CN108359015A

CN108359015A - Porcine rotavirus VP fusion protein reconstructed volumes and its preparation method and application

Info

Publication number: CN108359015A
Application number: CN201810271984.5A
Authority: CN
Inventors: 徐进平; 姚帅
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2018-03-29
Filing date: 2018-03-29
Publication date: 2018-08-03
Anticipated expiration: 2038-03-29
Also published as: CN108359015B

Abstract

The invention discloses porcine rotavirus VP fusion protein reconstructed volumes and its preparation method and application.Fusion protein VP8 VP7 TAT provided by the invention, including porcine rotavirus（PRV）11 Core amino acids of the TAT protein transduction peptide basic amino acid enrichment region for blocking segment VP8 and VP7 and being incorporated in its C-terminal of outer capsid structural proteins VP4.Mouse is immunized in fusion protein VP8 VP7 TAT by way of intraperitoneal injection or oral administration gavage, body can effectively be induced to generate humoral immune response and mucosal immune response, there is good immunogenicity.Therefore, porcine rotavirus albumen VP8, VP7 and TAT amalgamation and expression is provided a kind of new method, the also exploitation for porcine rotavirus new oral vaccine is laid a good foundation by the present invention to prevent PRV infection.

Description

Porcine rotavirus VP fusion protein reconstructed volumes and its preparation method and application

Technical field

The invention belongs to gene biological field of engineering technology, and in particular to porcine rotavirus albumen VP8 and VP7 and TAT structures At fusion protein and its preparation method and application.

Background technology

Porcine rotavirus is one of the important pathogen for causing piglet virus diarrhea.Caused by porcine rotavirus infection Grice diarrhoea is in the equal generally existing of each pig-raising countries in the whole world.It is reported that Britain piglet group incidence more than 80%, The death rate is 0-50%.The investigation of piglet PRV infection shows sense before Fujian Province of China Foochow, 10 age in days of Nanping Prefecture to wean Dye rate respectively reaches 82.3%, 91.7%, and (Zhang Lei defends wide gloomy porcine rotavirus infection research progress [J] .2005 (4), 42- 44), the virus either simple infection or mixed infection equal generally existing in swinery, thus causes to pig aquaculture huge Big economic loss.

Porcine rotavirus (Porcine Rotavirus, PRV) is Reoviridae (Reoviridae) rotavirus (Rotavirus) member, for no cyst membrane, double-stranded rna virus.Porcine rotavirus infection is main caused by porcine rotavirus The disease of property symptom characterized by piglet apocleisis, vomiting, diarrhea, dehydration and disturbance of acid-base balance.Woode and Bridge in 1975 For the first time rotavirus is isolated from pig.Hereafter, China veterinarian is again successively in many animals such as piglet, calf, lambs Detected in excrement and be isolated to rotavirus (investigation of Cheng Jianpin, Li Changming, Wei Jin dragon Piglet Diarrhea Caused by Porcine rotavirus with Prevent the Gansu [J] animal and veterinary, 2005,35 (5):28-29).Rotavirus particle is slightly rounded, has double capsid, diameter For 65-75nm, core diameter 37-40nm, no edge film has double capsid.PRV genomes by 11 segments Double-stranded RNA group At the molecular weight of total length 18Kb, each segment differ.In a replication process, RNAs plays two：1. being directly translated as egg White matter；2. lifting plate acts on, strand RNA s is synthesized, dsRNAs is further synthesized.Existing oneself identifies 11 kinds of albumen, respectively by 11 Genetic fragment coding synthesis.Belong to PRV underwears glutelin, outer capsid proteins and non-structural protein, they determine the sense of virus Metachromia, immunogenicity, and play an important role in terms of influencing virus structure and function.Wherein 6 kinds for structural proteins (VP1, VP2, VP3, VP4, VP6, VP7), 5 kinds are non-structural protein (NSPl, NSP2, NS34, NS35 and NS28).In this 11 kinds of albumen, VP1, VP2, VP3, NSP2, NS34, NS28 are rna binding protein, they are related with the duplication of genome, synthesis and expression (Chen Shuhong, Wang is newborn, and Shi Dongfang waits separation identification and part of properties Study of China Preventive Veterinary Medicine report [J] .2004 of porcine rotavirus, 7:1)。

Immunoprophylaxis is to prevent the effective ways for causing grice diarrhoea by rotavirus infection.Although the existing inactivation of China Rotavirus traditional vaccine also has certain effect application is upper, but this traditional inactivated vaccine generate complete immunity compared with Slowly, immunizing dose is bigger than normal and passive immune protection rate is low, is unfavorable for urgent immunization campaign and reduction expense.In addition, attenuation seedling phase Than can effectively induce alimentary canal part mucosa-immune in inactivated vaccine, antibody-mediated and cell-mediated immune response is generated, Immune duration is longer than inactivated vaccine, and strong virus attack protective rate can reach 90%.But since attenuated live vaccine exists Virulence returns strong potential danger, at present not as the mainstream of vaccine research.Therefore the research of subunit viral vaccine is to preventing this Disease most important (investigation of Cheng Jianpin, Li Changming, Wei Jin dragon Piglet Diarrhea Caused by Porcine rotavirus and prevention [J] Gansu herding beasts Doctor, 35 (5):28-29).Rotavirus outer capsid is made of structural proteins VP4 and VP7, wherein VP7 by genetic fragment 9 or 7,8, It is different according to different strains) it encodes, molecular weight 37K is made of 326 AA, accounts for the 30% of virus protein total amount, is outer virionic membrane Important glycoprotein and it is main neutralize antigen, and determine the G serotypes of virus, and be between the different strains of phase homologous serotype It is highly conserved.Studies have shown that the synthetic peptide (275-295 amino acids) of recombinant protein and VP7, which can generate protection animal, avoids RV The neutralizing antibody of infection.VP4 is encoded by genetic fragment 4, and the virus polypeptide for being 88K by the molecular weight that 776 AA are formed accounts for disease The 1.5% of toxalbumin total amount.VP4 is related with viral virulence, so that virus is had pathogenic.VP8 belongs to VP4 and blocks segment, contains 247 amino acid, VP8 contains the major antigenic sites of VP4, and is responsible for VP4 specificity neutralization reactions.Its VP4 with overall length Segment is the same, body can be stimulated to generate neutralizing antibody, to excite immanoprotection action (Bridges J C.Detection by electron microscopy of caliciviruses,astroviruses and rotavirus-like Particles in the faeces of piglets with diarrhoea [J] .Veterinary Record, 1980, 107(23):532-533)。

Protein transduction domains (Protein Transduction Domain, PTD) be one section can effectively transport it is various Substance enters the peptide fragment of cell and nucleus.PTD can by carry substance, as protein, DNA, chemicals, oligonucleotides, They are transported to corresponding position by liposome etc..Present most study, most widely used protein transduction domain turn from HIV-1's Record activity factor (Trans-activating Transcriptional Activator, TAT).TAT protein has transduction work( The structural domain of energy shares 86 amino acid, wherein the minimum peptide fragment with protein transduction is deposited and is made of 11 amino acid, sequence It is classified as YGRKKRRQRRR.Wherein 6 arginine and 2 lysine residues, isoelectric point 12.7, therefore be to carry height positive electricity The polypeptide of lotus.Substituting any one alkaline amino acid residue therein with uncharged alanine can all make to wear film activity drop It is low, and wearing film activity then when other amino acid residues in alternative sequence will not change, and illustrate TAT cell-penetrating peptides institute band just Charge is necessary to its transmembrane ability, therefore speculates that these positive charges are likely to the cell membrane of eukaryocyte to occur Strong electrostatic interaction wears membrane process to mediate.TAT can the transduction in several minutes by the polypeptide being attached thereto or protein It into cell, and can be transported in vivo to brain tissue by blood circulation, enter neuron or colloid across blood-brain barrier Into the cell, transduction rate is fast, efficient.Have tens kinds of compounds of studies have shown that and protein enters difference by Successful transductions Cell or cross over blood-brain barrier, and show corresponding bioactivity (Yi Zhang, Jian-Fang Ning, Xing- qin Qu,X iao-Lin Meng,Jin-Ping Xu.TAT-mediated oral subunit vaccine against white spot syndrome virus in crayfish,Journal of Virological Methods,2012, 181:59-67. patent No.s：CN 102206660B).

Therefore, it is merged with destination protein using TAT, by oral administration or piglet is immunized in the mode of injection, is prevented by colyliform Virus infection causes the effective ways of grice diarrhoea, has important scientific meaning and application value.

Invention content

In order to expand the research range of the prior art, the object of the present invention is to provide porcine rotavirus albumen VP8 and VP7 with The fusion protein reconstructed volume VP8-VP7-TAT of TAT and the Escherichia coli base containing recombinant expression carrier pGEX-VP8-VP7-TAT Because of engineered strain.The fusion protein VP8-VP7-TAT yield of bacterial strain expression is larger, is easy to industrialized production, at low cost, safety Property it is good, it is particularly possible to be applied to large-scale pig aquaculture in.TAT protein transduction peptide can carry VP8-VP7 albumen and pass through Intestinal wall cell and enter blood, by blood circulation, fusion protein VP8-VP7-TAT is made to reach different tissues, induces body Humoral immune response and mucosal immune response are generated, the protective effect that body resists rotavirus infection is enhanced.

Above-mentioned purpose that the invention is realized by the following technical scheme：

First aspect present invention provides a kind of fusion protein VP8-VP7-TAT, amino acid sequence such as SEQ ID NO.2 institutes Show.

Second aspect of the present invention provides a kind of nucleotide sequence that fusion protein VP8-VP7-TAT is corresponding, optimized encoding Nucleotide sequence is as shown in SEQ ID NO.1.

Third aspect present invention provides a kind of Recombinant organism containing fusion protein VP8-VP7-TAT genes Strain, Classification And Nomenclature：Escherichia coli BL21 (DE3) (pGEX-VP8-VP7-TAT), e. coli bl21 (DE3) (pGEX-VP8-VP7-TAT), deposit number：CCTCC NO:2018047.The bacterium is sent to Chinese allusion quotation on January 19th, 2018 Type culture collection carries out preservation, address：The Chinese Wuhan Wuhan Universitys.Later referred to as E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT)。

Fourth aspect present invention provides a kind of preparation method of fusion protein VP8-VP7-TAT, and its step are as follows：

1, the recombinant expression plasmid pGEX-VP8-VP7-TAT for peptide gene of transduceing containing VP8, VP7 gene and TAT is obtained：

Artificial synthesized recombination VP8-VP7-TAT genetic fragments, sequence are shown in SEQ ID NO.1.VP8-VP7- TAT genetic fragments and plasmid vector pGEX-6p-1 are respectively through EcoR I and Xho I digestions, agarose gel electrophoresis, glue recycling, Finally target fragment is connected on pGEX-6p-1 carriers, is ligated and transformed into bacillus coli DH 5 alpha competent cell, it is identified, Obtained positive recombinant is the Escherichia coli containing VP8-VP7-TAT genes, that is, constructs plasmid pGEX-VP8-VP7- TAT。

2, the preparation of Recombinant organism：

Recombinant expression plasmid pGEX-VP8-VP7-TAT is converted into E. coli BL21 (DE3) competent cell, It is energy expressed fusion protein VP8-VP7- that obtained positive transformant is accredited as positive bacterium colony through bacterium solution PCR and gene sequencing The recombination engineering bacteria E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT) of TAT.

3, the preparation of fusion protein VP8-VP7-TAT：

The single bacterium colony of recombination engineered strain E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT) is inoculated in and is contained In the 20ml LB liquid mediums of 100 μ g/ml Amp, 37 DEG C of incubator overnight culture 10-12h.Second day, 1ml bacterium solutions is taken to transfer Into LB liquid mediums of the 100ml containing 100 μ g/ml Amp, 37 DEG C of shaking table culture 3h arrive bacterium solution OD₆₀₀Value about 0.6 or so When, add IPTG derivants to final concentration of 0.5mM, in 37 DEG C of shaking table 250rpm induced expressions 4-5h.By 4 DEG C of bacterium solution, 12000rpm centrifuges 5min, abandons supernatant, collects thalline.With appropriate PBS buffer solution washing thalline, it is used in combination and appropriate lysozyme is added Thalline is resuspended in cell pyrolysis liquid, and 300W ultrasonications 20min, 8000rpm centrifuge 20min.Broken precipitation becomes through 8M urea Property after 4 DEG C, 8000rpm, centrifuge 10min, collect supernatant.By GST-Resin purification step purified fusion albumen VP8-VP7- After TAT, the fusion protein VP8-VP7-TAT purified, after carrying out renaturation dialysis, as there is the gene of biological activity Engineered fusion protein VP8-VP7-TAT.The sequence of fusion protein VP8-VP7-TAT is shown in SEQ ID NO.2.

A kind of fusion protein VP8-VP7-TAT of fifth aspect present invention offer prevents preparing or treats by porcine rotavirus Application in caused grice diarrhoea medicine.

In one particular embodiment of the present invention with 0.9% physiological saline suspension VP8-VP7-TAT, VP8-TAT, VP7- The suspension of about 500 μ g/ml is made in TAT inclusion bodys.By the dosage of every 100 μ g albumen of mouse through intraperitoneal injection or gavage Mouse is immunized in mode；Simultaneously using the only mouse of injection or gavage PBS buffer solution as negative control.Continuous immunity three times, every time Interval 14 days.Blood is taken respectively at immune preceding docking every time, and collects stool in mice.Using ELISA method, detection is immune small respectively Specific antibody IgA in specific antibody IgG and night soil-treatment liquid in mouse serum.ELISA the experimental results showed that, with negative control Group compares, through be injected intraperitoneally and three kinds of recombinant proteins of intragastric after induction of mice serum in specific antibody IgG production It is raw.IgG levels in intraperitoneal injection group mice serum are higher than gavage group.In addition, after three kinds of recombinant proteins of intragastric Produce the specific antibody IgA of higher level in inducing mouse body, and intraperitoneal injection group and negative control group IgA antibody OD₄₉₀Value is then in low-level.Fusion protein VP8-VP7-TAT is through being injected intraperitoneally inducing producing specificity IgG antibody and through filling The level of stomach inducing producing specificity antibody I gA is above single albumen VP8-TAT and VP7-TAT.

In one particular embodiment of the present invention, using indirect ELISA method detection fusion albumen VP8-VP7-TAT's Gut function is worn, that is, measures fusion protein VP8-VP7-TAT sample sets as the time increases fusion protein VP8-VP7-TAT across small Mouse intestines intestinal wall enters the OD of parenteral solution₄₉₀Value.Experimental result shows, fusion protein VP8-VP7-TAT in the parenteral solution of sample sets OD₄₉₀Value is gradually increasing as time increases, and Cut-off value ratios, is positive in sample 2-5h.Experimental result table Bright fusion protein VP8-VP7-TAT, which has, wears intestines activity.

The VP8 of the present invention and existing report, VP7 subunit vaccine are compared, advantage in：

1) the fusion protein weight of Bacillus coli expression TAT protein transduction structural domain and PRV outer capsid proteins VP8-VP7 is utilized Body is immunized in structure body VP8-VP7-TAT.Fusion protein VP8-VP7-TAT and single VP8-TAT, VP7-TAT fusion protein phase Compare, VP8-VP7-TAT can induce body to generate stronger humoral immune response and mucosal immune response, be porcine rotavirus New generation vaccine development is laid a good foundation.

2) fusion protein VP8-VP7-TAT of the C-terminal according to the present invention with nexin transduction domain peptide T AT has Intestines activity is worn, TAT can carry destination protein VP8-VP7 and pass through small intestine intestinal wall cell, prevent VP8-VP7 albumen by Gastric juice digestion And enter directly into blood, induction body generates humoral immune response and mucosal immune response.Solving subunit vaccine can By without injection but by being immunized in a manner of oral, the feasibility with easier practical operation.

3) construction method of engineering strain disclosed by the invention, expression quantity is big, easy to operate, at low cost, safety Property it is good, it is particularly possible to be applied to large-scale pig aquaculture in.

Description of the drawings

Fig. 1 is the plasmid map of recombinant plasmid pGEX-VP8-TAT, pGEX-VP7-TAT, pGEX-VP8-VP7-TAT.

A：PGEX-VP8-TAT plasmid maps；B：PGEX-VP7-TAT plasmid maps；C：PGEX-VP8-VP7-TAT plasmids Collection of illustrative plates.

Fig. 2 is the PCR qualification figures of recombinant plasmid pGEX-VP8-TAT, pGEX-VP7-TAT, pGEX-VP8-VP7-TAT.

A. the PCR qualification figures of recombinant plasmid pGEX-VP8-TAT

M:DNA Marker IV；1:Using plasmid pGEX-6p-1 as the PCR product of template；2：With recombinant plasmid pGEX- VP8-TAT is the PCR product of template.

B. the PCR qualification figures of recombinant plasmid pGEX-VP7-TAT

M:DNA Marker IV；1:Using recombinant plasmid pGEX-VP7-TAT as the PCR product of template.

C. recombinant plasmid pGEX-VP8-VP7-TAT PCR qualification figures

M:DNA Marker IV；1:Using recombinant plasmid pGEX-VP8-VP7-TAT as the PCR product of template.

Fig. 3 is recombination engineering E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT), E.coli BL21 (DE3) (pGEX-VP8-TAT), the SDS-PAGE electrophoresis of E.coli BL21 (DE3) (pGEX-VP7-TAT) induced expression.

A. the SDS-PAGE electrophoresis of recombination engineering E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT) induced expression Figure

M:Albumen Marker；1：E.coli BL21 (DE3) (pGEX-6p-1) are crushed liquid precipitate after induction；2：After induction E.coli BL21 (DE3) (pGEX-6p-1) are crushed liquid supernatant；3：E.coli BL21 (DE3) (pGEX-VP8-VP7- is not induced TAT) it is crushed liquid precipitate；4：E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT) are not induced to be crushed liquid supernatant；5：After induction E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT) are crushed liquid precipitate；6：E.coli BL21 (DE3) (pGEX- after induction VP8-VP7-TAT) it is crushed liquid supernatant.

B. the SDS-PAGE electrophoresis of recombination engineering E.coli BL21 (DE3) (pGEX-VP8-TAT) induced expression

M:Albumen Marker；1：E.coli BL21 (DE3) (pGEX-6p-1) are crushed liquid precipitate after induction；2：After induction E.coli BL21 (DE3) (pGEX-6p-1) are crushed liquid supernatant；3：E.coli BL21 (DE3) (pGEX-VP8-TAT) are not induced Broken liquid precipitate；4：E.coli BL21 (DE3) (pGEX-VP8-TAT) are not induced to be crushed liquid supernatant；5：E.coli after induction BL21 (DE3) (pGEX-VP8-TAT) is crushed liquid precipitate；6：E.coli BL21 (DE3) (pGEX-VP8-TAT) are broken after induction Liquid supernatant.

C. the SDS-PAGE electrophoresis of recombination engineering E.coli BL21 (DE3) (pGEX-VP7-TAT) induced expression

M:Albumen Marker；1：E.coli BL21 (DE3) (pGEX-6p-1) are crushed liquid precipitate after induction；2：After induction E.coli BL21 (DE3) (pGEX-6p-1) are crushed liquid supernatant；3：E.coli BL21 (DE3) (pGEX-VP7-TAT) are not induced Broken liquid precipitate；4：E.coli BL21 (DE3) (pGEX-VP7-TAT) are not induced to be crushed liquid supernatant；5：E.coli after induction BL21 (DE3) (pGEXVP7-TAT) is crushed liquid precipitate；6：E.coliBL21 (DE3) (pGEX-VP7-TAT) is crushed liquid after induction Supernatant.

Fig. 4 is the Western blot qualification figures of fusion protein.

A：The Western blot qualification figures of fusion protein VP8-VP7-TAT

M:Albumen Marker；1:E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT) are crushed liquid precipitate after induction；2: E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT) are crushed liquid supernatant after induction.

B：The Western blot qualification figures of fusion protein VP8-TAT

M:Albumen Marker；1:E.coli BL21 (DE3) (pGEX-6p-1) are crushed liquid precipitate after induction；2：After induction E.coli BL21 (DE3) (pGEX-VP8-TAT) are crushed liquid supernatant；3：E.coliBL21 (DE3) (pGEX-VP8- after induction TAT) it is crushed liquid precipitate.

C：The Western blot qualification figures of fusion protein VP7-TAT

M：Albumen Marker；1：After induction liquid supernatant is crushed after E.coli BL21 (DE3) (pGEX-VP7-TAT) inductions； 2:It is crushed liquid precipitate after E.coli BL21 (DE3) (pGEX-VP7-TAT) inductions.

Fig. 5 is the SDS-PAGE electrophoresis of fusion protein VP8-VP7-TAT, VP8-TAT, VP7-TAT purifying

M：Albumen Marker；1-3：Purifying protein VP8-VP7-TAT；4-6：Purifying protein VP8-TAT；7-9：Purifying protein VP7-TAT。

Fig. 6 is that ELISA measures immune serum specific IgG antibodies.

A：Intraperitoneal injection group mice serum specific IgG antibodies detect；B：Gavage group mice serum specific IgG antibodies are examined It surveys.

Fig. 7 is that ELISA measures immune mouse Specific IgA antibody.

A：Intraperitoneal injection group mouse specific antibody IgA antibody detects；B：Gavage group mouse specific antibody IgA antibody is examined It surveys.

Fig. 8 is that ELISA detection fusion albumen VP8-VP7-TAT wears gut function Activity determination.

Fig. 9 is fusion protein VP8-VP7-TAT to mouse safety testing.

Specific implementation mode

By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.The implementation provided Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.

The involved molecular biology method of this experiment is conventional method, is known to those skilled in the art.In the present invention not The content elaborated refers to《Molecular Cloning:A Laboratory guide》, J. Pehanorm Brookers, the chief editors such as D.W. Russells.

【Embodiment 1】The preparation of fusion VP8-VP7-TAT

VP8 [the sequence numbers of ripe PRV are obtained from U.S.'s biotechnology center (NCBI) gene pool： AY523636.1] and VP7 [sequence numbers：AER25320.1] albumen amino acid sequence, intercept VP8, VP7 protein sequence in have The primary structure functional areas for having immunogenicity, are converted into the nucleotide sequence containing Escherichia coli preferred codons, and according to egg The gene of white transduction structural domain peptide T AT codings can carry out the characteristic of amalgamation and expression with foreign protein genes, after optimization The nucleotide sequence of 3 ' end connection TAT of gene order, i.e. VP8-VP7-TAT, sequence is SEQ ID NO：Nucleosides shown in 1 Acid sequence.VP8-VP7-TAT sequences are obtained by the way that Tian Yihuiyuan companies are artificial synthesized, are connected to expression vector pGEX-6p-1, structure Build recombinant expression plasmid pGEX-VP8-VP7-TAT.Through the sequencing of Tian Yihuiyuan companies, comparison and PCR identifications, the results showed that structure Fusion VP8-VP7-TAT sequences it is correct, electrophoretic band be consistent with VP8-VP7-TAT theoretical value 1069Kb sizes to get To recombinant plasmid pGEX-VP8-VP7-TAT.Recombinant plasmid pGEX-VP8-VP7-TAT collection of illustrative plates is shown in attached drawing 1, plasmid PCR electrophoresis mirror Determine result and sees attached drawing 2.

【Embodiment 2】The structure of recombinant plasmid pGEX-VP8-TAT, pGEX-VP7-TAT

Using recombinant expression plasmid pGEX-VP8-VP7-TAT as template, using the method for recombinant PCR, VP8- is cloned respectively TAT, VP7-TAT sequence, through EcoR I and Xho I digestions, the electrophoresis on Ago-Gel cuts rapidly be intended to back in the UV lamp The band of receipts, with health be century Ago-Gel DNA QIAquick Gel Extraction Kit purify, single target DNA band is put into clean In Eppendorf pipes, weight is weighed.Into blob of viscose, (gel weight is 0.1g to the sol solutions PG of addition three times volume, and volume is visual For 100 μ l, and so on).60 DEG C of water-baths 10 minutes mildly spin upside down Eppendorf pipes, to ensure at intervals of two minutes therebetween Blob of viscose fully dissolves.The Buffer PS of 250 μ l are added into adsorption column, 12000rpm centrifuges 2min after standing 2min, outwells receipts Liquid in collector.750 μ L are taken to be added in an adsorption column (adsorption column is put into collecting pipe) acquired solution, room temperature (20- 25 DEG C) it places 2 minutes, 12000rpm centrifuges 2min, outwells the waste liquid in collecting pipe, adsorption column is put into collecting pipe.It will be molten Glue is transferred in adsorption column, and 12000rpm centrifuges 1min after the static 2min of room temperature, outwells the liquid in collecting pipe.To absorption 500 μ l Buffer PG are added in column, after acting on 1min at room temperature, 12000rpm centrifuges 1min, outwells the liquid in collecting pipe. 650 μ l Buffer PW are added into adsorption column again, after standing 1min at room temperature, 12000rpm centrifuges 1min, outwells collecting pipe In liquid.It repeats and adds a 650 μ l Buffer PW, after standing 1min at room temperature, 12000rpm centrifuges 1min, outwells receipts Liquid in collector.12000rpm centrifuges 2min again, and a few minutes are placed at 37 DEG C and are thoroughly dried to ethyl alcohol.Adsorption column is put into In sterile Eppendorf pipes, the ddH that 60 DEG C of water-bath the pre-heat treatments of process of 30 μ l are crossed vacantly is added among film₂O, room temperature Lower placement 2min centrifuges 2min in 12000rpm, and it is digestion products to collect the solution of acquisition in Eppendorf pipes, and will be pure The digestion products of change are connected with plasmid pGEX-6p-1.

Double digestion system is as follows：

Linked system is as follows：

Calcium Chloride Method prepares competent escherichia coli cell, and competent escherichia coli cell uses E.coli DH5 α, step Rapid and method instructs to carry out according to Molecular Cloning: A Laboratory.Prepared by competent escherichia coli cell, step is：

1. with the E.coli DH5 α single bacterium colonies newly activated on oese picking solid LB tablets, it is inoculated into 20ml liquid LB In culture medium, 37 DEG C, 250rpm shakes activation overnight.(following steps are both needed to sterile working)

2. take the Escherichia coli of the 200 above-mentioned activation of μ l in fresh 20ml LB liquid mediums, 37 DEG C, 300rpm shaking tables 2-3 hours are cultivated to OD₆₀₀Value about 0.6.

3. taking the above-mentioned bacterium solutions of 1.5ml in sterile Eppendorf pipes, 30min is placed on ice.4000rpm, 4 DEG C of centrifugations 10 Minute, abandon supernatant.

4. the 0.1M CaCl of 200 μ l ice precooling are added₂Bacterial sediment, ice bath 30 minutes, 4000rpm, 4 DEG C is resuspended in solution Centrifugation 10 minutes, abandons supernatant.

5. the 0.1M CaCl of 100 μ l ice precooling are added₂Precipitation, as competent cell is resuspended in solution, is placed in 4 DEG C of preservations, Use is advisable in for 24 hours.

10 μ l connection products are taken to distinguish Transformed E .coli DH5 α competent cells.PCR evaluation and screenings go out positive transformant, The gained positive colony as respectively E.coli DH5 α containing fusion VP8-TAT, VP7-TAT.

Competent escherichia coli cell is converted, step is：

1. taking 10 μ l of connection product under aseptic condition, it is added in 100 μ l E.coli DH5 α competent cells, gently mixes It is even, ice bath 30 minutes (above step is both needed to sterile working).

2. heat shock 90 seconds in 42 DEG C of water-baths, moves to rapidly ice bath 90 seconds in ice.

3. 800 μ l LB liquid mediums are added, 37 DEG C, 150rpm jogs, incubating 1h makes cell recovery.

4. 4000rpm is centrifuged 10 minutes, draw 800 μ l supernatants and discard, by remaining bacterium solution liquid-transfering gun gently mixing.

5. above-mentioned bacterium solution is uniformly coated on the solid LB tablets containing 100 μ g/ml ampicillins with sterile triangle glass rod On, forward direction is placed 1-2 hours, until liquid is all absorbed, is inverted tablet overnight incubation in 37 DEG C of incubators.It respectively obtains The bacterium colony of E.coli DH5 α (pGEX-VP8-TAT), E.coli DH5 α (pGEX-VP7-TAT).

The bacterium colony PCR identifications of positive transformants bacterium：

Under aseptic condition, LB tablets are observed, picking single bacterium colony is inoculated in the final concentration of containing ampicillin of 200 μ l In the LB liquid medium of 200 μ g/ml, the universal sequencing primer object on pGEX-6p-1 carriers is used after 37 DEG C of shaken cultivation about 3h Carry out bacterium colony PCR identifications.

Reaction system is as follows：

PCR response procedures are：94 DEG C of pre-degeneration 4min；Then following cycle (30cycles) is carried out：

1)VP8-TAT:94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 50s；72 DEG C re-extend 10min；

2)VP7-TAT:94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 5s；72 DEG C re-extend 10min.

After reaction, product is identified through agarose gel electrophoresis, and electrophoretic band is theoretical with VP8-TAT, VP7-TAT respectively Value 1013bp, 221bp size is consistent.See attached drawing 2.The experimental results showed that successfully constructing recombinant plasmid pGEX-VP8- respectively TAT、pGEX-VP7-TAT。

【Embodiment 3】Genetically engineered E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT), E.coli BL21 (DE3) structure of (pGEX-VP8-TAT), E.coli BL21 (DE3) (pGEX-VP7-TAT)

1 μ l plasmids pGEX-VP8-VP7-TAT, pGEX-VP8-TAT, pGEX-VP7-TAT conversion Escherichia coli are taken respectively BL21 (DE3) competent cell.Go out positive transformant through PCR evaluation and screenings.Gained positive colony is respectively engineering strain E.coli BL21(DE3)(pGEX-VP8-VP7-TAT)、E.coli BL21(DE3)(pGEX-VP8-TAT)、E.coli BL21 (DE3)(pGEX-VP7-TAT)。

Engineering strain E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT) are sent on January 19th, 2018 into State's Type Tissue Collection preservation, Classification And Nomenclature：Escherichia coli BL21(DE3)(pGEX-VP8-VP7- TAT), e. coli bl21 (DE3) (pGEX-VP8-VP7-TAT), deposit number：CCTCC NO:2018047, address：Chinese Wuhan Wuhan Universitys.

【Embodiment 4】The expression of fusion protein VP8-VP7-TAT, VP8-TAT, VP7-TAT.

(1) recombination engineered strain E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT), E.coliBL21 (DE3) (pGEX-VP8-TAT), the single bacterium of E.coli BL21 (DE3) (pGEX-VP7-TAT) is fallen respectively is inoculated in containing 100 μ g/ml ammonia benzyls In the 20ml LB liquid mediums of penicillin, 10-12h is incubated overnight in 37 DEG C of shaking table 250rpm.

(2) 1 is pressed：100 ratio is inoculated into the fresh LB Liquid Cultures containing 100 μ g/ml ampicillins of 100ml respectively In base, 37 DEG C, 250rpm culture 3h or so, until OD₆₀₀For 0.6-0.8 when, add derivant IPTG to final concentration of 0.5mM, in 37 DEG C shaking table 250rpm induced expressions 4-5h.

(3) bacterium solution 12000rpm is centrifuged into 5min, abandons supernatant, collect thalline.Thalline is resuspended with appropriate PBS buffer solution, 3s is broken in 300W ultrasonications, stops 5s, 20min, then 4 DEG C, and 12000rpm centrifuges 2min, take 50 μ l supernatants with isometric 2 × Broken Supernatant samples are made in SDS-PAGE sample buffer mixings.It discards supernatant, is resuspended and is precipitated with appropriate amounts of sterilized water, take 50 μ l With isometric 2 × SDS-PAGE sample buffer mixings, broken deposit sample is made.

(4) sample is placed in boiling water and boils 5-10min, carry out SDS-PAGE electrophoresis, whether detection recombinant protein expresses, and And it is solubility expression or formation inclusion body.After electrophoresis, gel is unloaded, (2.0g examines horse in coomassie brilliant blue staining liquid This brilliant blue R-250,450ml methanol, 450ml ddH₂O, 100ml glacial acetic acid) 3 hours of middle dyeing, then use destainer (40ml Methanol, 10ml acetic acid, 50ml ddH₂O it) decolourizes, was changed the liquid once every 30 minutes, until background is de- totally.

(5) with 12% SDS-PAGE electrophoresis detections, find have and expected purpose albumen in precipitation after bacterial cell disruption The protein band that size 59.9kDa, 56.3kDa and 31.4kDa are consistent, and largely exist with inclusion bodies, it expresses Amount is higher, and destination protein is seldom in the supernatant after bacterial cell disruption.Attached drawing 3 is shown in the SDS-PAGE identifications of three kinds of fusion proteins. The experimental results showed that expression plasmid pGEX-VP8-VP7-TAT, pGEX-VP8-TAT, pGEX-VP8-VP7-TAT of recombination to construct Fusion protein VP8-VP7-TAT, VP8-TAT, VP7-TAT are correctly expressed respectively, and are expressed in the form of inclusion body.

【Embodiment 5】Fusion protein Western blot analyses

1. by genetically engineered E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT), E.coli BL21 (DE3) (pGEX-VP8-TAT), E.coli BL21 (DE3) (pGEX-VP7-TAT) are inoculated in the 20ml LB containing 100 μ g/mlAmp respectively In fluid nutrient medium, after 37 DEG C are incubated overnight 10-12h, it is transferred to the fresh LB Liquid Cultures of the 20ml containing 100 μ g/ml Amp In base, 37 DEG C of culture 3h or so to OD₆₀₀Value is 0.6-0.8 or so, the IPTG derivants of final concentration of 0.5mM is added, at 30 DEG C Induced expression 4h.After induction, it is crushed supernatant deposit sample after preparing induction, carries out PAGE gel electrophoretic analysis.

2. after electrophoresis, removing the gel after electrophoresis, 6 filter paper and 1 NC film (one are cut out according to the size of protein versus glue Surely to wear gloves, avoid albumen on hand by fouling membrane), NC films are placed in transferring film buffer solution together with cotton pad, filter paper, gel In, impregnate 15min.

3. the clip of transferring film opened, with the gel after cotton pad, three layers of filter paper, electrophoresis, NC films, three layers of filter paper, cotton pad Sequentially, above-mentioned apparatus (paying attention to avoiding generating bubble between each layer) is completed successively in black one side, by putting down on one side for white To close clip (filter paper on film both sides cannot contact with each other, and short circuit otherwise can occur).

4. clip is slowly put into slot, (albumen is of different sizes, and the transferring film time is not yet by transferring film 2h or so under 20V voltages Together).

5. taking out NC films with tweezers, after clean 1-2min with PBST on shaking table, 5% skimmed milk power of addition is to being completely covered NC films, 4 DEG C of closings are overnight.

6. next day outwells confining liquid, NC films are shaken to cleaning 3 times, each 10min soon with PBST buffer solutions, are added by appropriate NC films are shaken cleaning 5 times, every time by the good GST primary antibodies of dilution proportion soon after room temperature shakes be incubated 2h slowly with PBST buffer solutions 10min。

Proper proportion 1 is pressed 7. being added：The secondary antibody of the 5000 HRP labels diluted, shakes after being incubated 2h, uses PBST slowly at room temperature NC films are shaken cleaning 5 times, each 10min by buffer solution soon.

8. ECL chemiluminescence detections carry out x-ray film exposure in darkroom.

Western blot immunoblotting analysis is the results show that band occurs in the specific region in pvdf membrane.Show that three kinds melt Hop protein VP8-VP7-TAT, VP8-TAT, VP7-TAT specific reaction can occur with anti-GST antibody and band is more visible, nothing Miscellaneous band.See attached drawing 4.The experimental results showed that recombination engineering bacteria E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT), E.coli BL21 (DE3) (pGEX-VP8-TAT), E.coli BL21 (DE3) (pGEX-VP7-TAT) respectively successful expression Destination protein VP8-VP7-TAT, VP8-TAT and VP7-TAT.

【Embodiment 6】The preparation of inclusion body:

(1) genetically engineered E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT), E.coli BL21 (DE3) (pGEX-VP8-TAT), E.coli BL21 (DE3) (pGEX-VP7-TAT) are fallen respectively is inoculated in containing 100 μ g/ml ampicillins 20ml LB liquid mediums in, be incubated overnight 10-12h in 37 DEG C of shaking table 250rpm.By 1：100 ratio is inoculated into respectively In the LB liquid medium containing 100 μ g/ml ampicillins fresh 100ml, 37 DEG C, 250rpm culture 3h or so, until OD₆₀₀ For 0.6-0.8 when, add derivant IPTG to final concentration of 0.5mM to induce, 37 DEG C, 250rpm induced expressions 4-5h.4 DEG C, Thalline were collected by centrifugation by 6000rpm.Thalline is resuspended with 20ml cell pyrolysis liquids, -20 DEG C of freeze thawing twice, then add 30ml cell pyrolysis liquids With appropriate lysozyme, 30 DEG C of water-bath 30min, ultrasonication (working time 3sec, intermittent time 5sec, power 300W, when whole Between 30min), 4 DEG C, 12,000rpm centrifugation 10min, abandon supernatant.

(2) inclusion body 4 DEG C of the inclusion body cleaning solution of precipitation obtained, level concussion washing 30min, this process repeat three Secondary, 4 DEG C later, 12,000rpm centrifugation 10min obtain inclusion body precipitation.Floating parts precipitate, and boil and carry out SDS-PAGE, inspection Survey destination protein.

【Embodiment 7】The purifying of fusion protein

(1) inclusion body is formed sediment with 2M urea washes twice, 4 DEG C, after 12000rpm centrifugations 10min abandons supernatant, is added appropriate Inclusion body lysate, overnight, solution becomes clarification, and by supernatant through 0.45 μm of membrane filtration, obtained solution is both for 4 DEG C of cracking The protein solution being denaturalized.

(2) 1ml GST Agarose are drawn with pipettor and is packed into chromatography column bottom, washed with the deionization of 10 times of column volumes Pillar is washed, then pillar is balanced with the Lysis Buffer of 10 times of column volumes.

(3) the good protein solution of above-mentioned denaturation is added to the nickel column balanced with the rate of 0.2ml/min with peristaltic pump In, 4 DEG C recycle loading three times, and the GST of fusion protein front end is made fully to be combined with glutathione.

(4) column is washed with the rate of 1ml/min with 50ml Lysis Buffer, with elute in lower prop not with glutathione knot The albumen of conjunction.

(6) successively with the Wash Buffer of Imidazole containing 20mM, 40mM Imidazole, 60mM Imidazole Pillar is eluted with the rate of 0.5ml/min, elutes the non-destination protein combined on glutathione column.It is examined with nucleic acid-protein detector Survey the OD of the efflux after elution every time₂₈₀, until stopping washing when baseline is steady.

(6) pillar is eluted with the rate of 0.2ml/min with the Elution Buffer of the Imidazole containing 250mM, used Eppendorf pipes collect destination protein, until OD₂₈₀Stop collecting when being continuously 0.SDS-PAGE electrophoresis is carried out, collected by detection Albumen purification effect.

(7) the above-mentioned destination protein being collected into is fitted into the bag filter handled well, bag filter both ends are clipped with clip, are set 4 DEG C of dialysis renaturations are for 24 hours in protein renaturation liquid.For the urea except deproteinized middle and high concentration, bag filter is put into PBS buffer solution In, 4 DEG C of gradient dialysis are about for 24 hours.Period replaces elution buffer once or twice, you can removes urea completely.

(8) protein solution after dialysis is concentrated into the 1/3 of about original volume to get to purified with PEG20000 Destination protein, take part carry out SDS-PAGE electrophoresis, remaining packing after be stored in -20 DEG C it is spare.

SDS-PAGE electrophoresis results show, three kinds of fusion proteins VP8-VP7-TAT, VP8-TAT, VP7-TAT can and paddy The sweet peptide of Guang-agarose resin combines, and obtains purifying protein VP8-VP7-TAT, VP8-TAT and VP7-TAT respectively after elution. See attached drawing 5.

【Embodiment 8】The detection of specific antibody IgG in immune serum

The preparation of Serum Antibody.Kunming mice is randomly divided into intraperitoneal injection group, gavage group and negative control group.Abdominal cavity Injection group and gavage group use fusion protein VP8-VP7-TAT, VP8-TAT, VP7-TAT respectively, are only immunized with 100 μ g/, cloudy Property control group is immunized with the PBS of same volume.It is 3 times immune, every minor tick 14 days.The the 7th, 21,35 day after immune with Machine takes 3 mouse dockings to take blood, and after 37 DEG C stand 1h, 4 DEG C stand overnight, 4 DEG C of next day, and 2000g centrifuges 20min, by layering Serum be sucked out and dispense freeze it is spare in -20 DEG C.

Using indirect elisa method detection serum specific antibody IgG.Respectively by the fusion of the purifying of a concentration of 10ug/ml Albumen VP8-VP7-TAT, VP8-TAT, VP7-TAT overnight, 3 are washed with PBST with 100 μ l coated elisa plates of every hole, 4 DEG C of coatings Secondary, plank is patted dry after washing on paper and (is paid attention to by each 5min：Every part of sample to do 3 it is parallel, finally measure when be averaged Value).200 μ l, 5% skimmed milk powers are added per hole, after 37 DEG C are closed 2h, PBST is washed 3 times.100 μ l are added per hole by appropriate times The serum to be checked (positive serum to be measured, the negative serum that include preparation) that number has diluted, after 37 DEG C are incubated 2h, PBST is washed 5 times. 100 μ l are added per hole and press 1:5000 diluted HRP mark sheep anti-mouse igg antibody, and after 37 DEG C are incubated 2h, PBST is washed 5 times.Per hole 100 μ l OPD substrate solutions are added, are protected from light colour developing 5min.50 μ l 2M H are added per hole₂SO₄Terminate liquid, 20min is interior to measure OD Value.The measurement of OD values：Using ELISA microplate reader OD is measured under wavelength 490nm₄₉₀Value.

Immune serum specific IgG antibodies testing result is shown, after 7 days immune, intraperitoneal injection group and gavage group The OD of mice serum specific IgG antibodies₄₉₀Value has notable raising compared to negative control group, it is seen that intraperitoneal injection group and Gavage group mouse just produces specific antibody IgG on 7th day after immune.After two exempt from, three exempt from, intraperitoneal injection group and filling The OD of IgG in the serum of stomach group mouse₄₉₀Value is lasting to be increased, compared with negative control group significant difference, and negative control group OD₄₉₀Value variation is little.In the entire experiment process, the OD of the serum specific antibody IgG of intraperitoneal injection group₄₉₀Value is consistently higher than Gavage group, and the OD of fusion protein VP8-VP7-TAT group serum specific antibodies IgG₄₉₀Value be consistently higher than VP8-TAT groups and VP7-TAT groups, and the OD of negative control group₄₉₀Value then maintains always reduced levels.See attached drawing 6.

【Embodiment 9】The detection of specific antibody IgA in immune stool in mice

The preparation of antibody in excrement.7th, 21,35 day intraperitoneal injection group, gavage group, negative control group are small after acquisition is immune The excrement of mouse is mixed well per 0.1g excrement with the PBS of 200 μ l 0.01mol/L, and upper liquid is collected by centrifugation in 4 DEG C of effect 1.5h Body, -20 DEG C of preservations are to be checked.

Using indirect elisa method detection specific antibody IgA.According to IgA detection methods, respectively by three kinds of fusion proteins The excrement diluted by suitable multiple is added after being coated with, closing in VP8-VP7-TAT, VP8-TAT, VP7-TAT in ELISA Plate Just treatment fluid washs 5 times after being incubated 2h at 37 DEG C, and by 1:5000 diluted HRP labels sheep anti mouse IgA antibodies are incubated at 37 DEG C 2h, after by OPD develop the color and terminate react, measure OD under wavelength 490nm using ELISA microplate reader₄₉₀Value.

Immune mouse Specific IgA antibody testing result is shown, after 7 days immune, gavage group mouse specific antibody IgA OD₄₉₀Value has a notable raising compared to negative control group, and the OD of intraperitoneal injection group mouse specific antibody IgA₄₉₀It is worth phase It is more not notable than being increased in negative control group.After two exempt from, three exempt from, the OD of the IgA antibody of gavage group mouse₄₉₀Value is lasting It increases, the significant difference compared with negative control group group, and the OD of intraperitoneal injection group and negative control group₄₉₀Value variation is little.Whole In a experimentation, the OD of the specific antibody IgA of gavage group₄₉₀Value is consistently higher than intraperitoneal injection group, and fusion protein VP8- The OD of VP7-TAT group-specific antibodies IgA₄₉₀Value is consistently higher than VP8-TAT groups and VP7-TAT groups, and the OD of negative control group₄₉₀ Value is then low always.See attached drawing 7.

【Embodiment 10】ELISA detection fusion albumen VP8-VP7-TAT's wears gut function Activity determination

The preparation of sample：Laboratory mice purchase is 4 week old kunming mices from Disease Control and Prevention Center of Hubei Province.It is raised after buying back In this laboratory, 28 DEG C or so of raising temperature.The mouse newly bought is fed 15 days in laboratory, after mouse is in stable condition Extremely, it takes 5cm mouse intestinal tubes, after being rushed with PBS buffer solution, intestinal tube one end is tightened, the fusion protein of purifying is drawn with liquid-transfering gun VP8-VP7-TAT ties the other end of intestinal tube to the mouse intestinal tube tied, puts into the test tube equipped with 10ml PBS buffer solution In, so that PBS buffer solution is totally submerged mouse intestinal tube.As a control group with the PBS of equivalent.Stood at 30 DEG C, respectively at 0,1,2, 3,4 and 5h draws the PBS solution in 100ul test tubes.

Wear gut function detection：Measuring samples are added to the holes 100ul/ in ELISA Plate, while right using PBS as feminine gender According to the fusion protein VP8-VP7-TAT of purifying repeats 3 parallel, 37 DEG C of coating 3h as positive control.It outwells in ELISA Plate Solution, by 250 holes μ l/ be added PBST solution, wash 5min on the oscillator, buckle on paper do, be repeated 3 times with this.It presses again 5% skimmed milk power is added in 200 holes μ l/, and 4 DEG C of closings are overnight.Confining liquid is outwelled, is washed repeatedly 3 times by above-mentioned steps.100 μ are pressed again The holes l/ are added 1:1000 diluted GST tag antibodies, 37 DEG C of incubation 2h.After primary antibody is incubated, washed 3 times with PBST, by 100 The holes μ l/ are added 1:The secondary antibody of 5000 diluted horseradish peroxidase (HRP) labels, 37 DEG C of incubation 2h.After secondary antibody is incubated, It is washed repeatedly 3 times with PSBT, then OPD developing solutions is added by 100 holes μ l/, be protected from light colour developing 5min at room temperature.50 μ l/ are pressed after taking-up Hole, which is added, terminates reaction solution, uses OD of the microplate reader at 490nm₄₉₀Value.

Fusion protein VP8-VP7-TAT wears intestines experimental result and shows, VP8-VP7-TAT sample sets detect melts in parenteral solution The OD of hop protein VP8-VP7-TAT₄₉₀Value keeps lasting during 4h and rises, in 4h, fusion protein VP8-VP7-TAT's OD₄₉₀Value reaches maximum value, and 5h is begun to decline.OD measured by negative control₄₉₀It calculating, Cut-off values are 0.15, The OD of the parenteral solution of VP8-VP7-TAT sample sets₄₉₀Value is being above 0.15 in 2-5h, and be positive result.It can be seen that TAT protein Transduction peptide can be carried to be entered across mouse intestinal wall cell outside intestinal wall in solution with the VP8-VP7 albumen of its amalgamation and expression, and And in a period of time, the concentration level of fusion protein VP8-VP7-TAT increases as time went in the outer solution of intestinal wall.Experiment The result shows that fusion protein VP8-VP7-TAT, which has, wears intestines activity.See attached drawing 8.

【Embodiment 11】Mouse safety testing

The mouse safety testing about fusion protein VBP8-VP7-TAT is carried out by monitoring the variation of mouse weight. In 42d, intraperitoneal injection group, gavage group, the average weight of PBS control group mouse are weighed in 0,14,28,42d respectively, observation is melted Influences of the hop protein VP8-VP7-TAT to mouse physical signs.

From fig. 9, it can be seen that the weight of 3 groups of mouse does not have significant difference, illustrate fusion protein VP8-VP7-TAT not shadows The growth indexes of mouse are rung, are safe and reliable immunizing antigen.See attached drawing 9.

Sequence table

<110>Wuhan University

<120>Porcine rotavirus VP fusion protein reconstructed volumes and its preparation method and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 888

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

atggcttcgc tcatttatag acaactactt actaattcat acacagtcaa tctttctgac 60

gaaattcaag agattggatc ggctaagtca caggatgtta ctataaatcc tggtccattc 120

gcacaaacag gttatgcacc agttaattgg ggagcaggtg agactaatga ctccacaact 180

gtcgagccgt tattagatgg tccataccaa ccaaccactt tcaatccacc aacaagctat 240

tgggtactac ttgcgccaac tgtagagggc gtaattgttc aaggaacaaa caataccgat 300

agatggttgg ccactatact aattgaacca aacgtacaaa caactaacag aatatacaat 360

ctttttggtc agcaagtaac tttatcggtg gagaatacgt cacagacaca atggaagttc 420

attgatgtga gtacaactac gccaacagga agttatacgc agcacggacc attgttctct 480

acaccaaaat tatacgctgt aatgaaattc agtggtagaa tatatacata taatggaacc 540

acaccaaacg caacaacagg atactattca actactaatt atgacacagt aaatatgaca 600

tcattttgtg atttttatat tataccaaga aatcaagaag aaaaatgtac tgagtatatc 660

aatcatggat tacctcctat acaaaataca aggaatgttg tgccagtatc tttatcggct 720

agagagatag tgcacacaag agctggtggc ggtggctcgg gtggcggtgg ctcgggtggc 780

ggtggctcgc caacaactgc accacaaact gaaagaatga tgcgtataaa ttggaagaga 840

tggtggcaag tctatggccg taagaaacgt cgtcagcgtc gtcgttag 888

<210> 2

<211> 295

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 2

Met Ala Ser Leu Ile Tyr Arg Gln Leu Leu Thr Asn Ser Tyr Thr Val

1 5 10 15

Asn Leu Ser Asp Glu Ile Gln Glu Ile Gly Ser Ala Lys Ser Gln Asp

20 25 30

Val Thr Ile Asn Pro Gly Pro Phe Ala Gln Thr Gly Tyr Ala Pro Val

35 40 45

Asn Trp Gly Ala Gly Glu Thr Asn Asp Ser Thr Thr Val Glu Pro Leu

50 55 60

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

65 70 75 80

Trp Val Leu Leu Ala Pro Thr Val Glu Gly Val Ile Val Gln Gly Thr

85 90 95

Asn Asn Thr Asp Arg Trp Leu Ala Thr Ile Leu Ile Glu Pro Asn Val

100 105 110

Gln Thr Thr Asn Arg Ile Tyr Asn Leu Phe Gly Gln Gln Val Thr Leu

115 120 125

Ser Val Glu Asn Thr Ser Gln Thr Gln Trp Lys Phe Ile Asp Val Ser

130 135 140

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

145 150 155 160

Thr Pro Lys Leu Tyr Ala Val Met Lys Phe Ser Gly Arg Ile Tyr Thr

165 170 175

Tyr Asn Gly Thr Thr Pro Asn Ala Thr Thr Gly Tyr Tyr Ser Thr Thr

180 185 190

Asn Tyr Asp Thr Val Asn Met Thr Ser Phe Cys Asp Phe Tyr Ile Ile

195 200 205

Pro Arg Asn Gln Glu Glu Lys Cys Thr Glu Tyr Ile Asn His Gly Leu

210 215 220

Pro Pro Ile Gln Asn Thr Arg Asn Val Val Pro Val Ser Leu Ser Ala

225 230 235 240

Arg Glu Ile Val His Thr Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gly Gly Gly Gly Ser Pro Thr Thr Ala Pro Gln Thr Glu Arg

260 265 270

Met Met Arg Ile Asn Trp Lys Arg Trp Trp Gln Val Tyr Gly Arg Lys

275 280 285

Lys Arg Arg Gln Arg Arg Arg

290 295

Claims

1. a kind of porcine rotavirus VP fusion protein reconstructed volumes VP8-VP7-TAT, it is characterised in that：Its amino acid sequence such as SEQ Shown in ID NO.2.

2. nucleotide sequence corresponding fusion protein reconstructed volume VP8-VP7-TAT described in claim 1, it is characterised in that： Coding nucleotide sequence is as shown in SEQ ID NO.1.

3. a kind of Recombinant organism strain containing fusion protein VP8-VP7-TAT genes, it is characterised in that：It is classified Name：Escherichia coli BL21 (DE3) (pGEX-VP8-VP7-TAT), e. coli bl21 (DE3) (pGEX-VP8- VP7-TAT), deposit number：CCTCC NO:2018047.

4. the preparation method of fusion protein VP8-VP7-TAT described in claim 1, including steps are as follows：

1) the recombinant expression plasmid pGEX-VP8-VP7-TAT for peptide gene of transduceing containing VP8, VP7 gene and TAT, is obtained：

Artificial synthesized recombination VP8-VP7-TAT genetic fragments, sequence are VP8-VP7-TAT bases shown in SEQ ID NO.1 Because segment and plasmid vector pGEX-6p-1 are respectively through EcoR I and Xho I digestions, agarose gel electrophoresis, glue recycling finally will Target fragment is connected on pGEX-6p-1 carriers, is ligated and transformed into bacillus coli DH 5 alpha competent cell, identified, is obtained Positive recombinant is the Escherichia coli containing VP8-VP7-TAT genes, that is, constructs plasmid pGEX-VP8-VP7-TAT；

2), the preparation of colibacillus engineering：

Recombinant expression plasmid pGEX-VP8-VP7-TAT is converted into E. coli BL21 (DE3) competent cell, is obtained Positive transformant through bacterium solution PCR and gene sequencing be accredited as positive bacterium colony be can expressed fusion protein VP8-VP7-TAT Recombination engineering bacteria Escherichia coli BL21 (DE3) (pGEX-VP8-VP7-TAT), e. coli bl21 (DE3) (pGEX-VP8-VP7-TAT), i.e. E.coli BL21 (DE3) (pGEX-VP8-VP7-TAT)；

3), the preparation of fusion protein VP8-VP7-TAT：

The single bacterium colony of recombination engineered strain Escherichia coli BL21 (DE3) (pGEX-VP8-VP7-TAT) is connect Kind is in the 20ml LB liquid mediums containing 100 μ g/ml Amp, 37 DEG C of incubator overnight culture 10-12h；Second day, take 1ml bacterium Liquid is transferred in LB liquid mediums of the 100ml containing 100 μ g/ml Amp, and 37 DEG C of shaking table culture 3h are about 0.6 to bacterium solution OD values When, add IPTG derivants to final concentration of 0.5mM, in 37 DEG C of shaking table 250rpm induced expressions 4-5h.By 4 DEG C of bacterium solution, 12000rpm centrifuges 5min, abandons supernatant, collects thalline；With appropriate PBS buffer solution washing thalline, it is used in combination and appropriate lysozyme is added Thalline is resuspended in cell pyrolysis liquid, and 300W ultrasonications 20min, 8000rpm centrifuge 20min；Broken precipitation becomes through 8M urea Property after 4 DEG C, 8000rpm, centrifuge 10min, collect supernatant；By GST-Resin purification step purified fusion albumen VP8-VP7- After TAT, the fusion protein VP8-VP7-TAT purified, after carrying out renaturation dialysis, the gene with biological activity is obtained Engineered fusion protein VP8-VP7-TAT；Fusion protein VP8-VP7-TAT sequences are shown in SEQ ID NO.2.

5. fusion protein reconstructed volume VP8-VP7-TAT described in claim 1, which is characterized in that by oral administration, can induce body production Raw humoral immune response and mucosal immune response.

6. fusion protein reconstructed volume VP8-VP7-TAT described in claim 1 prevents or treats to be drawn by porcine rotavirus in preparation Application in the grice diarrhoea medicine risen.