the balance solution containing 1.0% (w/v) Triton-X100 and 1.0% (w/v) DDM of 10 times of column volumes after loading, again with 3 times of column volumes not containing decomposition agent Triton-X100, the balance solution washing of DDM and EDTA, finally with solution (the 20mM Tris containing 10mmol maltose, pH 8.0, 0.2M NaCl, 10mM maltose) eluting, Fraction collection, measure protein content, purity,
Wherein, described level pad contains 20mM Tris-HCl, pH 8.0,300mM NaCl, 1mM 2 mercapto ethanol, 1mM EDTA;
C enzyme action
Add the Xa factor normal-temperature reaction 36-48h of 1 unit by 75-100ug albumen, make enzyme action degree reach 90-95%;
D anti-maltose antibody Sepharose 4 affinitive layer purification
After endonuclease reaction liquid fully mixes with anti-maltose antibody coupling Sepharose 4 affinity media, hatch 18-24 hour for 4 DEG C, absorption removing maltose-binding protein, collect stream and wear liquid, stream wears the centrifugal rear results supernatant of liquid chamber temperature 4000rpm, obtain the hepatitis E virus Δ ORF2 albumen of purification at supernatant, purity detecting can reach 90%;
E sieve chromatography purification
Upper step stream wear liquid centrifugal after supernatant in, adding Triton X-100 makes its final concentration be 0.031% (w/v), be that the filter centrifugal concentrating of 20kDa is to containing 5-10mg/mL albumen by cutoff value, 2500mLSuperdex 20002/150 post is used to carry out sieve chromatography, protein concentrate injection loading, chromatographic column balance liquid eluting, Fraction collection eluent, collect liquid and detect purity and content, merge and collect eluent and concentrate with the ultrafilter dialysis that cutoff value cutoff value is 20KDa, obtain highly purified Δ ORF2 albumen, purity detecting can reach 99.0%;
Wherein, described balance liquid contains 10mM HEPES, pH 7.2,150mM NaCl, 0.3mM TCEP, 0.031% (w/v) Trioton X-100;
(5) hepatitis E virus sample particle vaccines preparation
According to the difference using object, adopt dissimilar adjuvant, when people is as use object, the Δ ORF2 albumen of purification adds adjuvant aluminum hydroxide absorption by 0.6-0.7mg/mL, to obtain final product; When pig, dog and chicken are as use object, the Δ ORF2 albumen of purification adds W/O/W adjuvant emulsion by 1: 3 weight ratio, to obtain final product.
In the present invention, preferably, the primer described in step (2) is:
Forward primer:
5’-AA GGATCCATGGCGGTCGCTCCAGCCCATGACACCCCGCCAGT-3’；
Downstream primer:
5’-GG TCTAGACTATAACTCCCGAGTTTTACCCACCTTCATCTT-3’。
In the present invention, preferably, the escherichia coli described in step (3) are B834-pRARE2.
In the present invention, preferably, in the decomposition agent described in step (4), Triton X-100 and DDM concentration are 5-10% (w/v); Described total protein and the ratio of decomposition agent are 1: 4 (w/w).
In the present invention, preferably, described wild type hepatitis E virus total length ORF2 gene refers to the hepatitis E virus total length ORF2 gene deriving from people, pig, dog, fowl, cattle, sheep, camel and rabbit.
Present invention also offers by described in above any one the hepatitis E virus sample particle vaccines for preparing of preparation method.
Further, the invention provides described hepatitis E virus sample particle vaccines and prepare the application in the capsid protein polyclonal antibody that hepatitis E virus blocks; And
The application of described hepatitis E virus sample particle vaccines in the medicine of the disease caused by preparation prevention hepatitis E virus.
In the present invention, preferably, the capsid protein polyclonal antibody adopting affinitive layer purification hepatitis E virus to block.
The present invention blocks capsid protein-2 genes to hepatitis E virus and to carry out codon optimized, screen employing prokaryotic expression system and can improve hydrophobin expression coli strain, merge raising hydrophobin solubility expression, facilitate separation and purification label maltose-binding protein MBP, the decomposition agent different to escherichia coli membrane Protein Extraction ability screens and combines, pass through amalgamation and expression, bacteria breaking, bacterial membrane extracting, maltose amylose affinity chromatograph, and by external enzyme action, affinity chromatograph, sieve chromatography removing maltose-binding protein and non-viral structural protein, and impel the assembling of virus-like particle, the virus-like particle purity of the capsid protein HEV-Δ ORF2 that hepatitis E virus is blocked reaches more than 99.0%, overcome the problem of the inclusion body that the hydrophobic and escherichia coli expression of HEV-ORF2 albumen generates, and without any protein tag there is biological activity, the capsid protein HEV-Δ ORF2 that hepatitis E virus of the present invention blocks exists with non-inclusion bodies, without the step of denature and renature in purge process, and there is solubility.
Except adopting gene codon optimization to improve except the expression of hepatitis E virus sample granule in the present invention, preferably high-caliber expression coli strain, the escherichia coli B834-pRARE2 that rare codon supplements is high level, solubility expression preparation has bioactive Hepatitis E Virus Capsid Protein best antibacterial.PMBP-HEV Δ ORF2 conversion rare codon supplements bacterium BL21 (DE3)-RILP and does not improve fusion rotein solubility and high level expression, the level that other rare codon embodying strain C41 (DE3)-pRARE2 and C43 (DE3)-pRARE2 express MBP-Δ ORF2 is the same with E.coli B834-pRARE2, and the albumen of expression does not form inclusion body.
The consumption of decomposition agent needed for the extraction that the mixing of Triton X-100 and DDM greatly reduces total protein, reduce the negative effect of decomposition agent agent to Hepatitis E Virus Capsid Protein biology, physicochemical property and post processing, improve the Isolation and purification yield of Hepatitis E Virus Capsid Protein fusion rotein.The fusion rotein that in subsequent step, affinitive layer purification and molecular sieve gel chromatography purification eliminate molecular label MBP and do not cut off, creates the hepatitis E virus sample granule of the high-purity of self assembly, high yield simultaneously.
Utilize preparation method of the present invention, the ferment tank inducing culture of 20L (working volume 18L) finally all can obtain the hepatitis E virus sample granule protein of 4 ~ 6mg, purity more than 99.0%, can be used for the preparation of hepatitis E virus sample particle vaccines and hepatitis E is treated, the preparation of diagnosis antibody.
The protected effect to the different pathogenic HEV strain of fowl is highlighted in the present invention's preferred embodiment.First prepare infectious fowl HEV, and establish its pathogenic on fowl.For the immune protective effect of the fowl vaccine of assessment preparation, 5 week age, 72 chickens were divided into 4 groups, often organized 18.The 1-2 group immune fowl vaccine of W/O/W adjuvant, containing antigen 2.5 microgram, immunity 2 times, interval January.1 group of intravenous injection 5 × 10 after June 2.5precursor virus before the chicken hepatitis E of 50% chicken infective dose, 2 groups of intravenous injections 5 × 10 2.550% chicken infective dose chicken HEV-ZL strain, 3 groups and 4 groups are as not immune negative control.The chicken often organized is dissected in infection attack for 2,3,4 weeks.Whole immune chickens antibody male rotary after immunity, the whole negative antibody of negative control.
Before infecting, the Mean antibody titers of precursor virus 3 groups of chickens is higher than infecting the viral antibody organized fowl and produce of fowl HEV-ZL, but without significant difference; 3-4 group in viremia, feces toxin expelling without significant difference.In hemanalysis, except the serum creatine phosphokinase of precursor virus group before chicken HEV is higher than except HEV-ZL strain infected group, all the other indexs and rather infected group are without significant difference.Result discloses that HEV-ZL is pathogenic weakens, and the generation of HS syndrome is with other interactional causes of disease.
Chicken HEV causes the scorching splenomegaly syndrome of Hepar Gallus domesticus, due to the chicken HEV of standard rareness and at present cannot cell culture, adopt the chicken being separated HEV prototype-strain and virulence attenuation of recovery strain of going down to posterity to carry out counteracting toxic substances.First infectiousness titration is carried out to the HEV be separated.Adopt commercial W/O/W fowl adjuvant, be emulsified into the vaccine containing 2.5 μ g antigens, immune programme for children 2 doses, intramuscular injection, interval January, the counteracting toxic substances time is latter 6 months of immunity, infects pathogenic having compared in clinical course, pathology damage of attack poison determine immune group chicken and not immune chicken.Namely to immunogenicity, the protected effect of vaccine, comprise antibody-producing capacity, HEV is infected cause viremia, feces toxin expelling, the protection of liver splenic trauma and CPK level compares.
Vaccine group is attacked antibody horizontal in latter a week in infection and is significantly improved, and the vaccine group antibody horizontal that different pathogenic strain is attacked is without significant difference.Protection is produced to viremia, feces toxin expelling and pathology damage that the recovery infective virus on fowl HEV prototype virus and chicken causes.
In another preferred embodiment of the present invention; from seroepidemiological survey; pet dog, cat infect hepatitis E virus; without clinical symptoms; there is hepatitis histopathologic change; but dog HEV virus does not have suitable cell culture system so far; and separating clone is difficult; fail to set up before the dog HEV infection virales of appropriate criteria; by setting up dog immunoinfective HEV model; understand dog and infect the process of Hepatitis E virus and pathology (infected dogs), assess the safety of vaccine of the present invention and whether dog infection, clinical, disease are protected and immune period.HEV of the present invention blocks capsid protein vaccine; immune 10-100 μ g vaccine antigen on pig; 2 weeks, interval; booster immunization; attack with the HEV intravenous injection of separate sources after December; within 4 weeks, perform an autopsy on sb. after attacking, observe the change of viremia, faecal viruses toxin expelling situation and hepatic tissue pathology, the safety of assessment HEV vaccine, immunogenicity, immunological cross protectiveness.Immune animal reacts induction of strong IgG anti-HEV, infects to attack to produce protect completely animal derived people, pig hepatitis E virus.Show that vaccine of the present invention can the animal derived hepatitis E of prevention and control.
In another preferred embodiment of the present invention, because HEV fails to breed on cell so far, make HEV virus breeding and In vivo infection abnormal difficult.So far find pig, animal model that chicken, rabbit can be used as tentative vaccine, but the attack adopted poison is for must adapt to cell line proliferates or molecular cloning, grasps and understands the pathogenic and immunologic mechanism of HEV on animal model.For the vaccine of assessment invention, animal model is assessed with pig as vaccine in embodiment, vaccinate is containing 10-100 μ gHEV antigen (W/O/W adjuvant for animals, people is with containing 0.75mg/mL aluminium hydroxide), and pig is divided into 8 groups, often organize 6, respectively immune 1-3 antigenic content vaccine, after immunizing agent, interval January, booster immunization, uses the type III pig HEV of homogenic type, type III people HEV, Genotype IV people HEV counteracting toxic substances respectively after December after immunity completes.Matched group PBS immunity, counteracting toxic substances respectively.After poison, every treated animal gets weekly hematometry HEV RNA and anti-HEV IgG antibody; all pigs are collected weekly fecal sample and detect HEV RNA; because HEV infected pigs produces inferior clinical symptom, the special index adopting viremia generation, viremia persistent period, faecal viruses and the loss of micro-liver to infect as assessment protection in invention.The evidence of pig counteracting toxic substances postoperative infection is that feces has virus excretion and viremia.All pigs are all virus-free mass formed by blood stasis after immune December, and all creates anti-HEV IgG antibody, discloses the protection that vaccine infects not homology, different genotype HEV, the broad immune that vaccine infects animal HEV and protected effect.Vaccine, on HEV animal model pig, can infect to produce and protect completely people, pig HEV.
Registering and tentative HEV vaccine at present; be all the HEV capsid protein of single Strain be antigen; the discovery of the animal HEV of many different genotype and identification; can the vaccine of single HEV strain produce protection to the virus of different genotype; particularly the infection of the animal derived HEV of different genotype produces enough protections, and vaccine provided by the invention produces cross-protection to pig, people HEV viral infection on pig body.
In embodiment, each group 6 animals rather continue to have for 2 weeks the pig of 100% to there occurs feces to fall apart malicious phenomenon after HEV virus attack.Contrary in each group that attacks with the pig HEV of vaccine immunity, the loose poison of feces significantly reduces.Disclose vaccine and the protection creating certain level is attacked to people, pig HEV.Generally, pig HEV infects and produces slight liver tissue injury, feature lymph plasmarrhexis inflammation.After postmortem, the liver of all non-immunity-attack groups loss mean scores, all higher than immune group attack group, illustrates that 5 kinds of HEV to animal produce the cross protection of certain levels, and the fall apart detection of poison of feces suffices to show that the protective effect of vaccine of the present invention.
The pig of all infection gene type III pig HEV, anti-HEV IgG is positive, show to infect, toxin expelling is there is in feces, after infecting virus, performance is healthy without clinical symptoms, start and continue toxin expelling 2 weeks, and the time of origin of viremia, antibody male rotary, feces toxin expelling, persistent period are different because pig HEV difference.Feces toxin expelling, the viremia persistent period of some pigs are longer.The phenomenon occurred with HEV infected chicken is similar.People HEV chronic patient also has the similar phenomenon of long-time toxin expelling.The pig of gene type III pig HEV natural infection also has lasting toxin expelling phenomenon, and toxin expelling was for up to 12 weeks, and what judge that HEV infected pigs has thus develops into persistent infection.Whole anti-HEV IgG is positive for pig after 2 doses of immunity simultaneously, and antibody horizontal is higher than threshold value.Illustrate humoral immune reaction vaccine-induced, attacked at December postoperative infection, protection has been created to gene type III, Genotype IV people HEV virus and homologous genes IV type pig HEV, viremia and feces toxin expelling do not occur.Vaccine infects to produce protect completely the homologous genes type of people, pig, the HEV of heterologous gene type.
HEV is popular on people, pig, dog, fowl, cat, sheep, cattle, camel, rabbit, there is I-IV genotype HEV at home, as Genotype I, gene type III, Genotype IV popular, but because these virus purification, cultivation and animal experiment fail across kind of an infection, the viral infection thing of example is limited.Confirm immunogenicity and the broad spectrum activity of invention vaccine in example, create cross protection to homologous genes type and heterologous gene type HEV viral infection, at least this vaccine produces to infect to pig and people's gene I type, gene type III, Genotype IV virus and produces protection.
These embodiments illustrate, the complete protectiveness of clinical symptoms (feces toxin expelling and viremia) that hepatitis E virus sample particle vaccines of the present invention infects people, pig HEV, to the protection completely of dog fowl HEV viral infection toxin expelling, viremia.Also illustrate except non-human primate is upper outside, pig, fowl are the best model of HEV vaccine assessment simultaneously.
Compared with prior art, beneficial effect of the present invention is embodied in:
The preparation method of hepatitis E virus sample particle vaccines of the present invention is simple, be easy to amplify and transform, and is applicable to setting up scale, is easy to quality control, Escherichia coli fermentation with low cost, purifying process; The method of quality control of relative maturity and quality standard, high-purity does not add the virus-like particle antigen of any molecular label, is applicable to the development of the interior biological product of body; Method can use object product by GMP requirement production 2 class difference, and people is with hepatitis E biological product and hepatitis E biological product for animals.Hepatitis E virus sample granule protein, purity 99.0%, for hepatitis E structure, as X-ray, mass spectrum, crystal structure, hepatitis E virus immunity and research of causing a disease.Hepatitis E virus sample particle vaccines of the present invention infects to produce protect completely pig, dog, chicken HEV, has cross-protective antigen between different genotype HEV virus.Animal can be used as a kind of vaccine, the disease that prevention pig, dog, fowl HEV cause and infection.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1 Hepatitis E Virus Capsid Protein gene codon is optimized
Agents useful for same and enzyme: T4DNA ligase and restricted enzyme equal purchased from American NEB company, Isopropyl-β-D-thiogalactopyranoside (IPTG), polymerase, dNTP and protein labeling are all purchased from German Roche Holding Ag, bioreactor is purchased from Lithuania company, and all chemical drugss are purchased from German Merck (Germany) and Sigma Co., USA.
HEV gene ORF2 genome sequence is separated poison (accession no.AF444002.1) from gene bank China HEV.The ORF2.1 coding HEV capsid protein 1-660 amino acids that wild type blocks, carries out rare codon analysis with the ORF2 genome sequence of GenScript software to wild type, places 2 restricted enzyme NdeI and XbaI at ORF2 gene two ends.By the restriction enzyme site digestions pGEM-T plasmid shown in table 1, synthesis wild type hepatitis E virus total length ORF2 gene, after gene chemical synthesis, inserts cloned plasmids pGEM-T.
ORF2 (coding HEV capsid protein 112-660 amino acids sequence) is blocked for building, BamHI restriction enzyme site is added in 112 amino acids front ends, XbaI enzyme recognition site and termination codon is added, with software simulation enzyme action and the accuracy of expressing confirmation implementation sequence after 660 amino acids sequences.Design and synthesis primer ORF2-F (5 ' AA gGATCCaTGGCGGTCGCTCCAGCCCATGACACCCCGCCAGT-3 ') and ORF2-R (5 ' GG tCTAGAcTATAACTCCCGAGTTTTACCCACCTTCATCTT-3 ').
Polymerase chain reaction 25 μ L reactant mixture: the gene outcome that 10 μ LORF2 synthesize, 2mM MgCl2,1 × standard buffer solution II (Perkin Elmer Corp.), 0.2mM dNTP, 0.375 unit Taq enzyme, 0.3 μM of 5 ' primer MF1,0.3 μM of 3 ' primer MR1, primer is in table 1.Polymerase chain reaction condition: 95 DEG C of 5min degeneration, enters 30 circular response, and each circulation 95 DEG C of degeneration 30s, 50 DEG C of annealing 30s, 72 DEG C of extension 45s, 30 circulation afterproducts, 72 DEG C of extension 10min, are placed in 4 DEG C.PCR primer purification can carry out with commercial kits by specification.
With wild type hepatitis E virus total length ORF2 gene for template, primer ORF2-F and ORF2-R in table 1 is adopted to synthesize Δ ORF2 gene.Use ORF2 gene (orf2.1) codon of GenScript software analysis truncate, do not changing the codon all using escherichia coli to like under amino acid whose prerequisite, original and optimize codon tailored index (codon adaptation index, CAI) compare, protein expression level and CAI value closely related, when CAI is greater than 0.8, albumen can have good representation, during CAI=1, express ideal.The CAI of optimized gene of the present invention is 0.85.
Table 1HEV protein gene amplimer, restriction enzyme site, product and protein expression
The solubility of the capsid protein HEV-Δ ORF2 albumen that embodiment 2 hepatitis E virus blocks, great expression and extraction
1 test method
1.1 solubility expression plasmid construction and screenings
The Δ ORF2 gene outcome of embodiment 1 being synthesized connects with the pMAL-p4x expression plasmid fragment of same enzyme action.For increasing the solubility of expression product, select applicable fusion partner and purification tag, the fusion partner particularly optimized is that N holds maltose-binding protein, and C end does not add fusion partner or purification tag, ensures that recombination expression product can do vaccine antigen.MBP (maltose-binding protein) label protein size is 40kDa, by the malE gene code on pMAL-p4x expression plasmid.MBP can be increased in the dissolubility of the fusion rotein of overexpression in antibacterial, especially eukaryotic protein.MBP label detects easily by immunoassay.
The screening of 1.2 expression antibacterials
Different expression plasmid transformation of E. coli BL21 (DE3) of the MBP-Δ ORF2 built, B834-pRARE2, BL21 (DE3)-RILP, C41 (DE3)-pRARE2, C43 (DE3)-pRARE2.BL21 (DE3) and B834 activating cell are all purchased from German Novagen company, BL21 (DE3)-RILP purchased from American Stratagene company, C41 (DE3) and C43 (DE3) is all purchased from Lucigen company, rare codon supplements plasmid pRARE2 from escherichia coli Rosetta2, proceed to suitable antibacterial Enhanced expressing in the application, purchased from Novagen company, culture medium adds 100 μ g/mL ampicillin, chloromycetin is added containing in rare codon Enhanced expressing plasmid (pRARE2, RILP) transformed bacteria by 34 μ g/mL in cultivation.Antibacterial seed 3mLLB culture medium, 37 DEG C are cultured to OD600 when reaching 0.5-0.6, be inoculated in the LB culture medium of 100mL, 25 DEG C of shaken overnight are cultivated, inoculation 1L is when to have LB culture medium 37 DEG C to be cultured to OD600 be 0.7, add 0.7mM IPTG and cultivate 4-6 hour at 37 DEG C, collected by centrifugation antibacterial ,-80 DEG C of preservations.Get 0.1g antibacterial and carry out electrophoretic analysis as sample.
The screening of 1.3 decomposition agents and combination
Resuspended 35mL chromatography buffer (the 20mM NaH of 0.2g bacterial precipitation thing 2pO 4, pH 7.5,100mM NaCl, 1 μM of protease inhibitor E-64,0.3mM tricarboxylic methyl acid phosphate), on ice bath ultrasonic 15 minutes, ultrasonic 2s, pause 2s, exported and be set to 7, Ultrasound Instrument Misonix 3000.Ultrasonic liquid 75,000g, 4-8 DEG C centrifugal 1 hour, precipitation 800 μ L lysates (20mM Tris-HCl, pH 8.0,300mM NaCl, 1mM 2 mercapto ethanol) dissolving.The total protein content BCA kit measurement going back original reagent compatible type, in test kit, diluent distilled water replaces, and all standards and diluent deionized water are prepared, and other operation by specifications carry out.
Selected decomposition agent is: Triton X-100 (5% or 10%w/v), N-ethyl-N-perfluorooctyl sulfonyl-ethylaminoethanol (FC-10), 2-hydroxy-n; N, N-trimethyl-2-phosphono-1-myristyl ammonium (FC-12), lauryl dimethyl amine oxide (LDAO), 3-[(3-gallbladder amidopropyl) dimethylammonio]-1-propane sulfonic acid salt (CHAPS), dodecyl-β-D-Maltose glycosides (DDM), sodium cholate, eight alkyl-glucosides (β-OG), digitonin (Digitonin).
With 96 orifice plate screenings, select 5 kinds of decomposition agents to dilute in 96 orifice plates at every turn, 45 μ L transfer to another analysis and use 96 orifice plates, control wells only adds decomposition agent, every hole adds the resuspended antibacterial liquid of 5 μ L, 4 DEG C are spent the night or place 3 hours in room temperature, analysis plates 10 DEG C 5, centrifugal 1 hour of 500rpm, another new microwell plate is transferred to containing soluble protein supernatant, originally plate hole precipitation adds 50 μ L and dissolves buffer, gets 10 μ L supernatants lysate (S) and non-soluble protein liquid (P-pellet) carries out SDS-PAGE electrophoresis.By comparing precipitation and lysate obtains the dissolved efficiency of each decomposition agent, when the dissolved efficiency of supernatant lysate (S) is greater than 50%, then decomposition agent uses alone or in combination, improves the efficiency of the dissolving film albumen of optimization decomposition agent.
1.4HEV protein purification
2.2cm diameter is containing balance solution (the 20mM Tris-HCl of post containing 1.0% (w/v) Triton X-100 and 1.0% (w/v) DDM of 10mL amylose medium, pH 8.0, 300mM NaCl, 1mM 2 mercapto ethanol, 1mMEDTA) balance, containing the supernatant of fusion rotein MBP-Δ ORF2 at 4 DEG C with 2mL/min flow velocity loading, wash with the balance solution containing 1.0% (w/v) Triton X-100 and 1.0% (w/v) DDM of 10 times of column volumes after loading, again with 3 times of column volumes not containing decomposition agent Triton-X100, the balance solution washing of DDM and EDTA, finally use 10 times of column volume elute soln (20mM Tris-HCl, pH 8.0, 300mM NaCl, 1mM 2 mercapto ethanol, 10mM maltose) eluting, collect 5mL eluent 4-20% gradient SDS-PAGE glue and detect purity, the protein content BCA test kit going back original reagent compatible type detects.Immunoblotting determining molecular weight, concrete steps: bacterial lysate (100mM Tris-HCl, pH7.2,1mM EDTA, 2%Triton-X 100,0.5M KCl), film extracts thing, MBP affinitive layer purification product, gel chromatography is through 12%SDS-PAGE electrophoresis, forward CAPS (10mM 3-(cyclohexylamine)-1-propane sulfonic acid) to, pH10.5,0.5%W/V DTT, 15%V/V methanol) on buffer pvdf membrane (Milliporo, 0.45 μm), voltage 65V, time 1h.Pvdf membrane spends the night with Superblock PBS (Pierce product, containing 0.05%Tween 20) is closed at 4 DEG C, with PBST (PBS is containing 0.05%Tween 20) washing 3 times.Primary antibodie is the anti-HEV purified polyclonal antibodies of rabbit, with PBS dilution 5000 or 10000 times, with next 1h that reacts of pvdf membrane room temperature, 3 times are washed again with PBST, react 1h under using the goat anti-rabbit igg antibody of Radix Cochleariae officinalis per-compound enzyme labelling (1: 10000 dilution) room temperature again, HRP (Radix Cochleariae officinalis peroxidating synthase) ECL (Enhanced chemiluminescence) reagent detects.
2 result of the tests
The selection result of 2.1 expression plasmids and expression antibacterial
After design and synthesis primer, adopt PCR method to build the different expression plasmids carrying Δ ORF2, escherichia coli separately or and (glutathione-S-transferase, GST), Intein (intein) amalgamation and expression, produce insoluble inclusion body, structure and activity is lost.For making Hepatitis E Virus Capsid Protein solubility expression, carry out MBP-Δ ORF2 expressing fusion protein, utilize primer that HEV Δ ORF2N is held and MBP fusion, at the encoding gene MalE that HEV Δ ORF2N end upstream is maltose-binding protein MBP, centre is connected by Factor Xa restriction enzyme site, under they are co-located at same promoter Ptac, be convenient to amalgamation and expression soluble product, also the capsid protein HEV-Δ ORF2 albumen be conducive to blocking gets off from enzyme action fusion rotein, insert plasmid pMal p4X and plasmid pMalc4x, transformation of E. coli E.coli BL21 (DE3), find that HEV creates solubility expression at pMAL p4XMBP-Δ ORF2, pMLl c2xMBP-M expresses and creates insoluble inclusion body.
PMAL-p4XMBP-Δ ORF2 expresses MBP-Δ ORF2 at BL21 (DE3) and B834-pRARE2, and with detection fusion albumen MBP-Δ ORF2 after amylose column chromatography, it is 100kDa that SDS-PAGE analyzes its molecular weight.PMAL-p4XMBP-Δ ORF2 expresses the amount of MBP-Δ ORF2 higher than BL21 (DE3) at B834-pRARE2, reach 5-6mg/L, and the amount that BL2 (DE3) expresses MBP-Δ ORF2 is 1mg/L.
PMAL-p2XMPB-Δ ORF2 expresses at C41 (DE3)-pRARE2, C43 (DE3)-pRARE2, C41 (DE3), C43 (DE3), amount after purification and B834-pRARE2 expression and purification compare, and the expression of MBP-Δ ORF2 does not improve.Therefore the expression plasmid of hepatitis E virus Δ ORF2 albumen is pMAL-p4XMPB-M, and preferred escherichia coli are B834-pRARE2.Selected culture medium is LB culture medium (1.0% peptone, 0.5% yeast extract, 0.5% sodium chloride, 0.2% sucrose, 100 μ g/m ammonia benzyls), the final concentration 0.7mM of induction IPTG.
PMAL-p4XMPB-Δ ORF2/B834-pRARE2 is by 1: 1000 volume ratio inoculation 20L fermentation tank, working volume 18L, rotating speed 250rpm, 37 DEG C of cultivations, when OD600 reaches 0.7, adding (isopropyl-β-D-thiogalactoside, IPTG) makes final concentration reach 0.7mM, continuation cultivation is 4000rpm centrifugal 25 minutes collection thalline after 4 hours, obtain thalline and claim weight in wet base 110-120g/L.
The optimal conditions expressed: expression plasmid pMAL-p4XMBP-Δ ORF2, the Host Strains B834-pRARE2 of expression, derivant IPTG, concentration 0.7mM, induce 4 hours for 37 DEG C, thalline claims weight in wet base 110-120g/L, and the amount of MPB-Δ ORF2 reaches 5-6mg/L.
2.2 screening of escherichia coli membrane decomposition agent and combined result
Decomposition agent CHAPS (3-[(3-gallbladder amidopropyl) dimethylammonio]-1-propane sulfonic acid salt, CHAPS), LDAO (lauryl dimethyl amine oxide, LDAO), TritonX-100, digitonin (Digitonin) can effective extracted fusion protein, decomposition agent and protein ratio 4: 1 (w/w), wherein the efficiency of TritonX-100 extracting MBP-Δ ORF2 albumen is high and cheap, and therefore the present invention adopts decomposition agent TritonX-100.
Triton X-100 has Stabilization to activated HEV memebrane protein, the Tot Prot being used alone MBP-Δ ORF2 on Triton X-100 extraction escherichia coli membrane is less than 50%, the ability that Triton X-100 mixes other decomposition agent extracting films detects and screening shows: Triton X-100 (5% or 10%w/v) and other decomposition agent (FC-10, FC-12, CHAPS, LDAO, β-OG, DDM, sodium cholate) there is several combination to improve the Stabilization of MBP-Δ ORF2 in mixture, Triton X-100/DDM, the extracting ability that Triton X-100/FC-10 combines reaches 90%, but the critical micelle concentration of DDM is 0.009%w/v, FC-10 critical micelle concentration is 0.35%w/v, and it is cheap, easy crystallization, so the present invention selects Triton X-100/DDM mixture to be the decomposition agent of extracting HEV memebrane protein, the namely mixture of 5-10% (w/v) Triton X-100 and 5-10%DDM (w/v).
2-3mL protease inhibitor is added by every gram of thalline.With Branson Ultrasound Instrument 250 thallus suspension liquid ultrasonication 15 minutes in ice bath, 1cm pops one's head in output 50%, ultrasonic 2s, pause 2s.100000g ultracentrifugation 1h separating granular and soluble protein after bacterial cell disruption, precipitation is resuspended in the column chromatography buffer of 240-300mL containing appropriate decomposition agent or decomposition agent combination, the ratio of total protein and decomposition agent is 1: 4 (w/w), and 4 DEG C are stirred 2h extracting memebrane protein.The centrifugal 1h of 100000g obtains soluble upper, makes the concentration of TritonX-100 and DDM be respectively 0.5-1.0%w/v with chromatography buffer dilution.
Preferred decomposition agent by 4: 1 (w/w) join ultrasonic after thalline centrifugation suspension in, 100000g ultracentrifugation 1 hour, MBP-Δ ORF2 is just present in ultracentrifugal supernatant, loading amylose affinity column, the non-specific binding albumen of chromatographic column is removed after the buffer solution of 10-15 times of column volume, 10mM maltose eluting, eluent is 63-64kDa through 12%SDS-PAGE detection molecules amount, and purity is more than 90%.
Purification MBP-Δ ORF2 is summarized description, it is 23g antibacterial that 18L E. coli broth produces weight in wet base, through cellular lysate, the lysate that 2 kinds of decomposition agent mixture that are centrifugal, that optimize are made extracts, the bacterioprotein of removing 75%, active raising 5 times, through amylose affinity chromatograph column purification, active raising 30 times, purity is more than 90%.
The capsid protein HEV-Δ ORF2 proteolytic cleavage that embodiment 3 hepatitis E virus blocks and affinitive layer purification
1 test method
The enzyme action of 1.1MBP-Δ ORF2 fusion rotein
Xa factor buffer (20mM Tris, pH 8.0,0.2M NaCl, 5mM CaCl 2), add 1 unit Xa factor by 100 μ g albumen, HEV-Δ ORF2 albumen embodiment 2 prepared adds Xa factor, room temperature effect 36-48 hour, the proteolytic cleavage digestion of 95%.
1.2 anti-maltose antibody Sepharose 4 affinity media purification
Endonuclease reaction liquid and latter 4 DEG C of 5mL anti-maltose antibody coupling Sepharose 4 affinity media mixing hatch 18-24 hour, every 1mL media adsorbs 0.5mg MBP, can with the specific site covalent bond on MBP, for the albumen of purification and MBP amalgamation and expression.The centrifugal rear results supernatant of room temperature 4000rpm.
In enzyme action, purification, albumen adopts 12%SDS-PAGE, coomassie brilliant blue staining analysis and embodiment 1 immunoblotting assay, with embodiment 2 adopts method mensuration protein content.
2 result of the tests
Room temperature carries out proteolysis reaction, 48 hours response time, has 90-95%MBP-Δ ORF2 to be cut into Δ ORF2 albumen.Xa factor cleavage reaction thing by Sepharose 4 chromatographic column of MBP antibody coupling, due to Δ ORF2 can not with MBP antibodies, many parts Δ ORF2 wears from chromatographic column stream, MBP albumen and do not digest the MBP-Δ ORF2 protein binding of shearing on chromatographic column on a small quantity.The endonuclease reaction liquid that 30mL or 50mL dialyses fully mixes with 5mL affinity chromatography medium, put 4 DEG C of shaking table concussions to spend the night, collect stream and wear liquid, stream wears the centrifugal rear results supernatant of liquid chamber temperature 4000rpm, obtain the HEV-Δ ORF2 of purification at supernatant, purity detecting can reach 90%.Chromatographic column binding buffer liquid (the 75mM Tris of 3 times of column volumes, pH7.5) after fully washing, use 10ml eluent (0.1M glycine again, pH2.7) eluting is collected, collect liquid containing main containing MBP albumen and a small amount of MBP-Δ ORF2 albumen, add immediately (3M Tris, the pH8.8) of the alkaline buffer of 30 μ l, make pH to 6.8 ~ 7.2.Can be used for the preparation of antibody.
The capsid protein HEV-Δ ORF2 sieve chromatography purification that embodiment 4 hepatitis E virus blocks and structure preliminary study
The stream of embodiment 3 wear liquid centrifugal after supernatant in add Triton X-100, and make its final concentration be 0.031% (w/v), the filter centrifugal concentrating by cutoff value being 20kDa, to containing 5-10mg/mL albumen, uses 2500mL column volume Superdex 20002/150 post to carry out sieve chromatography.Protein concentrate 500 μ L injects loading, with balance liquid (10mM HEPES, pH 7.2,150mM NaCl, 0.3mM trichloroethyl phosphate (TCEP), 0.031% (w/v) Trioton X-100) eluting, Fraction collection eluent, collects liquid and detects purity and content.Merge and collect eluent and concentrate with the ultrafilter dialysis that cutoff value is 20KDa.
The stream of embodiment 3 wears liquid after sieve chromatography purification, and Δ ORF2 purity improves 175 times, reaches 99.0%, and preserves stable at-80 DEG C, and preserve 4 months at 4 DEG C, its activity is still original 98%.Show capsid protein HEV-Δ ORF2 fusion rotein that hepatitis E virus blocks simultaneously, MBP-Δ ORF2 and Δ ORF2 exists with multimeric forms, MBP-Δ ORF2 forms 6 aggressiveness complexs in Triton X-100 or DDM, and the capsid protein HEV-Δ ORF2 that hepatitis E virus blocks forms 4 aggressiveness virus-like particles in Triton X-100 or DDM.
Determine the molecular weight 650kDa of MBP-Δ ORF2 albumen-decomposition agent complex in gel-filtration purified, Δ ORF2 decomposition agent complex molecules amount is 250kDa.
The preparation of the capsid protein HEV-Δ ORF2 affinitive layer purification polyclonal antibody that embodiment 5 hepatitis E virus blocks
1.1 sero-fast preparations
Preparation employing purification MBP-Δ ORF2 (the concentration 10mg/ml that initial immunity is former, prepared by embodiment 3)+Freund's complete adjuvant, make by 1: 1, immunizing antigen adopts purification Δ ORF2 albumen (concentration 1mg/ml, prepared by embodiment 4)+incomplete Freund's adjuvant emulsifying to form again.Only, the initial immunity of intravenous injection 1ml is former, injects immunizing antigen again at 7,14,22,36,50,64,78,92 days for 4-5kg New Zealand white rabbit 5-6, and last immunity is after 12 days, and carry out Culling heart blood to rabbit, separation of serum ,-20 DEG C for subsequent use.
1.2 antibody or antiserum titre measure
Serum titer measures and adopts ELISA method, specific as follows:
100 μ l contain 1 μ g purification HEV-Δ ORF2 albumen sodium carbonate liquor bag by 96 orifice plates, put 4 DEG C to spend the night, washing liquid PBST (PBS is added by 150 μ/hole, 05%Tween-20) wash 3 times, with PBS (pH7.4) the 37 DEG C of closed 1h containing 3% bovine serum albumin, add different dilution factor (1: 100,1: 1000,1: 10 4, 1: 10 5) antiserum at 37 DEG C, 5%CO 22h is hatched in incubator, with wash liquid 3 times, add the goat anti-rabbit igg antibody of horseradish peroxidase, this antibody dilutes with the PBS containing 1% bovine serum albumin by 1: 5000 or 1: 1000, at 37 DEG C of effect 1h, PBST washs 3 times, adds tetramethyl benzidine (TMB) substrate and carries out chromogenic reaction 10 minutes.2M sulphuric acid cessation reaction, reads OD value with ELISA readout instrument at 450nm.
The mensuration of serum titer also can use the method for Western blot, specific as follows:
With miniature electrophoresis (Miniprotein II, Bio-Rad) electrophoresis (200V voltage is carried out to purification HEV-Δ ORF2 albumen, room temperature 45min), 1h is shifted under 100V voltage 4 DEG C of conditions, the film of transfer is with containing the PBS (pH7.4) of 3%BSA at 37 DEG C of closed 1h, 3 times are washed with the PBST containing 0.05%Tween-20, add 1: 5000 respectively, 1: 10000, 1: 20000, the antiserum of 1: 50000 dilution is at incubated at room 1h, PBST washs 3 times, add the goat anti-rabbit igg antibody of the alkali phosphatase coupling by 1: 5000 or 1: 3000 dilution, room temperature effect 1h, wash 3 times, add substrate colour developing.
1.3 sero-fast preliminary purifications
Get 5ml serum, 4 DEG C with the centrifugal 30min of 10000rpm, supernatant proceeds in the centrifuge tube of 50ml, stirs gently, while add saturated ammonium sulfate to final concentration 50%, after placing 4h on ice with film sealing, supernatant, with the centrifugal 30min of 10000rpm, is moved in the pipe of clean 50mL by 4 DEG C, obtain polyclonal antibody, then be bag filter or pipe 4 DEG C of dialyzed overnights of 12-14kD by cutoff value, dialysis solution (PBS, pH7.5) volume should be 1000 ~ 10000 times of antibody.
The preparation of 1.4 Δ ORF2 albumen affinity columns
Get the medium Sepharose 4B that 1-2g CNBr activates, with the 1mM HCl solution washing of 250 ~ 500ml, the distilled water inflating medium using 500ml again, with binding buffer liquid (the 0.1M NaHCO of 500mL 3, 0.5M NaCl, pH8.3) and washing, add and carry out coupling containing purification HEV 3.4M albumen or MBP albumen or MBP-M protein solution, 4 DEG C are spent the night, and coupling condition is 1ml medium in conjunction with 2mg purification Δ ORF2 albumen or MBP albumen or MBP-Δ ORF2 albumen.Wash away in conjunction with Δ ORF2 albumen with binding buffer liquid, add 0.2M glycine liquid (pH8.0) 4 DEG C of closed 12h, binding buffer liquid (pH 8.5) is used to wash again 1 time, with acetate buffer solution (0.1M acetic acid, 0.5M NaCl, pH4.0) wash 4 times, after the SDS adding 0.1% carries out degeneration, for antibody purification or add appropriate Hydrazoic acid,sodium salt put 2 ~ 8 DEG C for subsequent use.
1.5 Δ ORF2 protein antibodies purification
The antibody that 3mL or 5mL dialyses fully mixes with 3mL affinity chromatography medium, put 4 DEG C of shaking table concussions to spend the night, with binding buffer liquid (the 75mM Tris of 3 times of column volumes, pH7.5) after fully washing, use 10mL eluent (0.1M glycine, pH2.7) eluting to collect again, collect (the 3MTris that liquid adds the alkaline buffer of 30 μ L immediately, pH8.8), pH to 6.8 ~ 7.0 are made.Add rear 30%v/v glycerol after antibody purification solution is concentrated, make antibody purification concentration be 250 μ g/mL, for subsequent use-20 DEG C of preservations.
The application of embodiment 6HEV-Δ ORF2 virus sample particle vaccines in the HEV infection of prevention chicken and hepatitis splenomegaly
1 materials and methods
1.1. fowl HEV prototype-strain infective virus
Have 5 week age the bile of HS syndrome (Hepar Gallus domesticus scorching splenomegaly syndrome) feces be separated, chicken farm, Wei County, Hebei, place, makes suspension with PBS, and with the titration of SPF chickling, this seed is containing 5 × 10 2.550% chicken infective dose/mL.
1.2 healthy chicken HEV-ZL strain infective virus
Healthy chicken HEV-ZL strain is located away from the healthy chicken flock of Wei County, Hebei chicken house, the titration of tentative infection SPF chickling, and feces and serum HEV RT-PCR detect positive sample collection and make infectious virus.First the amplification of intravenous injection SPF chicken has infective viral material, the chicken manure collecting every day detects HEV RNA, the chicken postmortem of the HEV RNA positive in feces, collects bile, intestinal makes 10% suspension (w/v) containing thing PBS, as fowl HEV virus seed, titration.
1.3 the infectious titration of fowl HEV-ZL strain on SPF chicken
24 SPF chickens are divided into 6 groups, often organize 4, the PBS 10 times dilution 10 of fowl HEV-ZL infectiousness seed -1-10 -5. the different diluent of each group every chicken intravenous injection 200 μ L in isolator, 4 chickens of contrast inject 200 μ LPBS.Get weekly blood and Faecal swabs, detect serum Anti-HEV antibody with ELISA, RT-PCR detects the HEV RNA of serum and feces, and continuous observation detects for 10 weeks, calculate 50% chicken infective dose/mL (50%chicken infectiousdose, CHID50/mL).
1.42 kinds of chicken HEV viruses (vaccine challenge poison, matched group and normal non-immune group, non-counteracting toxic substances group)
Hepatitis E virus sample particle vaccines is prepared:
According to use object, adopt dissimilar adjuvant, people purifies Δ ORF2 albumen with by every agent containing 15 μ g, adjuvant aluminum hydroxide absorption is added by 0.6-0.7mg/mL, total protein is no more than 100 μ g, and endotoxin is not higher than 10EU, and DNA of bacteria residual quantity is not less than 1ng, Host Strains albumen is not higher than 0.02% of total protein, active without residues of antibiotics.Animal is mainly pig, dog, W/O/W adjuvant emulsion is added by 1: 3 weight ratio, wherein every agent purification Δ ORF2 proteantigen is not less than 20 μ g, total protein is not higher than 120 μ g, endotoxin is not higher than 100EU, bacterium DNA residual quantity is not less than 10ng, and Host Strains albumen is not higher than 0.05% of total protein, active without residues of antibiotics.
In 72 5 week ages, SPF chicken is divided into 4 groups, often organizes 18.The 1-2 group immune fowl vaccine of W/O/W adjuvant, containing antigen 2.5 μ g, immunity 2 times, interval January.1 group of (n=18) intravenous injection 5 × 10 after June 2.5the prototype chicken HEV virus of CHID50,2 groups of (n=18) intravenous injections 5 × 10 2.5the chicken HEV-ZL virus of CID50.3-4 group injects PBS as a control group, after June, and 3 groups of (n=18) intravenous injections 5 × 10 2.5the prototype chicken HEV virus of CHID50,4 groups of (n=18) intravenous injections 5 × 10 2.5the chicken HEV-ZL virus of CID50,3 groups (n=18) is as non-infected chicken.Often organize independent isolated rearing, strictly carry out feeding and drinking-water by bio-safety regulations.
1.5 sample collection and process
Counteracting toxic substances collects weekly blood and the anal swab of every chicken before and after infecting, weekly blood plasma test biochemical indicator, and serum sample ELISA detects Anti-HEV antibody, and the fowl HEV RNA RT-Nested PCR of serum and feces detects.6 chickens often organized respectively after infection 2,3,4 weeks solutions take organ, organ comprises liver, thymus, spleen, the heart, pancreas, duodenum, jejunum, ileum, caecum, Colon and rectum, fixedly carries out Histopathological Studies with neutral formalin solution.
1.6 macropathology and histopathological evaluation
The tissues such as liver or organ 10% neutral formalin solution are fixed, and pathology damage judges by the hierarchy system of standard.Hepatic injury classification 0-4,0 represents not damaged; 1 indicates and is less than 5 focuses; 2 indicate 5-8 focus; 3 indicate 9-15 focus; 4 indicate the focus being greater than 15.Injury of thymus gland classification 0-4,0 indicates without focus; 1, indicate 1-5 focus; 2 represent 5-10 focus; 3 indicate 10-20 focus; 4 have 20 to reach above focus.Splenic injury classification 0-3,0 represents normal; 1 represents slight damage; 2 represent moderate lesion; 3 represent major injury.
1.7 blood biochemical analyses
Often group gets 6, amount to 18, analyze from infection 2 weeks-4 blood testing biochemical indicator weekly, detect aspartate aminotransferase (Aspartate aminotransferase, AST), lactase dehydrogenase (Lactatedehydrogenase, LDH), creatine phosphokinase (Creatine phosphokinase, CPK), cholic acid (Bile acids, and total protein (Total protein, TP) BA).Fowl HEV prototype-strain and fowl HEV-ZL capsid protein nucleotide homology are 90.7%
1.8ELISA detects anti-HEV IgG
The Δ ORF2 albumen that restructuring purification blocks is as envelope antigen, and detect chicken HEV IgG, the anti-chicken horseradish peroxidase of rabbit is as second antibody (available from Sigma).OD 405nm is greater than 0.3 for positive.Chicken HEV acute stage of infection serum and normal SPF serum are respectively positive and negative control.
1.9HEV RT-PCR detects
100 μ L serum or 10% fecal suspension TRI reagent (MRC) extract RNA, be resuspended in 12.25 μ LDNase, without RNA enzyme, in the water of protease, 42 DEG C of 60min carry out reverse transcription reaction: with the special P2 primer of 1 μ L chicken HEV (5 '-ACAGTTTCACCTCAGGCTCG-3 ') or 11 μ L chicken HEV-ZL special primer YR (5 '-CTGCGCAACAGTATCCATTAAG-3 '), the super reverse transcriptase II of 0.25 μ L [50U] (purchased from American Invitrogen company), 1 μ L 0.1M DTT, 4 μ L 5x RT buffer, 0.5 μ l [20U] RNA enzyme level (purchased from American Promega company), 1 μ l 10mM dNTPs.
Add 5 μ L cDNA at 50 μ L reaction system amplified reactions.First round amplification chicken HEV prototype virus 595bp fragment, first round reaction forward primer P1 (5 '-ACAACATCCACCCCTACAAG-3 ') and reverse primer P2.Second takes turns forward primer P3 (5 '-AGAACAATGGTTGGCGGTCC-3 ') and reverse primer P4 (5 '-GAGGGCAAGCCACCTAAAAC-3 '), the 394bp fragment of the chicken HEV prototype poison of amplification expection.The parameter of amplified reaction comprises: 94 DEG C of degeneration 9min, enters 39 circulations, and each circulation comprises 94 DEG C of degeneration 0.5min, 52 DEG C of annealing (second takes turns PCR 56 DEG C) 0.5min, and 72 DEG C extend 1min, and last 72 DEG C extend 7min.
Detect chicken HEV-ZL strain, the first round reacts, forward primer YF (5 '-GCTGCCCTTGGGATGTTTGCAT-3 ') and reverse primer YR expection amplification generation 712bp fragment.Second takes turns reaction, is contemplated to the fragment of 578bp with forward primer YF2 (5 '-AGTTTTGCGGTCTGTCGTGTTT-3 ') and reverse primer YR2 (5 '-AGCGTGTTAATCACCGCAAGGC-3 ') amplification.Reaction condition is except first round reaction annealing temperature 48 DEG C, and second takes turns outside reaction annealing temperature 54 DEG C, and all the other parameters are the same with amplification HEV prototype virus.
The feces of each group of infected chicken and the 0.8% agarose separation and purification of serum amplified production are checked order.
1.10 statistical analysis
Histopathological lesions lesion scores represents, mean scores user difference analysis, antibody titer GLIMMIX program carries out Gauss distribution (Gaussian distribution) analysis, biochemical indicator liver enzyme, total protein, cholic acid GLIMMIX program analysis.
2 immunity and the result of the tests of infected group chicken
The preparation of 2.1HEV-ZL strain infectious virus seed
The raw manure that field obtains and bile suspension inoculation 2 SPF chickling, after 3 days, feces has fowl HEV RNA to detect, and infects after 23 days, and the feces collecting the HEV RNA positive makes 10%PBS suspension.Another is in infection after 30 days, and collection feces and bile make 10%PBS suspension.
The infectiousness of 2.2 use SPF chicken titration HEV-ZL strain virus seeds
All inoculations 10 -1, 10 -2chicken antibody all sun turn of HEV-ZL strain virus seed diluent, in table 2, but matched group chicken antibodies of other groups are negative all the time.Inoculate the highest infective dose (10 -1dilution) 4 chickens, have 1 infection after 5 days antibody just sun turn, other chickens are 6 days antibody male rotaries after infection.Inoculation dosage (10 -2dilution) chicken in, 1 chicken infects latter 1 day antibody male rotary, 2 antibody male rotaries after 4 days, other 2 at infection 6-7 days antibody male rotaries.There is viremia and feces toxin expelling in the chicken that maximum dose level infects, but in inoculation 10 -3sight is not had to find viremia, feces toxin expelling, in table 3 in the group of dilution infective dose.Inoculation 10 -4dilution infective dose, 10 -5the chicken of dilution infective dose, PBS infected group detects HEV RNA feminine gender at duration of test RT-PCR, and anti-HEV-IgG is negative, therefore calculates end with antibody male rotary as infectious virus infection titer.So HEV-ZL strain virus seed transmission titre is 5 × 10 2.5cHID50/mL.
2.3 chicken HEV prototype-strains and the microdamage of HEV-ZL strain on non-immune chicken compare
Only have 2 chickens infected to observe obvious damage in infection after 2 days, one 1/18 is infected chicken HEV prototype-strain, and in liver lobus sinister circular spot district, another 1/18, for infecting HEV-ZL strain infective virus infected chicken, has film bleed bottom at liver lobus dexter.2 chickens are higher in histopathology classification, are respectively 3 and 4, the damage that other chickens of 17/18 are not significantly observed visually after infecting.The damage be observed visually is not seen after the chicken infection of immune group.
HEV-ZL strain infective virus infected chicken liver microdamage comprises lymphocyte phlebitis, lymphocyte and addicted to different in nature periphlebitis, fiber-like albumen granuloma, but it is slight, film bleed bottom, does not observe large area granuloma or amyloid lake (amyloid-likeLakes).
The chicken tissues damage mean scores that infects of 3 days chicken HEV prototype-strains is higher than HEV-ZL strain infective virus infected chicken after infection, and HEV-ZL strain infective virus infected chicken tissue injury mean scores is higher than matched group, but the 4th day, their score value zero difference, in table 3.In a word, hepatic injury score value difference group variant (P=0.03), HEV prototype-strain hepatic injury (minimum card side average LSM2.722) is greater than HEV-ZL strain infected group (minimum card side average LSM2.61), the hepatic injury of HEV-ZL strain infected group is greater than matched group (minimum card side average LSM1.877, but hepatic injury there is no significant difference (P=0.48) at time of origin.Chicken spleen lesion scores is different in the group of each infection, P=0.0006, but damage time of origin zero difference (P=0.32).Thymus has slight crust hypertrophy to damage, and the lesion scores of different infected group does not have difference (P=0.27).The damage and microdamage that are observed visually is not seen after the chicken infection of immune group.
2.4 anti-chicken HEV IgG transform
Before inoculation HEV virus, immune chicken HEV antibody total positives, whole immune chicken 3 days after infection, anti-chicken HEV IgG level significantly promotes.
3 days after infection, 1 group of chicken HEV prototype-strain infected chicken had the anti-HEV IgG of 100% (12/12) chicken, has 75% (12/12) anti-HEV IgG significantly to rise in 2 groups of HEV-ZL strain infective virus infected chickens.Counteracting toxic substances infects latter 3 days, 1 group and 2 groups of chicken serum ELISA OD values peak, it is 6.89 ± 0.21 that 1 group of chicken of immunity infects HEV prototype-strain ELISA OD meansigma methods (mean OD ± SEM), HEV-ZL strain infective virus infected group ELISA OD meansigma methods (mean OD ± SEM) is 8.45 ± 0.26, and the ELISA OD value of vaccine immunity 1 group and vaccine immunity 2 groups does not have significant difference (P=0.11).There were significant differences (P=0.0001) for vaccine 1 group and matched group 3ELISA OD value, and the antibody tormation time is different (GRP X WPI, P=0.0001) also.There were significant differences (P=0.0001) for vaccine 2 groups and contrast 4 groups of ELISA OD values, and the antibody tormation time is different (GRP X WPI, P=0.0001) also.
Before inoculation HEV virus, not immune chicken HEV negative antibody, the chicken that great majority infect 3 days after infection, anti-chicken HEV IgG sun turns, in table 4 and table 5.
3 days after infection, 3 groups of chicken HEV prototype-strain infected chickens had the anti-HEV IgG sun of 83% (10/12) chicken to turn, and have 75% (9/12) anti-HEV IgG sun to turn in 4 groups of HEV-ZL strain infective virus infected chickens.At 4 days that infect, 3 groups and 4 groups of all chicken 100% (6/6) sun turn, there were significant differences (P=0.0001) for the ELISA OD value of different infection counteracting toxic substances 3,4 groups, and the antibody tormation time is different (GRP X WPI, P=0.0001) also.HEV prototype-strain infected group (P < 0.0001) and HEV-ZL strain infective virus infected group (P < 0.0001) GRP X WPI Be very effective, but negative control group (P=0.11) GRP X WPI effect is not remarkable.Infect latter 3 days, 2 groups of ELISA OD values peak, HEV prototype-strain infected group is higher than HEV-ZL strain infective virus infected group, HEV prototype-strain infected group ELISA OD meansigma methods (mean OD ± SEM) is 0.847 ± 0.081, HEV-ZL strain infective virus infected group ELISA OD meansigma methods (mean OD ± SEM) is 0.721 ± 0.1.
2.5RT-PCR detects chicken HEV RNA in feces, serum
The blood gathered weekly, Faecal swabs adopt chicken HEV RNA primer, carry out RT-Nested PCR detection, and serum, the feces of 1-2 group chicken all do not have HEV RNA any time.
The serum of 3 groups, 4 groups chickens, feces have HEV RNA to exist, in table 5.After infecting 1 week, serum, feces start just have HEV RNA to detect, and infect 2 weeks most of chickens and occur viremia, feces toxin expelling.
After HEV prototype-strain infects 1 week, there is the chicken manure just toxin expelling of 15/18, infect chicken manure just whole toxin expelling of after 2 weeks 18/18, have the chicken manure just toxin expelling of 8/12 after infecting 3 weeks, have 1/6 chicken to infect 4 weeks toxin expellings.
HEV-ZL strain infective virus infected group, have 17/18 chicken at infection 1 week feces toxin expelling, 17/18 chicken infects 2 weeks feces toxin expellings, and the chicken of 7/12 infects feces toxin expelling after 3 weeks, within 4 weeks, has the chicken manure just toxin expelling of 1/6 in infection.
In HEV prototype-strain infected group, after infecting 1 week, 11/18 Sanguis Gallus domesticus just has HEV RNA to detect, and infects and occurs HEV RNA in 2 weeks 17/18, infects 3 weeks 5/12 serum HEV RNA positive, infects and within 4 weeks, have 1/6 chicken serum HEV RNA to detect the positive.
HEV-ZL strain infective virus infected group, after infecting 1 week, 11/18 Sanguis Gallus domesticus just has HEV RNA to detect, and infects and occurs HEV RNA in 2 weeks 15/18, infects 3 weeks 3/12 serum HEV RNA positive, infects and within 4 weeks, have 0/6 chicken serum HEV RNA to detect the positive.
The chicken of vaccine immunity group, at whole duration of test, Faecal swabs, serum HEV RNA detect feminine gender.The different pathogenic feces of chicken HEV infected chicken of 3-4 group, the RT-PCR product sequencing result of serum confirm HEV virus and cause a disease on chicken; recovery process has attenuation in various degree; describe the safety of HEV-Δ ORF2 vaccine, immunogenicity and protectiveness, protection chicken is from HEV viral infection, the HEV viremia decreasing chicken and feces toxin expelling.
2.6 hemanalysis
Each group 6, amount to 24 Sanguis Gallus domesticus AST, BA, TP to detect, after 1 group of immune group chicken HEV prototype-strain infected chicken counteracting toxic substances and 2 groups of immunity chicken HEV-ZL strain infective virus infected chicken counteracting toxic substances, blood LDH detected value zero difference (P=0.52), LDH leveled time is also without significant difference (P=0.1).One group of LDH (least square mean (LSM)) is 1532.21, SEM be 68.47,2 groups of LSM be 1538.31, SEM is 74.39.
3 groups of HEV prototype-strain infected chickens and 4 groups of HEV-ZL strain infective virus infected chicken LDH detected values also zero difference (P=0.21), but at time of occurrence variant (P=0.001).HEV prototype-strain infected group and the response of HEV-ZL strain infective virus infected group serum CPK are at infection (P=0.049) and time (P=0.043) upper significant difference.The average CPK level of HEV prototype-strain infected group is higher (P < 0.0001), successively higher than HEV-ZL strain infective virus infected group (P < 0.0001), vaccine 1 group (P < 0.0001) or vaccine 2 groups (P < 0.0001).It is that to infect 4 groups of LSM be 1402.66 to 1686.15, HEV-ZL strain infective virus that HEV prototype-strain infects 3 groups of LSM.
Illustrate that different pathogenic chicken HEV infects the rising causing LDH, be the biochemical indicator that chicken infects HEV, vaccine injection 2 doses can protect chicken from the infection of chicken HEV, illustrates that vaccine does not affect change and the safety of the normal physiological parameter of chicken simultaneously.
Chicken HEV causes the scorching splenomegaly syndrome of Hepar Gallus domesticus, due to the chicken HEV of standard rareness and at present cannot cell culture, adopt the chicken being separated HEV prototype-strain and virulence attenuation of recovery strain of going down to posterity to carry out counteracting toxic substances.First infectiousness titration is carried out to the HEV be separated.Adopt commercial W/O/W fowl adjuvant, be emulsified into the vaccine containing 2.5 μ g antigens, immune programme for children 2 doses, intramuscular injection, interval January, the counteracting toxic substances time is latter 6 months of immunity, infects pathogenic the comparing in clinical course, pathology damage of attack poison determine immune group chicken and not immune chicken.To immunogenicity, the protected effect of vaccine, comprise antibody-producing capacity, HEV is infected cause viremia, feces toxin expelling, the protection of liver splenic trauma and CPK level analyzes.
Table 2HEV prototype-strain infectivity titer (chicken is evaluated and attacks poison and model foundation, attacks the antibody male rotary that poison produces)
Table 3HEV-ZL strain infectivity titer (chicken is evaluated and attacks poison and model foundation, feces toxin expelling and viremia after attacking)
Table 4 immunity and not immune chicken counteracting toxic substances after liver spleen microdamage classification results
Feces HEV RNA, serum HEV-RNA, Serum Antibody Detection (immune group should in feces toxin expelling, viremia 1-2 week feminine gender, and antibody occurred in 1-2 week, as similar in infection 3-4 week situation) after table 5 not immune chicken chicken infection counteracting toxic substances
The capsid protein HEV-Δ ORF2 that embodiment 7 hepatitis E virus blocks is to the protected effect of dog HEV viral infection
1 materials and methods
Inoculation material: the liver of infected dogs immediately-20 DEG C freezing after, then place-70 DEG C and save backup.For preparation liver inoculation liquid, make homogenate with 10%PBS (w/v) grinding, move into 15mL test tube, vortex concussion 1min, 4 DEG C, the centrifugal 20min of 4000 × g, supernatant proceeds to new test tube also with pin type frit (0.22 μm gPfilter unit, Millipore Products).Supernatant is divided into many parts, and every part of 2.5mL is stored in-70 DEG C, comprises 2 × 10 4hEV RNA copy/μ L RNA.
The nested PCR product of dog is rna replicon enzyme gene 261bp fragment, the homology of 98%-100% is had with gene bank (registering sequence KC802090, KC802091, KC802092, KC802093) ferret, Mongoose sequence, there is 1 nonsynonymous mutation, isoleucine becomes valine, be respectively 76% and 69% with G3-G4HEV nucleotide homology, be respectively 87% and 78% with G3-G4HEV amino acid sequence homology.
Sample and dog grouping:
Test requires with dog: detect and confirm that the anti-anti-HEV of serum is negative, feces HEV RNA is negative, and temperature check is normal.Isolated rearing.Immune programme for children, intramuscular injection immunity 2 vaccinating agents, every agent is containing HEV-Δ ORF2 antigen 20 μ g (preparing hepatitis E virus sample particle vaccines according to the method for embodiment 6), and interval immunity in January, sets up negative control (immune PBS and infection are attacked).Immunity is carried out infection and is attacked after 1 year, infect cranium side, the position caval vein attacked, consumption 2.0mL, every day observes, and grouping is in table 6.2 groups are being infected the time point 0,1,3,5,8,11,14,17 attacked, and within 21,24,27,29 days, measure body weight, body temperature, collect blood sample, fecal sample.Within continuous 2 days, body temperature >=40.0 DEG C are fever, and all serum samples are kept at-20 DEG C in order to antibody and biochemistry detection, and fecal sample is diluted in PBS, preserve-70 DEG C and extract in order to RNA.Postmortem collection organization sample (liver, hepatic lymph nodes, mesentery and mandibular lymph node, bile, bladder, large intestine, small intestinal, pancreas, kidney, spleen, tonsil, heart, brain, gonad, uterus or prostate, stock four quadratus) is studied for virusology, histopathology and SABC Epidemiological Analysis, wherein a part of organ-tissue 4% neutral formalin solution is fixed, and a part is stored in-70 DEG C in order to extracting RNA.
1.1 clinical-chemical analysis
With automatic analyzer (VetScan, U.S. Abaxis Products), ultraviolet spectrophotometry serum analysis sample (longitudinally), use special rotor (VetScan Mammalian Liver Profile reagent rotor, Abaxis) alanine aminotransferase (Alanine aminotransferase in detection by quantitative serum, ALT), albumin (Albumin, ALB), alkali phosphatase (Alkaline phosphatase, ALP), cholic acid (Bile acids, BA), total bile red pigment (Total bilirubin, TBIL), T-CHOL (Total cholesterol, CHOL), gamma glutamyl transferase (Gamma-glutamyl transferase (γ GT)), blood urea nitrogen (Blood ureanitrogen, BUN), calculate the high-order reference range of biochemical parameter and the value of negative control.
1.2 antibody and RNA detect
The restructuring HEV-Δ ORF2 albumen bag of preparation is detected the antibody of serum or blood plasma by ELISA method, OD450nm value is equal to or greater than 1 for positive.
1.3 serum and fecal suspension extract the method for viral RNA
Serum RNA adopts viral RNA Mini Kit (German QIAGEN GmbH Products), provides by producer and recommends description to carry out.In a organized way in viral RNA extraction RNeasy Mini Kit (German QIAGEN GmbH Products), adopt this 2 kinds of methods, all set up internal control RNA (IC2).The quantitative PCR in real time of HEVRNA detects (RT-qPCR), institute's instrument CFX96TM real-time system that makes (German Bio-RadLaboratories product).Primer and probe in table 7, quantitative probe RT-PCR kit (QIAGEN GmbH Products) carry out RT-qPC, reaction system 25 μ L, primer ultimate density 0.8 μM, probe ultimate density 0.1 μM, add 5 μ L extract RNA.After the condition 50 DEG C reaction 30min of reverse transcription reaction, 95 DEG C of degeneration 15min, enter 45 cyclic amplifications, circulate: 95 DEG C (10s), 55 DEG C (25s), 72 DEG C (25s) at every turn.HEV copy number is determined as the standard curve that calibration object is drawn according to the RT-qPCR amplicon containing 81bp.
1.4 histopathologies and SABC
The tissue samples that formalin is fixed is by the dyeing of S.O.P. hematoxylin-eosin (HE), and the observation sample for SABC is cut into 3 μm of sections, dewaxing, water suction.Pre-treatment step comprises 3%H 2o 2/ methanol solution closes inherent peroxidase 30 minutes, and microwave oven 600W power lower 10 minutes antigen are remedied.The anti-human CD3 polyclonal antibody of 1: 200 dilution rabbit, in conjunction with dog CD3 antigen, detects the inflammatory reaction of liver; The polyclonal antibody (HEV-Δ ORF2, gt3) 1: 1000 that the anti-HEV of virus antigen rabbit surpasses the affinitive layer purification of exempting from serum dilutes, and the preparation of this antibody and purification are shown in embodiment 5.Section and biotin hatch (purchased from German VectorLaboratorie) in conjunction with goat anti-rabbit igg, add avidin/biotin combined enzyme agent ( aBC reagent, purchased from German Vector Laboratories), add again 3,3-diaminobenzidine (DAB) substrate (purchased from American Sigma-Aldrich company) observe, virus antigen concentration graded as: 0 represent do not observe antigen dyeing; + represent slightly immune labeled, be less than the cellular antigens positive of 20%; ++ represent moderate immune labelling, 20-40% cellular antigens are positive; +++ represent obviously immune labeled, be greater than 40% cellular antigens positive, each section point 2 independent operations are observed.
2 hepatitis e virus infection dogs and pathological observation result
2.1 clinical biochemical parameters
The domesticated dog body temperature that intravenous injection is infected significantly does not rise, there are 2 clinical symptoms just occurring slight depression, mild diarrhea, slight anorexia, have a serum BA and γ GT to infect in intravenous injection within latter 21 days, to occur rising, but 25 days after infection, serum alt and γ GT recovered normal every other day afterwards.Not heating and other clinical symptoms are infected in the domesticated dog intravenous injection of all immunity, and BA keeps normal level always, and have 2 to infect latter 29 days γ GT levels at counteracting toxic substances and rise, wherein a boss dog ALT level rises.Table 8 illustrates the time dependent rule of BA, ALT and γ GT level in immune domesticated dog and negative control man dog serum, and other biochemical indicators or parameter keep normal.
2.2 serology and HEV RNA testing result
PBS negative control domesticated dog 17 days anti-HEV-IgG sun turn after infection of 2/4, during the test of other negative control domesticated dogs of 1/6, anti-HEV-IgG is always negative.Negative control domesticated dog 3/6 after infection 14 days HEV-IgG antibody detect the positive, within 13 days, there is HEV-IgG antibody, in table 9 after infection in 1/6 dog.
2/6 dog 28 days after infection, 21 days in negative control domesticated dog, the HEV RNA of serum is greater than 10 copies/μ L.3-5 days after infection, has HEV RNA in 3/6 dog feces to be greater than 10 5copy/μ L.2/6 domesticated dog of antibody male rotary is in 28 days of test, and HEV copy number remains high level, until decline a little after 28 days.In antibody male rotary 2/6 domesticated dog feces, HEV toxin expelling significantly reduces, and is less than < 10 1copy s/ μ L RNA.
HEV RNA is had to detect in negative control group all infection liver of domesticated dog, gallbladder, caecum, colon, spleen positive.> 20 copies/μ L RNA to have 1/6 to detect in brain, muscle, uterus, obviously positive.In feces toxin expelling in domesticated dog and serum, RNA copy number is suitable with domesticated dog, but antibody male rotary 2 domesticated dogs, at 14 days that infect, the HEV toxin expelling of feces obviously reduced.Have HEV RNA to detect in most infection liver of domesticated dog, gallbladder, caecum, colon, spleen positive, infect and detect a large amount of RNA copy numbers in 1/6 liver after 1 day, in serum and feces, HEV RNA testing result is in table 9.Table 10 is the virus load result of selected tissue samples, has HEV RNA in the domesticated dog 4/12, the bile of domesticated dog.
The anti-HEV IgG antibody of all domesticated dogs of immune group significantly rose in 2-3 days; viewing duration maintains higher level; the HEV RNA of serum and feces is negative all the time; illustrate that vaccine excites the high-caliber antibody of dog and other immunoprotection reactions do not detected; at least maintain March; after infection counteracting toxic substances, antibody tormation significantly strengthens, and peaks in 1 week, reduces HEV viral infection and viremia, feces toxin expelling.The protected effect at least 3 month of dog HEV vaccine dog is described.
2.3 pathology results
In all dogs, the change of large particular virus hepatitis is not had in immune group and negative control group dog liver, but the large intestine lymphoid tissue of non-immune some domesticated dogs of infected dogs has the gross feature of light to moderate follicle hypertrophy, and hepatic lymph nodes has the gross feature of follicle hypertrophy.Moderate nematode infections and many stoves white point (milk shape point) is had, the feature of chronic hepatitis in liver in postmortem display intestinal.The liver of the dog of all non-immunoinfectives is observed, and the virus antigen distribution of inner lobe of the liver is different, causes the damage of different size, form, number of times, but the dog of immune group find no the damage of the inner lobe of the liver of liver.
In the dog that all non-immunonegative groups infect, find that having light to moderate periphery lymph matter to dissolve in liver invades profit (Periportal lymphoplasmacytic Infiltrates) and Kupffer cell hypertrophy (Kupffer cellproliferations), but detect without antigen around portal vein.Immunohistochemical observation, mainly finds that there is HEV virus antigen at Kupffer cell and sinus hepaticus epidermis cell, even hepatocyte in lobe of the liver, part hepatic injury and CD3 positive cell invade profit to. the HEV antigen of Kupffer cell and sinus hepaticus epidermis cell is relevant.The heterogeneity more aobvious with immune domesticated dog of the viral domesticated dog of concrete infection, light to moderate many stoves leaf endolymph histolysis that the liver of the domesticated dog of 2/6 shows the enlargement of many stoves hepatocyte and cavity sample, virus antigen distribution is diffused as feature is had to invade profit, other domesticated dogs of 2/6 there occurs medial liver lobes because lymphocyte, plasma cell, Kupffer cell invade the relevant heptocellular death of profit, and these damages are obviously relevant to visible marking HEV virus antigen.
It is that the dissolving of feature lymphoid interstitital is invaded the moderate diffusion of profit and spreads to the virus antigen of middle amount in a small amount that the dog of not immune infectable infection HEV shows the single granulation of intrahepatic leaf hepatocyte.At the different lymph node of some dogs infected with by rete discovery HEV antigen, not finding of other dogs.The pathology damage of dog, histopathology, group detection are in table 6.
Table 6 immunity, matched group dog test guide look (safety)
Virus antigen concentration graded as: 0 represent do not observe antigen dyeing; + represent slightly immune labeled, be less than the cellular antigens positive of 20%; ++ represent moderate immune labelling, 20-40% cellular antigens are positive; +++ represent obviously immune labeled, be greater than 40% cellular antigens positive.
Table 7 detects the primer (1 part is identical with pig 4HEV with pig 3) of dog HEV RNA
The biochemical parameter testing result of table 8 immune group dog and infection attack dog
The cross-protection of embodiment 8 hepatitis E virus vaccine on pig
1 materials and methods
1.1HEV viral
HEV infective virus, gene type III pig HEV, by swine excrement infection cell, then collects feces acquisition through infected pigs, titre 10 4.550% pig infective dose (PID50), the strain of the people's gene type III HEV U.S., people's gene IV type strain in China, from America NI H, feces PBS is diluted to 10% suspension, makes containing 3.4 × 10 8gene equivalent titre/mL (Genomic equivalent titer, GE), is infectious in the injection of pig upper vein.
1.2 test method
Anti-HEV IgG antibody is negative, and healthy, 5 week age, 48 pigs, were divided into 8 groups at random, often organizes 6, raises separately, strictly carry out feeding and raising by bio-safety regulations in the test room of BSL-2.
Vaccine, respectively containing ORF2VLP (virus-like particle) antigen/mL that 10,50,100 μ g HEV block, prepares (preparing hepatitis E virus sample particle vaccines according to the method for embodiment 6) with W/O/W adjuvant emulsion.Negative control group is immunity 3 dose vaccine and PBS only, attacks with PBS or does not attack.In the immunity of pig neck lateral, first dose of 1mL vaccine, intramuscular injection immunity, carries out second dose of injecting immune after January.After Full-access immunization completes December, first group the same with vaccine group program with PBS, immune secondary, attack with PBS, with PBS immunity secondary, attack with pig HEV3 for second group, be all negative control group, the 3rd group, the 4th group, the vaccine of other immunity three various dose of BSA, respectively intravenous injection 10 4.5the gene type III pig HEV of PID50; Six to the eight group of Full-access immunization 1mL contains the vaccine of the ORF2VLP antigen that 50 μ gHEV block, the 6th group of intravenous injection 3.4 × 10 8the gene type III people HEV (U.S.'s strain) of GE titre, the 7th group of intravenous injection 3.4 × 108GE titre Genotype IV people HEV (strain in China), the 8th group of intravenous injection 10 4.5the Genotype IV pig HEV of PID50.
After attacking, 4 weeks all pigs carry out the observation of liver loss virus.Lymphoid interstitital dissolves score value assessment, and 0 represents NIP; 1 represents 10 lobe of the liver has 1-2 lymphocytolysis to invade profit; 1 represents 10 lobe of the liver has 1-2 lymphocytolysis to invade profit; 2 represent 10 lobe of the liver has 3-5 lymphocytolysis to invade profit; 3 represent 10 lobe of the liver has 6-10 lymphocytolysis to invade profit; 4 or be greater than 10 and represent 10 lobe of the liver and have more than 10 lymphocytolysises to invade profit.
Collect weekly feces before and after immunity front and back, counteracting toxic substances, blood sample RT-Nested PCR detects HEV-RNA, measures anti-HEV-IgG with ELISA.
1.2.1 sample collection
Collect weekly serum and Faecal swabs before immunity, collect weekly serum ELISA method after immunity and detect IgGanti-HEV, after counteracting toxic substances, get weekly blood and Faecal swabs RT-Nested PCR detection HEV RNA.Collect liver, bile, little enteric contents after postmortem, bile and 10% small intestinal suspension detect HEV RNA, liver carries out pathological observation and gives a mark by classification.
1.2.2RT-PCR detect HEV RNA
The primer of each genotype HEV, in table 11, is only gene type III HEV here, and the RT-PCR method of the detection of other types is identical, is exactly that primer is different.
The special nest-type PRC of gene type III HEV, 100 μ L10% fecal suspensions or serum TriZol reagent (LifeTechnologies) extract, be resuspended in 30 μ L sterilized water, reaction system adds 1 μ L (10 μMs) outside reverse transcriptase primer HEVORF1-REV1, 1 μ L (200units/ μ l) Superscript II reverse transcriptase (purchase of life company), the 0.1M DTT of 1 μ L, 5 × RT buffer of 4 μ L, 0.5 μ L (40units/ μ l) RNasin nucleic acid inhibitor (Promega), 10mM dNTP 42 DEG C of 1h of 1 μ L carry out reverse transcription reaction.
First round PCR reacts, with primer HEVORF1-FWD1 and primer HEVORF1-REV1 amplification 471bp product, second time nest-type PRC, forward primer HEVORF1-FWD2 and reverse primer HEVORF1-REV2,5 μ L AmpliTaq Gold archaeal dna polymerases (purchase of AB company), 5 μ L first round PCR primer amplification 277bp fragments, in table 11.After loop parameter comprises 95 DEG C of degeneration 9min, carry out 39 circulations: 94 DEG C of degeneration 1min, 52 DEG C of annealing 1min, 72 DEG C extend 1min, have circulated for 39 times latter last 72 DEG C and have extended 7min.PCR primer adopts 1% agarose gel electrophoresis observation analysis.
1.2.3 Enzyme-linked Immunosorbent Assay (ELISA) detects anti-HEV IgG
The purification HEV-Δ ORF2 of porcine blood serum bag quilt wraps quilt, affinity chromatograph HEV-Δ ORF2 polyclonal antibody is first antibody, concrete grammar is see embodiment 5, the threshold value of ELISA is serum average OD ± 3 standard error SD before all animal immunes, and all serum detects by 1: the 100 multiple hole of dilution.
1.2.4 statistical analysis
Histopathological scores in table 12, with asymmetric t-test (Excel, Microsoft's software) statistical analysis.P is less than 0.05 for significant difference.
2 result of the tests
2.1 histopathological findings
Pig muscle injection various dose 10,50,100 μ gHEV-Δ ORF2 reacts induction of strong IgG anti-HEV, the ELISA testing result of antigen-specific shows the antibody horizontal no significant difference in each dosage group, show that HEV-Δ ORF2 can react by induction of antibodies at 10-100 μ g dosage on pig, vaccine dose is containing antigen 1 0-100 μ g, minimum containing HEV-Δ ORF2 antigen 10 μ g, the highest containing HEV-Δ ORF2 antigen 1 00 μ g.
Between duration of immunity, each pig blood sample ELISA weekly detects, January after immunity first dose, in first group and second group of pig, there is 12/12 anti-HEV-IgG of pig negative, 3/6 pig HEV-IgG sun is had to turn in 3rd group of pig, 4th group has the anti-HEV-IgG sun of 5/6 pig to turn, 4/6 anti-HEV-IgG sun of pig is had to turn in 5th group, have 4/6 antibody male rotary in 6th group of pig, the 7th group of whole anti-HEV-IgG sun of pig turns, and the 8th group of anti-HEV-IgG sun of pig 4/6 turns, vaccine first dose stimulates pig body to create anti-HEV-IgG, and Conversion rate is 72.2.%.After second vaccinating agent completes 1 week, the anti-HEV-IgG of pig of all immunity all sun turned, except the first matched group.Show vaccine 2 doses injection, interval January, the immune programme for children of intramuscular injection can induce pig to produce HEV-IgG antibody.
All immune swine body temperature compares without significant difference with weightening finish and matched group, confirms vaccine safety.
Counteracting toxic substances postmortem after 4 weeks; lymph kytoplasm hepatitis (LymphoplasmacyticHepatitis) pathological observation is carried out in each group of sample Hepar Sus domestica section; in table 12; the symptom score that non-immune swine is attacked is higher; pig gene type III viral infection is attacked and is produced subclinical light symptoms; show that vaccine effectively reduces the damage of the hepatic injury that pig gene type III HEV causes, also show to produce protection to the liver loss of pig HEV, people HEV.
2.2RT-PCR detects the HEV RNA in serum and feces
Carry out RT-Nested PCR with the primer of HEV ORF1 after counteracting toxic substances and detect HEV RNA, different genotype HEV positive serum, negative serum, positive feces 10% suspension, negative feces 10% suspension are all synchronous and sample carries out reverse transcription and nest-type PRC reacts to avoid operate miss.The results are shown in Table 13.
Second group of pig gene 3 type HEV counteracting toxic substances, the pig that the 1-3 week of infection period has 6/6 there occurs viremia, first week 2/6, infect 2 weeks afterwards 4/6 pig generation viremia, the porcine blood serum RNA infected 3 weeks 6/6 detects the positive.
Infect 1 week, the 3rd group has 1/6 detection to find that there is viremia, has HEVRNA to detect in the 4th group of 1/6 porcine blood serum, 5th group have 0/6 virus-free mass formed by blood stasis counteracting toxic substances after, 6th group has 1/6 pig to have viremia, and the 7th group has the pig of 1/6 to have viremia, and the 8th group does not have viremia.Infect attack after 2 weeks, all immune attack group serum HEVRNA detect negative.Show that vaccine reduces or shortens the viremia of pig HEV, pig infects from HEV in protection.
Second group of 2-4 week all toxin expelling in test, continues 2-3 week.After counteracting toxic substances one week, 3rd group of all swine excrement has 1/6HEV RNA to detect, 4th group has the swine excrement of 0/6 to detect HEV RNA to detect, 5th group has 0/6 swine excrement to detect HEV RNA, 6th group has 1/6 swine excrement HEV RNA positive, 7th group has 1/6 swine excrement HEV RNA to detect, 8th group has 1/6 swine excrement HEV RNA to detect the positive, after counteracting toxic substances two weeks, the 3rd group of whole HEVRNA of swine excrement is negative, 4th group has the swine excrement of 0/6 to detect HEV RNA to detect the positive, 5th group has 1/6 swine excrement to detect HEV RNA, 7th group has 0/6 swine excrement HEV RNA, 8th group has 0/6 swine excrement HEV RNA to detect.Infect attack except matched group pig after 3 weeks, whole immune swine feces there is no HEV-RNA and detects.After matched group pig infects March, feces has 1/6HEVRNA to detect the positive.
Table 11 RT-Nested PCR detects the primer of HEV virus
Liver tissue injury score value after the HEV counteracting toxic substances of table 12 different animals source property
* P < 0.05 is compared with negative control group, significant difference.
Table 13RT-PCR detects the viremia after the different HEV counteracting toxic substances of immune swine and faecal viruses discharge

Claims (9)

1. prepare a method for hepatitis E virus sample particle vaccines, it is characterized in that, comprise the following steps:
(1) synthesize wild type hepatitis E virus total length ORF2 gene, after gene chemical synthesis, insert cloned plasmids pGEM-T;
(2) the Δ ORF2 gene that hepatitis E virus blocks is synthesized
Use the codon of escherichia coli hobby, the Δ ORF2 gene blocked of design coding hepatitis E virus 112-660 amino acids, then with wild type hepatitis E virus total length ORF2 gene for template, design primer synthesis Δ ORF2 gene, the Δ ORF2 gene outcome of synthesis connects with the pMAL-p4x expression plasmid fragment of same enzyme action;
(3) expression of Δ ORF2 gene in escherichia coli blocked of hepatitis E virus
Connect product conversion escherichia coli, select positive colony, order-checking, obtains pMAL-p4X MBP-Δ ORF2 recombiant plasmid; Recombiant plasmid pMAL-p4xMBP-Δ ORF2 inoculates LB culture medium, be transferred in the fermentation tank of the 20L containing 18L LB culture medium by 1:200 ~ 1:1000 (V/V), 4 ~ 6h is cultivated with 250rpm rotating speed 37 DEG C, when bacterium liquid OD600 reaches 0.6 ~ 0.8, add IPTG by concentration 0.5 ~ 0.7mmol/L and induce 4h, the centrifugal 25min of 4000rpm, obtains the yeast culture thing that weight in wet base is 110 ~ 120g/L;
(4) Isolation and purification of capsid protein Δ ORF2 that blocks of hepatitis E virus
The screening of a decomposition agent and bacterial membrane protein extract
Yeast culture thing step (3) obtained melts, be resuspended in chromatography buffer, with Ultrasound Instrument fracturing cell walls in ice bath, ultrasonic liquid 75, 000g, 4-8 DEG C is separated upper cleer and peaceful precipitation for centrifugal 1 hour, precipitation lysate dissolves, add decomposition agent and stir 2h extraction escherichia coli memebrane protein at 4 DEG C, measure the activity of extract, to determine the ratio of total protein and decomposition agent, extract is with 100, 000g ultracentrifugation 1 hour, obtain soluble upper, be that 0.5-1.0% (w/v) is so that subsequent purification by the concentration that chromatography buffer is diluted to final lysate again,
Wherein, described chromatography buffer contains 20mM NaH 2pO 4, 100mM NaCl, 1 μM of protease inhibitor E-64,0.3mM tricarboxylic methyl acid phosphate, pH 7.5;
Described lysate contains 20mM Tris-HCl, 300mM NaCl, 1mM 2 mercapto ethanol, pH 8.0;
Described decomposition agent is the mixture of Triton X-100 and DDM;
B maltose-binding protein-Δ ORF2 purification
Under 4 DEG C of conditions, 20mL amylose medium dress post, balance with the balance solution containing 1.0% (w/v) Triton-X100 and 1.0% (w/v) DDM of 3-5 times of column volume, supernatant containing maltose-binding protein-Δ ORF2 carries out siphon loading with the speed of 2mL/min, wash with the balance solution containing 1.0% (w/v) Triton-X100 and 1.0% (w/v) DDM of 10 times of column volumes after loading, again with 3 times of column volumes not containing decomposition agent Triton-X100, the balance solution washing of DDM and EDTA, finally with solution (the 20mM Tris containing 10mmol maltose, pH 8.0, 0.2M NaCl, 10mM maltose) eluting, Fraction collection, measure protein content, purity,
Wherein, described level pad contains 20mM Tris-HCl, pH 8.0,300mM NaCl, 1mM 2 mercapto ethanol, 1mM EDTA;
C enzyme action
Add the Xa factor normal-temperature reaction 36-48h of 1 unit by 75-100ug albumen, make enzyme action degree reach 90-95%;
D anti-maltose antibody Sepharose 4 affinitive layer purification
After endonuclease reaction liquid fully mixes with anti-maltose antibody coupling Sepharose 4 affinity media, hatch 18-24 hour for 4 DEG C, absorption removing maltose-binding protein, collect stream and wear liquid, stream wears the centrifugal rear results supernatant of liquid chamber temperature 4000rpm, obtain the hepatitis E virus Δ ORF2 albumen of purification at supernatant, purity detecting can reach 90%;
E sieve chromatography purification
Upper step stream wear liquid centrifugal after supernatant in, adding Triton X-100 makes its final concentration be 0.031% (w/v), be that the filter centrifugal concentrating of 20kDa is to containing 5 – 10mg/mL albumen by cutoff value, 2500mLSuperdex 20002/150 post is used to carry out sieve chromatography, protein concentrate injection loading, chromatographic column balance liquid eluting, Fraction collection eluent, collect liquid and detect purity and content, merge and collect eluent and concentrate with the ultrafilter dialysis that cutoff value cutoff value is 20KDa, obtain highly purified Δ ORF2 albumen, purity detecting can reach 99.0%;
Wherein, described balance liquid contains 10mM HEPES, pH 7.2,150mM NaCl, 0.3mM TCEP, 0.031% (w/v) Trioton X-100;
(5) hepatitis E virus sample particle vaccines preparation
According to the difference using object, adopt dissimilar adjuvant, when people is as use object, the Δ ORF2 albumen of purification adds adjuvant aluminum hydroxide absorption by 0.6-0.7mg/mL, to obtain final product; When pig, dog and chicken are as use object, the Δ ORF2 albumen of purification adds W/O/W adjuvant emulsion by 1:3 weight ratio, to obtain final product.
2. preparation method according to claim 1, is characterized in that, the primer described in step (2) is:
Forward primer:
5’-AA GGATCCATGGCGGTCGCTCCAGCCCATGACACCCCGCCAGT-3’；
Downstream primer:
5’-GG TCTAGACTATAACTCCCGAGTTTTACCCACCTTCATCTT-3’。
3. preparation method according to claim 1, is characterized in that, the escherichia coli described in step (3) are B834-pRARE2.
4. preparation method according to claim 1, is characterized in that, in the decomposition agent described in step (4), Triton X-100 and DDM concentration are 5-10% (w/v); Described total protein and the ratio of decomposition agent are 1:4 (w/w).
5. preparation method according to claim 1, is characterized in that, described wild type hepatitis E virus total length ORF2 gene refers to the hepatitis E virus total length ORF2 gene deriving from people, pig, dog, fowl, cattle, sheep, camel and rabbit.
6. the hepatitis E virus sample particle vaccines prepared by the preparation method described in any one of claim 1-5.
7. hepatitis E virus sample particle vaccines according to claim 6 is preparing the application in the capsid protein polyclonal antibody that hepatitis E virus blocks.
8. application according to claim 7, is characterized in that, adopts the capsid protein polyclonal antibody that affinitive layer purification hepatitis E virus blocks.
9. the application of hepatitis E virus sample particle vaccines according to claim 6 in the medicine of the disease caused by preparation prevention hepatitis E virus.
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CN106699895A (en) * 2016-12-05 2017-05-24 上海科华生物工程股份有限公司 Novel fusion antigen and detection kit comprising same and application
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CN112164419A (en) * 2020-09-04 2021-01-01 云舟生物科技（广州）有限公司 Method for inserting protein tag into ORF and computer storage medium
CN113855795A (en) * 2021-11-16 2021-12-31 山东农业大学 Avian hepatitis E virus ORF2 subunit vaccine
CN113855795B (en) * 2021-11-16 2023-09-26 山东农业大学 Avian hepatitis E virus ORF2 subunit vaccine

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