CN102964434A

CN102964434A - Recombinant encephalitis B virus and application thereof

Info

Publication number: CN102964434A
Application number: CN2012104791544A
Authority: CN
Inventors: 秦成峰; 史佩勇; 李世华; 李晓峰; 赵慧; 秦鄂德
Original assignee: Institute of Microbiology and Epidemiology of AMMS
Current assignee: Institute of Microbiology and Epidemiology of AMMS
Priority date: 2012-11-22
Filing date: 2012-11-22
Publication date: 2013-03-13
Anticipated expiration: 2032-11-22
Also published as: CN102964434B

Abstract

The invention discloses a recombinant encephalitis B virus and application thereof. The invention provides a protein which is obtained by mutating a 218th-site amino acid residue on an N terminal of the protein shown as a sequence 3 of a sequence table from a polar amino acid to a non-polar amino acid. The invention also discloses a gene coding the protein, a recombinant expression vector containing the gene, an expression cassette, a transgenic cell line, and a recombinant virus or recombinant bacterium. The invention also discloses an encephalitis B virus vaccine of which the active ingredient is the recombinant virus. The recombinant encephalitis B virus provided by the invention can be used as the encephalitis B virus vaccine, and has a better application prospect in prevention of the encephalitis B virus infection.

Description

A kind of restructuring encephalitis b virus and application thereof

Technical field

The present invention relates to a kind of restructuring encephalitis b virus and application thereof, particularly Methyl transporters deficient encephalitis b virus and application thereof.

Background technology

Encephalitis b virus claims again japanese encephalitis virus, belongs to flaviviridae Flavivirus member, mainly bites propagation by culex, can cause serious encephalitis and nervous system disorders clinically.The encephalitis B Major Epidemic is in south east asia, and lethality rate is up to 30%, and the patient of about 30-50% stays serious sequela.Estimate that according to the World Health Organization the annual Patients with Encephalitis B of Asia surpasses 16000 examples, death toll surpasses 5000, and human health and public health are constituted a serious threat.And encephalitis B had spread to other area in the last few years, such as Pakistan, and the countries such as Australia.Its high lethality and regional diffusion seriously jeopardize human health, have caused the extensive concern of international community.

At present, the popularity encephalitis B can only be prevented by vaccine inoculation without effective methods for the treatment of.Mainly containing three kinds of Vaccinum Encephalitis Bs in the world wide is used for clinical, comprise former generation hamster kidney cell inactivated vaccine, Vero cell inactivated vaccine and former generation hamster kidney cell attenuated live vaccine (SA14-14-2), wherein Vero cell inactivated vaccine mainly uses in developed countries such as the U.S., Europe, and rear two kinds of vaccines are mainly used in the developing countries such as India, China.

The genome of encephalitis b virus is the sub-thread positive chain RNA of long 10,976nt, comprise 5 ' and 3 ' non-coding region and single opening code-reading frame.3 kinds of structural protein of this reading frame coding: capsid protein (C), membranin precursor/membranin (PrM/M) and envelope glycoprotein (E) and 7 kinds of Nonstructural Proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5).Virus genomic 5 ' end has cap sequence, but 3 ' terminal without the polyA tail.Nonstructural Protein NS5 is the protein of the maximum of its genome encoding, viral genome copy with modulate host antiviral response process in bring into play keying action.NS5 albumen is a kind of multifunctional protein, has RNA polymerase (RNA-dependent RNA polymerase, RdRp) and methyltransgerase (methyltransferase, MTase) activity that RNA relies on.Wherein about 600 amino-acid residues of carboxyl terminal are the RdRp active zone, mainly are responsible for copying of virus genome RNA.Methyltransgerase (methyltransferase, the MTase) active zone of NS5 albumen mainly is positioned at aminoterminal, with viral genome 5' end I type cap sequence (m ⁷GpppAm-pGp) formation is closely related.In the cap sequence forming process, (be GpppA-RNA → m ⁷GpppA-RNA → m ⁷GpppAm-RNA), MTase has participated in two ground beetle glycosylation reactions, with S-adenosylmethionine (S-adenosyl-methionine, SAM) be methyl donor, 2 '-O and the N-7 of adenylic acid (AMP) ribose are methylated, form product S-adenosine-1-homocystine (S-adenosyl-1-homocysteine, SAH).The MTase activity of NS5 for virus copy with infect most important, when N-7 methylates active defective, the virus infection replication completely loses, and after 2'-O methylates active defective, virus still has replication but its pathogenic remarkable attenuating.

Summary of the invention

The purpose of this invention is to provide a kind of restructuring encephalitis b virus and application thereof.

The invention provides a kind of protein, is the protein that protein shown in the sequence 3 of sequence table is obtained after N-terminal the 218th amino acids residue sports nonpolar amino acid by polare Aminosaeren.This sudden change makes the loss of activity that methylates of described protein 2 '-O position, affects encephalitis b virus and transcribes and add the cap process.

The gene of code for said proteins also belongs to protection scope of the present invention.

Described gene specifically can be following 1)-3) in arbitrary described dna molecular: 1) sequence 2 of sequence table is from the dna molecular shown in 5 ' the terminal 7677-10391 position Nucleotide; 2) under stringent condition with 1) dna molecular of described dna molecule hybridize and code for said proteins A; 3) with 1) or 2) described dna molecular has homology more than 90% and the dna molecular of code for said proteins.Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, hybridizes under 65 ° of C, then uses 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively to wash film once.

Described polare Aminosaeren specifically can be L-glutamic acid (E).Described nonpolar amino acid specifically can be L-Ala (A).

The recombinant expression vector, expression cassette, transgenic cell line, recombinant virus or the recombinant bacterium that contain described gene all belong to protection scope of the present invention.

Described recombinant expression vector can be the recombinant plasmid that obtains at the double chain DNA molecule shown in the sequence 2 of the multiple clone site insertion sequence table of expression vector.The recombinant plasmid pAJE70-E218A that double chain DNA molecule shown in the sequence 2 of the multiple clone site of the pANCR-L1 carrier that described recombinant expression vector specifically can be at expression vector (as between Asc I and the XhoI restriction enzyme site) insertion sequence table obtains.Plasmid shown in the sequence 4 that described pANCR-L1 carrier is sequence table.

Described recombinant virus specifically can be the restructuring encephalitis b virus of genomic dna shown in the sequence 2 of sequence table.

Described recombinant virus specifically can be above arbitrary described recombinant expression vector is imported stripped mammalian cell and cultivates the recombinant virus that mammalian cell obtains.Described mammalian cell specifically can be BHK-21 cell, Vero cell or C6/36 cell.

It is the recombinant virus that the encoding sequence of nonpolar amino acid obtains by the sequence encoding mutant of polare Aminosaeren that described recombinant virus specifically can be the Nucleotide of coding NS5 albumen the 218th amino acids residue in the encephalitis b virus.Described NS5 albumen is shown in the sequence 3 of sequence table.Described polare Aminosaeren specifically can be L-glutamic acid (E).Described nonpolar amino acid specifically can be L-Ala (A).The encoding sequence of described L-glutamic acid specifically can be " gag ", and the encoding sequence of described L-Ala specifically can be " gcc ".

The present invention also protects the application of above arbitrary described recombinant virus in the preparation Vaccinum Encephalitis B.Described vaccine specifically can be the vaccine that the prevention encephalitis b virus infects.

The present invention also protects a kind of Vaccinum Encephalitis B, and its activeconstituents is above arbitrary described recombinant virus.Described vaccine specifically can be the vaccine that the prevention encephalitis b virus infects.

The present invention also protects a kind of method that reduces the methyl transferase activity of encephalitis b virus, is to be the encoding sequence of nonpolar amino acid with the Nucleotide of coding NS5 albumen the 218th amino acids residue in the encephalitis b virus by the sequence encoding mutant of polare Aminosaeren.Described NS5 albumen is shown in the sequence 3 of sequence table.Described polare Aminosaeren specifically can be L-glutamic acid (E).Described nonpolar amino acid specifically can be L-Ala (A).The encoding sequence of described L-glutamic acid specifically can be " gag ", and the encoding sequence of described L-Ala specifically can be " gcc ".

Restructuring encephalitis b virus provided by the invention (encephalitis b virus E218A) is the Methyl transporters deficient, has following advantage: (1) is attenuation fully, and security is very high; (2) the attenuation feature is stable, and genetic stability is good, reply into the possibility of wild-type virus extremely low; (3) only copy (being that the processes such as the copying of rna virus cdna group, the assembling of virus, ripe release are all carried out in tenuigenin) in tenuigenin, its viral genome of carrying is without the danger that is incorporated in the host cell gene group; (4) can induce the immunne response that produces for the wild-type encephalitis b virus; (5) can in animal model, the watch for animals attack of the external source encephalitis b virus of avoiding lethal dose.In sum, restructuring encephalitis b virus provided by the present invention can be used as Vaccinum Encephalitis B, infects having a good application prospect for the prevention encephalitis b virus.

Description of drawings

Fig. 1 is structural representation and the mutational site synoptic diagram of the cDNA of wild-type encephalitis b virus full-length RNA.

Fig. 2 is the result of the step 3 of embodiment 1.

Fig. 3 is the result of embodiment 2 indirect immunofluorescences.

Fig. 4 is the result of embodiment 3 hollow patch tests.

Fig. 5 is the result at the intracellular Proliferation Characteristics of difference of encephalitis b virus among the embodiment 4.

Fig. 6 is each result who tests for the virus liquid plaque among the embodiment 5.

Fig. 7 is result's (survival rate) of the neural invasiveness feature of encephalitis b virus among the embodiment 7.

Fig. 8 is result's (virus titer) of the neural invasiveness feature of encephalitis b virus among the embodiment 7.

Fig. 9 be among the embodiment 8 encephalitis b virus E218A to the result of the sensitivity testing step 1 of Interferon, rabbit.

Figure 10 be among the embodiment 8 encephalitis b virus E218A to the result of the sensitivity testing step 2 of Interferon, rabbit.

Figure 11 is the immunogenic result of encephalitis b virus E218A among the embodiment 9.

Figure 12 is the result of the immune protective of encephalitis B E218A recombinant virus among the embodiment 10.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.

PET-28a (+), available from Novagen company, catalog number (Cat.No.) CB4746393.E. coli bl21 (DE3): available from Novagen company, catalog number (Cat.No.) CB1493450.The BHK-21 cell: available from ATCC, catalog number is CCL-10.The Vero cell: available from ATCC, catalog number is CCL-81.The C6/36 cell: available from ATCC, catalog number is CRL-1660.

Encephalitis b virus SA14-14-2 strain: available from Chengdu Inst. of Biological Products; Reference: Wu Yonglin, Zhao Yu, topaz celestial being etc. the DYNAMIC DISTRIBUTION of encephalitis b virus attenuated strain SA14-14-2 in Mice Body.Products in China is learned magazine, 2007,20(3) 204-205).Encephalitis B virulent strain SA14: reference: Ling Jingping, Zhu Yingeng, Du Guizhi etc., epidemic encephalitis type B virulent strain and attenuated vaccine strain SA14-14-2 are to the pathogenic and pathological research of monkey and mouse, and the microbiology immunology is made progress, and 2000,4).

The Construction and identification of embodiment 1, encephalitis b virus E218A

The purpose of present embodiment is for making up a kind of restructuring encephalitis b virus (claim again encephalitis b virus E218A, represent with E218A) of Methyl transporters deficient.The structural representation of the cDNA that the full-length RNA of wild-type encephalitis b virus (virulent strain represents with WT) is corresponding and the synoptic diagram in mutational site are seen Fig. 1.

The cDNA sequence corresponding with the full-length RNA of wild-type encephalitis b virus (virulent strain) is shown in the sequence 1 of sequence table, from 5 ' terminal the 1st-95 Nucleotide are 5 ' UTR, 96-476 position Nucleotide is the encoding gene (C) of capsid protein C, 477-2477 position Nucleotide is the encoding gene (prM-E) of prM-E albumen, encoding gene (NS1) the 3723-4607 position Nucleotide that the 2478th-3722 Nucleotide are non-structural protein NS 1 is the encoding gene (NS2) of Nonstructural Protein NS2,4608-6464 position Nucleotide is the encoding gene (NS3) of Nonstructural Protein NS3,6465-7676 position Nucleotide is the encoding gene (NS4) of Nonstructural Protein NS4,7677-10391 position Nucleotide is the encoding gene (NS5) of Nonstructural Protein NS5, and 10395-10976 position Nucleotide is 3 ' UTR.

One, the structure of carrier pAJE70

Double chain DNA molecule shown in the sequence 1 of sequence table is inserted the pANCR-L1 carrier, and (the pANCR-L1 carrier is the plasmid shown in the sequence 4 of sequence table; In the sequence 4, be the sp6 promotor from 5 ' terminal 2350-2575 position Nucleotide) Asc I and the XhoI restriction enzyme site between, obtain carrier pAJE70.

Two, the structure of recombinant plasmid pAJE70-E218A

1, take carrier pAJE70 as template, adopt the primer of J-BamHI – F and J-E218A-R composition to carrying out pcr amplification, obtain pcr amplification product (2777bp).

J-BamHI–F：5’-ggaaccac ggatccttttcctgactcaaat-3’；

J-E218A-R：5’-ctaacccaatacatggcgtgattggagtttc-3’。

2, take carrier pAJE70 as template, adopt the primer of J-E218A-F and J-XbaI-R composition to carrying out pcr amplification, obtain pcr amplification product (840bp).

J-E218A-F：5’-gaaactccaatcacgccatgtattgggttag-3’；

J-XbaI-R：5’-accccaaagcttcaaac tctagataccgtg-3’。

3, simultaneously with the pcr amplification product of the pcr amplification product of step 1 and step 2 as template, adopt primer that J-BamHI – F and J-XbaI-R form to carrying out pcr amplification, obtain pcr amplification product (3586bp).

4, the pcr amplification product that obtains with restriction enzyme BamHI and XbaI double digestion step 3 reclaims enzyme and cuts product.

5, with restriction enzyme BamHI and XbaI double digestion carrier pAJE70, reclaim carrier framework (about 9982bp).

6, the enzyme of step 4 is cut the carrier framework connection that product is connected with step, obtained recombinant plasmid pAJE70-E218A.According to sequencing result, it is as follows that recombinant plasmid pAJE70-E218A is carried out structrual description: inserted the double chain DNA molecule shown in the sequence 2 of sequence table between the AscI of pANCR-L1 carrier and XhoI restriction enzyme site.The sequence 2 of sequence table only is Nucleotide shown in the sequence 1 is suddenlyd change for " cc " by " ag " from 5 ' terminal 8329-8330 position Nucleotide with the difference of the sequence 1 of sequence table, thereby Nonstructural Protein NS5 is suddenlyd change for L-Ala (A) by L-glutamic acid (E) from N-terminal the 218th amino acids residue, and this sudden change is called for short E218A sudden change.

Three, the E218A sudden change is on the impact of methyl transferase activity

The methyltransgerase section of wild-type Nonstructural Protein NS5 (being called for short the wild-type methyltransgerase) is shown in the sequence 3 of sequence table.The methyltransgerase section of mutant Nonstructural Protein NS5 (being called for short the mutant methyltransgerase) only is sequence 3 is suddenlyd change for L-Ala (A) by L-glutamic acid (E) from N-terminal the 218th amino acids residue with the difference of wild-type methyltransgerase.

1, the preparation of recombinant plasmid first

(1) take carrier pAJE70 as template, the primer that forms with MTase-F and MTase-R obtains pcr amplification product (encoding wild type Methyl transporters enzyme fragment) to carrying out pcr amplification.

MTase-F：5’-CC CATATGGAAGGCCTGGGGGCAGGAC-3’；

MTase-R：5’-CC CTCGAGATGCTCAGGGTCTTTGTGCC-3’。

(2) with the pcr amplification product of restriction enzyme NdeI and XhoI double digestion step (1), reclaim enzyme and cut product.

(3) with restriction enzyme NdeI and XhoI double digestion pET-28a (+), reclaim carrier framework (about 5289bp).

(4) enzyme of step (2) is cut product and is connected 3 with step) carrier framework connect, obtain recombinant plasmid first (expression has histidine-tagged wild-type Methyl transporters enzyme fragment, and target molecular weight is 35kD).

2, the preparation of recombinant plasmid second

(1) take recombinant plasmid pAJE70-E218A as template, the primer that forms with MTase-F and MTase-R obtains pcr amplification product (encoding mutant type Methyl transporters enzyme fragment) to carrying out pcr amplification.

Step (2) and step (3) are with step (2) and the step (3) of step 1.

(4) enzyme of step (2) is cut product and is connected 3 with step) carrier framework connect, obtain recombinant plasmid second (expression has histidine-tagged mutant Methyl transporters enzyme fragment, and target molecular weight is 35kD).

3, the preparation of wild-type Methyl transporters enzyme fragment and mutant Methyl transporters enzyme fragment

(1) recombinant plasmid first or recombinant plasmid second are imported e. coli bl21 (DE3), obtain recombinant bacterium.

(2) recombinant bacterium is seeded to LB liquid nutrient medium (containing 60 μ g/ml penbritins), 37 ℃, 250rpm shaking culture are to OD _600nm=0.6, then adding IPTG(in culture system, to make its concentration be 0.4mM), 16 ℃, 250rpm shaking culture 18 hours.

(3) take into the culture system of step (2), the centrifugal 15min of 5000rpm collects bacterial sediment.

(4) bacterial sediment that obtains with lysate (25mM HEPES, pH 7.5,500mM sodium-chlor, 10% glycerine) the resuspended step (3) that contains the 2mM beta-mercaptoethanol, then the centrifugal 60min of 20000rpm collects supernatant liquor.

(5) supernatant liquor that step (4) is obtained is splined on the nickel affinity column, first with the elutriant first wash-out of 2 column volumes to remove foreign protein, solution with the elutriant second wash-out of 2 column volumes and after collecting post then.Elutriant first: contain 20mmol/L Tris-HCl, 10mM imidazoles, 0.5mol/L NaCl and 10g/100mL glycerine.Elutriant second: contain 20mmol/L Tris-HCl, 200mmol/L imidazoles, 0.5mol/L NaCl and 10g/100mL glycerine.

That (6) gets that step (5) collects crosses solution behind the post, and dialysis to be removing imidazoles, is stored in-80 ℃ after concentrated.

Adopting the recombinant plasmid first to carry out the concentrated solution that above-mentioned steps obtains is the protein liquid of wild-type Methyl transporters enzyme fragment.Adopting recombinant plasmid second to carry out the concentrated solution that above-mentioned steps obtains is the protein liquid of mutant Methyl transporters enzyme fragment.

The polyacrylamide gel electrophoresis figure of the protein liquid of the protein liquid of wild-type Methyl transporters enzyme fragment and mutant Methyl transporters enzyme fragment see Fig. 2 A(wherein WT represent the protein liquid of wild-type Methyl transporters enzyme fragment, E218A represents the protein liquid of mutant Methyl transporters enzyme fragment), all show electrophoretically pure purpose band, and purity is all greater than 95%.

4, methylation analysis

Synthetic m7G*pppA-RNA ₁₉₀And G*pppA-RNA ₁₉₀(* represents the phosphorus P 32 mark of back, RNA ₁₉₀Representative and the sequence 1 of sequence table are from RNA corresponding to the DNA shown in the 1st to 190 Nucleotide of 5 ' end).Step is as follows: sequence 1 is carried out in-vitro transcription from 5 ' terminal the 1st to 190 Nucleotide, and (the in-vitro transcription test kit is available from Promega, article No. is P1280, the by specification operation) and with the cap sequence analogue add cap (for adding the test kit of cap available from NEB, article No. is S1405L and S1406S, the by specification operation), the P32 mark utilizes the vaccinia virus capping enzyme (M2080S) of NEB company to finish, the inorganic pyrophosphatase (NEB, M0296L) that adds 0.004 unit in the process increases the output of m7G*pppA-RNA190 and the G*pppA-RNA190 of P32 mark.Product is carried out purifying with sephadex column, then use phenol chloroform extracting and purifying, and precipitate with ethanol.

The methyl transferase activity of the protein liquid of the wild-type Methyl transporters enzyme fragment of difference detecting step 3 preparations and the protein liquid of mutant Methyl transporters enzyme fragment.

The N-7 methylation analysis system of adenylic acid (AMP) ribose (20 μ l): contain 4pmol G*pppA-RNA ₁₉₀The unlabelled S-adenosylmethionine of 50 μ M (available from NEB company), 1 μ g Methyl transporters enzyme fragment, 2mM dithiothreitol (DTT) (DTT), the sodium-chlor of a certain concentration (100mM, 200mM, 300mM or 400mM), all the other are 50mM Bis-Tris damping fluid (pH6.0); Hatch 1h for 30 ℃.

The 2'-O methylation analysis system of adenylic acid (AMP) ribose (20 μ l): contain 2mM dithiothreitol (DTT) (DTT), 1mM magnesium chloride, the unlabelled S-adenosylmethionine of 50 μ M, 3-4pmol m7G*pppA-RNA ₁₉₀, 1 μ g Methyl transporters enzyme fragment, all the other are 50mM Tris-HCl damping fluid (pH9.0); Hatch 25min for 23 ℃.

In the above-mentioned system, the Methyl transporters enzyme fragment all adds with the form of the protein liquid of the protein liquid of wild-type Methyl transporters enzyme fragment or mutant Methyl transporters enzyme fragment, and the quality of Methyl transporters enzyme fragment is in the total protein quality in the protein liquid.

After above-mentioned reaction finishes, also use the ethanol precipitated rna with the phenol-chloroform extracting and purifying, carry out resuspended digest with nuclease P 1 (available from US Biological company) afterwards with the water without the RNA enzyme, then be dissolved in 0.65M LiCl solution and carry out radioanalysis at polymine cellulose thin-layer chromatography plate (JT Baker company), cap sequence is undertaken quantitatively by the mark of radio isotope P33.

When adopting wild-type Methyl transporters enzyme fragment, the N-7 methylation analysis of adenylic acid (AMP) ribose the results are shown in Figure 2B, and the suitableeest NaCl concentration of reacting initial system is 300mM.

When adopting wild-type Methyl transporters enzyme fragment or mutant Methyl transporters enzyme fragment, the N-7 methylation analysis result of adenylic acid (AMP) ribose (the NaCl concentration of reacting in the initial system is 300mM) sees Fig. 2 C.When adopting wild-type Methyl transporters enzyme fragment or mutant Methyl transporters enzyme fragment, 2 of adenylic acid (AMP) ribose '-the O methylation analysis the results are shown in Figure 2D.The result shows, the E218A sudden change is so that the N-7 position methylation level of adenylic acid (AMP) ribose is reduced to 15% of normal level, and adenylic acid (AMP) ribose 2 '-the O methylation level is reduced to 0, the 218th amino acids that is NS5 albumen be L-glutamic acid for 2 of adenylic acid (AMP) ribose '-methylating of O be absolutely necessary, sport behind the L-Ala 2 '-loss of activity that methylates of O.

Four, the rescue of encephalitis b virus E218A

1, cuts recombinant plasmid pAJE70-E218A with restriction enzyme XhoI enzyme, reclaim linearization plasmid.

2, take the linearizing fragment of step 1 as template, adopt SP6 RiboMAX Express Large Scale RNA Production Systems(Promega company product) and the step of by specification carry out in-vitro transcription, then adopt RNeasy mini kit(Qiagen company product) purifying transcription RNA and quantitative ,-80 ℃ of transcription RNA packing postposition behind the purifying are frozen stand-by.

3, with liposome Lipofectamine 2000(available from Invitrogen company) transcription RNA transfection individual layer BHK-21 cell that step 2 is obtained, method is as follows: at first with 50 μ l OPTI-MEM substratum and 4 μ l liposome mixings, room temperature is placed behind the 5min and 10 μ l transcription RNA(5 μ g) and 50 μ l OPTI-MEM substratum mix, room temperature is placed 20min, then mixed solution is joined in 1 hole of 6 orifice plates (having inoculated the BHK-21 cell in 6 orifice plates), then add 450 μ l OPTI-MEM substratum, 37 ℃, 5%CO ₂Hatch 6h under the condition, remove supernatant and add the DMEM substratum that contains 2%FBS, 37 ℃, 5%CO ₂Hatch under the condition to cytopathy occurring, centrifugal collection culture supernatant is re-seeded into the BHK-21 cell with culture supernatant and cultivates, and centrifugal collection supernatant after the pathology appears in cell, is encephalitis b virus E218A seed liquor, and is frozen in-80 ℃.

4, the RNA of encephalitis b virus E218A detects

(1) total RNA of extraction encephalitis b virus E218A seed liquor adopts sequencing primer R13 to carry out reverse transcription, obtains cDNA.Sequencing primer R13:5 '-AGATCCTGTGTTCTTCCTCACCACCAGCTACA-3 '.

(2) cDNA that step (1) is obtained checks order, and sequencing result is shown in the sequence 2 of sequence table.

Five, the preparation of wild-type encephalitis b virus (virulent strain)

Replace recombinant plasmid pAJE70-E218A to carry out step 3 with carrier pAJE70, obtain wild-type encephalitis b virus (virulent strain) seed liquor, frozen in-80 ℃.

Embodiment 2, encephalitis b virus are to the infection rate of BHK-21 cell

Respectively the 2 transcription RNA that obtain of the step 3 of embodiment 1 and the 2 transcription RNA that obtain of step 4 are determined as follows:

1, infect individual layer BHK-21 cell with transcription RNA, respectively at after infecting 24,48,72h collects cell, is laid on slide glass, 37 ℃, 5%CO after being resuspended in the DMEM nutrient solution that contains 10%FBS ₂Cultivate 8-12h under the condition, be the antigen sheet, the antigen sheet is placed fixedly 30-60min of-20 ℃ of acetone, it is stand-by to be put in-20 ℃ of refrigerator sealings after drying.

2, adopt the encephalitis b virus envelope E protein to resist (available from abcam company, catalog number is ab41671) more, by indirect immunofluorescence the viral differential protein in the BHK-21 cell is detected.Method is as follows: antibody by the suitable proportion dilution, is hatched 1-2h with the BHK-21 cell in the antigen sheet at 37 ℃, with PBS damping fluid (10mM K ₂HPO ₄, 2mM KH ₂PO ₄, 135mM NaCl, 2.7mM KCl, pH7.4) and the washing 3 times of vibrating, each 10min, room temperature is dried; The sheep anti-mouse igg antibody that adds the FITC mark of using 800 times of dilutions of PBS damping fluid at the virus antigen sheet, put 37 ℃ of effect 60min, then the virus antigen slide is put into PBS damping fluid vibration washing 3 times, each 10min, room temperature is dried, observations under the fluorescent microscope.

IIFly the results are shown in Figure 3.Among Fig. 3, WT represents wild-type encephalitis b virus (virulent strain), and E218A represents encephalitis b virus E218A.Encephalitis b virus E218A and wild-type encephalitis b virus (virulent strain) all can be at BHK-21 cells encephalitis b virus E albumen, and expressing quantity increases along with the prolongation of time, and encephalitis b virus E218A is close to the infection rate of BHK-21 cell with wild-type encephalitis b virus (virulent strain).

The plaque feature of embodiment 3, encephalitis b virus

Respectively encephalitis b virus E218A seed liquor and wild-type encephalitis b virus (virulent strain) seed liquor of embodiment 1 preparation are carried out following detection:

Be 1 * 10 with titre ⁸The encephalitis b virus E218A seed liquor of PFU/mL is carried out 10 times of gradient dilutions with the DMEM substratum that contains 2%FBS, and (extent of dilution is followed successively by 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6With 10 ^-7).Be 4 * 10 with titre ⁸Wild-type encephalitis b virus (virulent strain) seed liquor of PFU/mL (virulent strain) is carried out 10 times of gradient dilutions with the DMEM substratum that contains 2%FBS, and (extent of dilution is followed successively by 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6With 10 ^-7).Each diluent is inoculated in the individual layer BHK-21 cell that is laid on 6 orifice plates, 37 ℃, 5%CO with 500 μ l/ holes respectively ₂Leave standstill 1-2h under the condition, inhale and abandon culture supernatant, in each hole, add agar lid (the DMEM substratum that namely contains 2%FBS and 1% agar), 37 ℃, 5%CO ₂Cultivate 3d under the condition, then with the fixing 1h of 4% formaldehyde room temperature, abandon the agar lid, with Viola crystallina room temperature dyeing 10min, observe the plaque form, and calculate plaque forming unit (PFU).

Photo is seen Fig. 4.Among Fig. 4, WT represents wild-type encephalitis b virus (virulent strain), and E218A represents encephalitis b virus E218A.Encephalitis b virus E218A can form comparatively homogeneous of size, sharp-edged plaque, and diameter is 0.53 ± 0.16mm(n=20).The plaque that wild-type encephalitis b virus (virulent strain) forms is larger, and its diameter is 0.91 ± 0.18mm(n=20).The above results shows that the plaque diameter of encephalitis b virus E218A is significantly less than the encephalitis b virus virulent strain, so it has significant stigma (small plaque, sp) feature.

Embodiment 4, encephalitis b virus at the intracellular Proliferation Characteristics of difference

In order to observe the Proliferation Characteristics of encephalitis b virus in muroid, primates and mosquito class clone, respectively encephalitis b virus E218A seed liquor and wild-type encephalitis b virus (virulent strain) seed liquor of embodiment 1 preparation are carried out following detection:

The encephalitis b virus seed liquor is inoculated BHK-21 cell, Vero cell or C6/36 cell in 24 orifice plates, 37 ℃, 5%CO with the inoculum size of MOI=1 ₂Leave standstill 1h under the condition, culture supernatant is abandoned in suction, add the DMEM nutrient solution (BHK-21 cell, Vero cell) that contains 2%FBS or the RPMI-1640 maintenance medium (C6/36 cell) that contains 2%FBS, 37 ℃ (BHK-21 cell, Vero cell) or 28 ℃ (C6/36 cell), 5%CO ₂Cultivate under the condition, collected culture supernatant on the 24th, 48 and 72 hour after the inoculation, (method all adopts the BHK-21 cell to carry out Plaque assay with embodiment 3 with plaque titration measuring virus titer; The unit of result data is PFU/mL, i.e. viral level in every milliliter of supernatant liquor), draw one step growth.

The results are shown in Figure 5.Among Fig. 5, WT represents wild-type encephalitis b virus (virulent strain), and E218A represents encephalitis b virus E218A.The replication of encephalitis b virus E218A in three kinds of clones all is weaker than wild-type encephalitis b virus (virulent strain).Encephalitis b virus E218A reaches respectively at 48h, 72h after infecting and 72h and copies the peak in BHK-21 cell, Vero cell and C6/36 cell.The result shows that encephalitis b virus E218A can effectively copy in different clones, but duplicating efficiency is lower than wild-type encephalitis b virus (virulent strain).

The genetic stability of embodiment 5, encephalitis b virus

The encephalitis b virus E218A seed liquor of embodiment 1 preparation (the 0th generation virus liquid) is carried out following detection:

1, with the 0th generation virus liquid be inoculated in individual layer Vero cell with the inoculum size of MOI=0.01, collecting cell supernatant behind the 3d (f1 disease venom) is by plaque test determination virus titer.

2, the viral supernatant that previous step is obtained is inoculated in individual layer Vero cell with MOI=0.01, by plaque test determination virus titer.

Repeat step 29 times, collect the viral supernatant of every generation, frozen stand-by in-80 ℃.

3, extract respectively the RNA of viral supernatant of per generation, adopt sequencing primer R13 to carry out reverse transcription, obtain cDNA, take cDNA as template, adopt the primer of J-BamHI – F and J-XbaI-R composition to carrying out pcr amplification, pcr amplification product is checked order.In per generation,, the sequencing result of virus supernatant was all consistent, all such as the sequence 2 of sequence table from shown in 5 ' the terminal 5568-9153 position Nucleotide.The result shows that behind the Vero passage, the E218A sudden change all has the genetic stability of gene.

4, get the 0th generation virus liquid, the 5th generation virus liquid and the tenth generation virus liquid, carry out respectively plaque size measurement (method is with embodiment 3, adopts the BHK-21 cell to carry out Plaque assay).Photo is seen Fig. 6.Go down to posterity through some generations on the Vero cell, the plaque size of encephalitis b virus E218A is without considerable change, and homogeneous comparatively, shows that this recombinant virus has good genetic stability.

The neurovirulence feature of embodiment 6, encephalitis b virus

With encephalitis b virus with 8 * 10 ³PFU, 8 * 10 ²PFU, 8 * 10 ¹3 week of the dosage encephalic inoculation BALB/c mouse in age (available from Military Medical Science Institute's Experimental Animal Center) of PFU or 8PFU, 8 mouse of every kind of dose inoculation, observe mouse invasion and death condition in 21 days, inoculate the mortality ratio (death toll/inoculation number) of adding up afterwards this dosage group mouse in 15 days, mean survival time (to inoculation the 15th day also the survival time of the mouse of survival be designated as 15 days) and encephalitis b virus to the mld (LD of mouse ₅₀).Carry out repeated experiments three times, results averaged.

The results are shown in Table 1.

The neurovirulence feature result of table 1 encephalitis b virus

The above results shows that the mouse neurovirulence of encephalitis b virus E218A is significantly smaller than its parent's wild-type encephalitis b virus (virulent strain), has obvious attenuation feature.

The neural invasiveness feature of embodiment 7, encephalitis b virus

1, with encephalitis b virus with 4 * 10 ⁷3 week of the dosage intraperitoneal inoculation BALB/c mouse in age (available from Military Medical Science Institute's Experimental Animal Center) of PFU is observed mouse invasion and death condition in 15 days.Carry out repeated experiments three times, each 8 mouse of every kind of virus inoculation of repeated experiments, results averaged.

The results are shown in Figure 7.Among Fig. 7, WT represents wild-type encephalitis b virus (virulent strain), and E218A represents encephalitis b virus E218A.Wild-type encephalitis b virus (virulent strain) under this dosage can make mouse all dead, and encephalitis b virus E218A to mouse without lethality.The above results shows that the neural invasiveness of the mouse of encephalitis b virus E218A is significantly smaller than parent's wild-type encephalitis b virus (virulent strain), has obvious attenuation feature.

2, with encephalitis b virus with 4 * 10 ⁷3 week of the dosage intraperitoneal inoculation BALB/c mouse in age (available from Military Medical Science Institute's Experimental Animal Center) of PFU is put to death mouse to pluck the eyeball mode in inoculation after 1,3,5,7 day respectively, puts to death 4 at every turn, gets blood and cerebral tissue.Blood in 4 ℃ leave standstill 3h after the centrifugal serum of collecting, 56 ℃ of deactivation 30min measure virus titer in the serum (method adopts the BHK-21 cell to carry out Plaque assay with embodiment 3) with the plaque method.After cerebral tissue ground and homogenizes, suspension centrifugal 10min collections of 10000rpm in was equally with plaque method mensuration virus titer (method is with embodiment 3, and employing BHK-21 cell carries out Plaque assay).Carry out repeated experiments three times, each 16 mouse of every kind of virus inoculation of repeated experiments, results averaged.

The results are shown in Figure 8.Among Fig. 8, WT represents wild-type encephalitis b virus (virulent strain), and E218A represents encephalitis b virus E218A.Behind wild-type encephalitis b virus (virulent strain) the infecting mouse 24h, detect the virus of about 640PFU/ml in the serum, be reduced under the detection threshold subsequently, and the mice serum that encephalitis b virus E218A infects did not all detect virus at 1,3,5 day.Virus titer in wild-type encephalitis b virus (virulent strain) the infected group mouse brain tissue in 1,3,5,7 day all far above encephalitis b virus E218A infected group.The virus load of encephalitis b virus E218A infected group mouse brain tissue reached peak value at the 5th day and (is about 8.4 * 10 ³PFU/ml) and at the 7th day be reduced under the detection threshold, the virus titer in wild-type encephalitis b virus (virulent strain) the infected group mouse brain tissue is 1.4 * 10 the 5th day value ⁷PFU/ml, and continued to be increased to 3.6 * 10 at the 7th day ⁷PFU/ml.Above result shows that encephalitis b virus E218A gets neural invasiveness and is significantly smaller than wild-type encephalitis b virus (virulent strain) in mouse.

Embodiment 8, encephalitis b virus E218A are to the sensitivity testing of Interferon, rabbit

Respectively encephalitis b virus E218A seed liquor and wild-type encephalitis b virus (virulent strain) seed liquor of embodiment 1 preparation are carried out following detection (measuring virus to the susceptibility of Interferon, rabbit):

1, experimental group: the BHK-21 cell is inoculated in the 96 porocyte plates, treat that it grows to individual layer postoperative infection encephalitis b virus, multiplicity of infection is 5, then culture plate is hatched 1h at 37 ℃ of incubators, suction is abandoned supernatant and is washed three times with the PBS damping fluid, then the IFN-α A/D (Sigma) that adds respectively various dose (10,100 or 500U/ml), continue to place 37 ℃ of incubators to incubate 48h, (method is with embodiment 3 with the virus titer in the Plaque determination method mensuration supernatant liquor behind the collecting cell supernatant, adopt the BHK-21 cell to carry out Plaque assay), with MTS (Cell

Aqueous One Solution Cell Proliferation Assay, Promega) method measure cell viability (detected result passed through OD492 _NmNumerical value embodies).Arrange and do not add the blank group that IFN-α A/D only adds encephalitis b virus.Arrange and do not add each negative control group that encephalitis b virus only adds corresponding dosage IFN-α A/D.

Inhibiting rate=(the OD492 of experimental group cell _NmThe OD492 of-blank group cell _NmThe OD492 of the negative control group of this experimental group of)/( _NmThe OD492 of-blank group _Nm).

Virus titer in the virus titer of inhibition multiple=blank group cell conditioned medium/experimental group cell conditioned medium.

The results are shown in Figure 9.Among Fig. 9, WT represents wild-type encephalitis b virus (virulent strain), and E218A represents encephalitis b virus E218A.10,100 or the IFN-α A/D dosage of 500U/ml under, Interferon, rabbit is to the inhibiting rate of encephalitis b virus E218A (in the born of the same parents) and suppress multiple (born of the same parents are outer) and all be significantly higher than inhibiting rate and inhibition multiple to wild-type encephalitis b virus (virulent strain), has significant difference.E218A is more responsive to Interferon, rabbit for this explanation encephalitis b virus.

2, the BHK-21 cell is inoculated in 96 porocyte plates, treat that it grows to individual layer postoperative infection encephalitis b virus, multiplicity of infection is 0.1,5 or 10,37 ℃ of incubators are hatched 1h, supernatant is abandoned in suction, wash three times with the PBS damping fluid, the IFN-α A/D (Sigma) that then adds 10U/ml processes, and continues to place 37 ℃ of incubators to hatch 48h.With the virus titer in the Plaque determination method mensuration supernatant liquor (method adopts the BHK-21 cell to carry out Plaque assay with embodiment 3), measure cell viability with the method for MTS behind the collecting cell supernatant.

The results are shown in Figure 10.Among Figure 10, WT represents wild-type encephalitis b virus (virulent strain), and E218A represents encephalitis b virus E218A.Under different multiplicity of infection (0.1,5 or 10), Interferon, rabbit is to the inhibiting rate of encephalitis b virus E218A (in the born of the same parents) and suppress multiple (born of the same parents are outer) and all be significantly higher than inhibiting rate and inhibition multiple to wild-type encephalitis b virus (virulent strain), has significant difference.E218A is more responsive to Interferon, rabbit for this explanation encephalitis b virus.

The immunogenicity of embodiment 9, encephalitis b virus E218A

The encephalitis b virus E218A seed liquor of embodiment 1 preparation is carried out following detection:

Experimental group (10): with encephalitis b virus E218A subcutaneous abdomen inoculation BALB/c mouse in 4 age in week, every mouse inoculation dosage is 10 ⁴PFU.

Negative control group (10): will with the isopyknic PBS damping fluid of the encephalitis b virus E218A of experimental group inoculation BALB/c mouse in 4 age in week.

Cut tail respectively at 14d and 28d after the immunity and get blood.In 4 ℃ leave standstill 3h after the centrifugal serum of collecting, 56 ℃ of deactivation 30min ,-20 ℃ frozen stand-by.

1, the mensuration of the special IgG antibody titer of the encephalitis b virus in the immune serum

With the PBS damping fluid immune serum is prepared into the diluent of 1:20,1:40,1:80,1:160,1:320,1:640,1:1280,1:2560 and 1:5120, measures IgG antibody titer in the serum by indirect immunofluorescence assay again.Antigen in the indirect immunofluorescence assay is encephalitis b virus SA14-14-2 strain.Concrete grammar is as follows: with above-mentioned diluent respectively with antigen sheet (i.e. absorption has the slide glass of BHK-21 cell) in 37 ℃ in BHK-21 cell hatch 1-2h, PBS damping fluid (10mM K ₂HPO ₄, 2mM KH ₂PO ₄, 135mM NaCl, 2.7mM KCl, pH7.4) to wash 3 times, room temperature is dried; Add the sheep anti-mouse igg antibody with the FITC mark of 800 times of dilutions of 0.02% Evans blue solution, put 37 ℃ of effect 60min, PBS damping fluid washing 3 times, room temperature is dried; Observations under the fluorescent microscope will can be observed special green fluorescence and be judged to be the positive under field of microscope.

The IgG antibody titers namely can be viewed as positive maximum dilution multiple.Experimental mice the results are shown in Figure 11B.The immunity 14d after, experimental mice serum in IgG antibody remarkable rising is all arranged, antibody titer can reach 1:1280, the antibody titer of negative control group is less than 1:20.In the time of behind the immunity 28d, the IgG antibody titer of experimental mice can reach 1:2560.The above results shows, with 10 ⁴Behind the PFU encephalitis b virus E218A immune mouse, can effectively excite mouse to produce high level and the lasting special IgG antibody of encephalitis b virus, encephalitis b virus E218A has good immunogenicity.

2, the mensuration of the encephalitis b virus NAT in the immune serum

Adopt the NAT in the PRNT mensuration serum.Concrete grammar is as follows: with the PBS damping fluid serum is prepared into the diluent that 1:20,1:40,1:80,1:160 and 1:320 doubly dilute; Diluent is mixed (encephalitis b virus that contains about 100PFU in the mixed solution) with encephalitis B virulent strain SA14 equal-volume, hatch 1.5h for 37 ℃, then (method is with embodiment 3 to detect virus titer in the supernatant, adopt the BHK-21 cell to carry out Plaque assay), calculate 50% NAT according to the Reed-Muench method.Calculation formula is as follows:

Distance proportion=(among the 50-and plaque number be lower than 50% percentage ratio)/(in and the plaque number be higher than 50% percentage ratio-in and the plaque number be lower than 50% percentage ratio).

The antilogarithm of 50% NAT=(in and plaque number be lower than the logarithm of 50% dilution logarithm+distance proportion x dilution factor).

Experimental mice the results are shown in Figure 11A.Immune function brings out the mouse generation tires greater than the neutralizing antibody of 1:20, and wherein the highest NAT can reach more than the 1:100.In addition, high-caliber neutralizing antibody can be kept more than 4 weeks.Above-mentioned data show that encephalitis b virus E218A can also effectively bring out mouse and produce the neutralizing antibody with provide protection.

3, immune mouse spleen cell proliferation activity and secretion of gamma-IFN level determination thereof

28d puts to death the disconnected neck of mouse after the immunity, in 70% alcohol immersion moments later, opens the abdominal cavity, gets spleen.Push gently spleen with the syringe nook closing member at 200 order Stainless Steel Cloths, make unicellular in woven wire flows into plate (15ml has been housed has contained 10%FBS RPMI-1640 substratum).Single cell suspension after 400 order woven wires filter, with filtrate in the centrifugal 10min of 200g.Remove supernatant, in precipitation, add 5ml Tris-NH ₄Cl (pH7.2) hangs cell, and room temperature is placed 10min, to remove red corpuscle.The centrifugal 10min of 200g removes supernatant, uses Tris-NH ₄Cl (pH7.2) removes red corpuscle once again.Add 5ml in the most backward precipitation and contain 10%FBS RPMI-1640 substratum, hanged cell, counting cells concentration, it is for subsequent use to put ice bath.

With the PBS damping fluid splenocyte suspension is diluted to 2 * 10 ⁶Cell/ml adds to 96 orifice plates (100 μ l/ hole); The every hole of experimental group adds the heat-inactivated encephalitis b virus SA14-14-2 of 100 μ l strain (2.5 μ g/ml), and the every hole of concanavalin A control group adds 100 μ l concanavalin A (2.5 μ g/ml; Sigma), the every hole of blank group adds 100 μ l PBS damping fluids, cultivates 4 days in 37 ℃.

Adopt the MTS method to measure the propagation situation of splenocyte under virus antigen stimulates.The MTS method: every porocyte adds 30 μ lMTS, and behind the 5h, every hole adds 30 μ l 10%SDS(again and contains 0.01M HCl), 37 ℃ of night incubation, the light absorption value in each hole of mensuration, 492nm place.Stimulation index (SI)=OD _{Experimental group}/ OD _{The blank group}The results are shown in Figure 11C.

Adopt the IFN-γ factor content in IFN-γ ELISA test kit (available from Excell) the detection culture supernatant.The results are shown in Figure 11D.

The result shows that after stimulating with virus antigen, the proliferation of splenocytes of immune group mouse significantly increases, and has significant difference with control group.And the IFN-γ factor level of splenocyte secretion also increases significantly, and this shows with this recombinant virus immune mouse can produce stronger cellular immune level.

The immune protective of embodiment 10, encephalitis B E218A recombinant virus

Experimental group (10): with encephalitis b virus E218A subcutaneous abdomen inoculation BALB/c mouse in age in 20 4 weeks, every mouse inoculation dosage is 10 ⁴PFU, the encephalitis B virulent strain SA14 of subcutaneous vaccination 10LD50 after 4 weeks.

Control group: replace encephalitis b virus, other same experimental group with isopyknic PBS damping fluid.

Attack the rear survival condition of observing mouse every day of poison, totally 15 days.

Figure 12 is seen in result's demonstration, and the mouse of control group is all dead at the 9th day, and all survivals at 15 days of the mouse of experimental group, protection ratio is 100%.This shows, attacks with the lethal dose abdominal cavity in the situation of mouse, and this recombinant virus can be mouse fully protection is provided, and shows that further this recombinant virus has good immune protective.

Claims

1. a protein is the protein that protein shown in the sequence 3 of sequence table is obtained after N-terminal the 218th amino acids residue sports nonpolar amino acid by polare Aminosaeren.

2. the gene of protein shown in the coding claim 1.

3. gene as claimed in claim 1, it is characterized in that: described gene is following 1)-3) in arbitrary described dna molecular:

1) sequence 2 of sequence table is from the dna molecular shown in 5 ' the terminal 7677-10391 position Nucleotide;

2) under stringent condition with 1) dna molecular of described dna molecule hybridize and code for said proteins A;

3) with 1) or 2) described dna molecular has homology more than 90% and the dna molecular of code for said proteins.

4. the recombinant expression vector, expression cassette, transgenic cell line, recombinant virus or the recombinant bacterium that contain claim 2 or 3 described genes.

5. the recombinant virus shown in claim 4 is characterized in that: shown in recombinant virus be the restructuring encephalitis b virus of genomic dna shown in the sequence 2 of sequence table.

6. claim 4 or the 5 described recombinant viruses application in the preparation Vaccinum Encephalitis B.

7. Vaccinum Encephalitis B, its activeconstituents is claim 4 or 5 described recombinant viruses.

8. method that reduces the methyl transferase activity of encephalitis b virus is to be the encoding sequence of nonpolar amino acid with the Nucleotide of coding NS5 albumen the 218th amino acids residue in the encephalitis b virus by the sequence encoding mutant of polare Aminosaeren.