CN102964434B

CN102964434B - Recombinant encephalitis B virus and application thereof

Info

Publication number: CN102964434B
Application number: CN201210479154.4A
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: 2014-04-02
Anticipated expiration: 2032-11-22
Also published as: CN102964434A

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 by culex, bites propagation, can cause serious encephalitis and nervous system disorders clinically.Encephalitis B Major Epidemic is in south east asia, and lethality rate is up to 30%, and the patient of about 30-50% leaves serious sequela.According to the World Health Organization, estimate, the annual Patients with Encephalitis B in Asia surpasses 16000 examples, and 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, as Pakistan, 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, popularity encephalitis B, without effective methods for the treatment of, can only be prevented by vaccine inoculation.In world wide, mainly contain three kinds of Vaccinum Encephalitis Bs for clinical, comprise primary hamster kidney cell inactivated vaccine, Vero cell inactivated vaccine and primary hamster kidney cell attenuated live vaccine (SA14-14-2), wherein mainly developed country is used Vero cell inactivated vaccine in the U.S., Europe etc., and latter two vaccine is 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 ' end is without polyA tail.Nonstructural Protein NS5 is the maximum protein 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 approximately 600 amino-acid residues of carboxyl terminal are RdRp active zone, are mainly responsible for copying of virus genome RNA.Methyltransgerase (methyltransferase, the MTase) active zone of NS5 albumen is mainly positioned at aminoterminal, with viral genome 5' end I type cap sequence (m ⁷gpppAm-pGp) formation is closely related.In 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 of adenylic acid (AMP) ribose and N-7 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 defect, virus infection replication completely loses, and when 2'-O methylates after active defect, virus still has replication but its pathogenic remarkable attenuating.

Summary of the invention

The object 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 of sequence table 3 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 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 ' end 7677-10391 position Nucleotide; 2) under stringent condition with 1) described DNA molecule hybridize and the DNA molecular of code for said proteins A; 3) with 1) or 2) described DNA molecular has more than 90% homology 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, under 65 ° of C, hybridizes, and 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 that contains described gene, expression cassette, transgenic cell line, recombinant virus or recombinant bacterium all belong to protection scope of the present invention.

Described recombinant expression vector can be the recombinant plasmid obtaining 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 as 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 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 as shown in the sequence 2 of sequence table.

Described recombinant virus specifically can be above arbitrary described recombinant expression vector is imported in vitro 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.

The recombinant virus that the encoding sequence that it is nonpolar amino acid by the sequence encoding mutant of polare Aminosaeren that described recombinant virus specifically can be the Nucleotide of NS5 albumen the 218th amino acids residue of encoding in encephalitis b virus obtains.Described NS5 albumen is as 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 preparing Vaccinum Encephalitis B.Described vaccine specifically can be the vaccine that 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 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 the encoding sequence that is nonpolar amino acid by the Nucleotide of NS5 albumen the 218th amino acids residue of encoding in encephalitis b virus by the sequence encoding mutant of polare Aminosaeren.Described NS5 albumen is as 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 Methyl transporters deficient, and tool has the following advantages: (1) is attenuation fully, and security is very high; (2) attenuation feature is stable, and genetic stability is good, reply into the possibility of wild-type virus extremely low; (3) only in tenuigenin, 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), its viral genome of carrying is without the danger being incorporated in host cell gene group; (4) can induce the immunne response producing for wild-type encephalitis b virus; (5) can watch for animals in the animal model 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, for prevention encephalitis b virus, infects and has a good application prospect.

Accompanying drawing explanation

Fig. 1 is structural representation and the mutational site schematic 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 in embodiment 4.

Fig. 6 is each result of testing for virus liquid plaque in embodiment 5.

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

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

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

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

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

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

Embodiment

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

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

Encephalitis b virus SA14-14-2 strain: purchased 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 the SA14-14-2 pathogenic and pathological research to monkey and mouse, microbiology immunology progress, 2000,4).

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

The object of the present embodiment is for building a kind of restructuring encephalitis b virus (claim again encephalitis b virus E218A, represent with E218A) of Methyl transporters deficient.Fig. 1 is shown in by the corresponding structural representation of cDNA and the schematic diagram in mutational site of full-length RNA of wild-type encephalitis b virus (virulent strain represents with WT).

The cDNA sequence corresponding with the full-length RNA of wild-type encephalitis b virus (virulent strain) is as shown in the sequence 1 of sequence table, from the 1st-95 Nucleotide of 5 ' end, be 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, the 2478th-3722 encoding genes that Nucleotide is non-structural protein NS 1 (NS1) 3723-4607 position Nucleotide 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, 10395-10976 position Nucleotide is 3 ' UTR.

One, the structure of carrier pAJE70

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

Two, the structure of recombinant plasmid pAJE70-E218A

1, take carrier pAJE70 as template, the primer pair that adopts J-BamHI – F and J-E218A-R to form carries out pcr amplification, obtains pcr amplification product (2777bp).

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

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

2, take carrier pAJE70 as template, the primer pair that adopts J-E218A-F and J-XbaI-R to form carries out pcr amplification, obtains pcr amplification product (840bp).

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

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

3,, simultaneously using the pcr amplification product of the pcr amplification product of step 1 and step 2 as template, the primer pair that adopts J-BamHI – F and J-XbaI-R to form carries out pcr amplification, obtains pcr amplification product (3586bp).

4, the pcr amplification product obtaining by 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 carrier framework of the enzyme of step 4 being cut to product and step 5 is connected, and obtains recombinant plasmid pAJE70-E218A.According to sequencing result, recombinant plasmid pAJE70-E218A is carried out to structrual description as follows: between the AscI of pANCR-L1 carrier and XhoI restriction enzyme site, inserted the double chain DNA molecule shown in the sequence 2 of sequence table.The sequence 2 of sequence table is only Nucleotide shown in sequence 1 to be suddenlyd change for " cc " by " ag " from 5 ' end 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 impact of E218A sudden change on methyl transferase activity

The methyltransgerase section of wild-type Nonstructural Protein NS5 (being called for short wild-type methyltransgerase) is as shown in the sequence 3 of sequence table.The methyltransgerase section of saltant type Nonstructural Protein NS5 (being called for short saltant type methyltransgerase) is only sequence 3 to be 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, with the primer pair that MTase-F and MTase-R form, carry out pcr amplification, obtain pcr amplification product (encoding wild type Methyl transporters enzyme fragment).

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 to product and be connected with the carrier framework of step (3), 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, with the primer pair that MTase-F and MTase-R form, carry out pcr amplification, obtain pcr amplification product (encoding mutant type Methyl transporters enzyme fragment).

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

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

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

(1) recombinant plasmid first or recombinant plasmid second are imported to 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 in culture system, adding IPTG(to make its concentration is 0.4mM), 16 ℃, 250rpm shaking culture 18 hours.

(3) get the culture system of completing steps (2), the centrifugal 15min of 5000rpm, collects bacterial sediment.

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

(5) supernatant liquor step (4) being obtained is splined on 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: containing 20mmol/L Tris-HCl, 10mM imidazoles, 0.5mol/L NaCl and 10g/100mL glycerine.Elutriant second: containing 20mmol/L Tris-HCl, 200mmol/L imidazoles, 0.5mol/L NaCl and 10g/100mL glycerine.

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

Adopting 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 saltant type 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 saltant type 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 saltant type Methyl transporters enzyme fragment), all show electrophoretically pure object band, and purity is all greater than 95%.

4, methylation analysis

Synthetic m7G*pppA-RNA ₁₉₀and G*pppA-RNA ₁₉₀(* representative phosphorus P 32 mark below, RNA ₁₉₀represent with the sequence 1 of sequence table 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 to in-vitro transcription from the 1st to 190 Nucleotide of 5 ' end, and (in-vitro transcription test kit is purchased from Promega, article No. is P1280, by specification operation) and with cap sequence analogue add cap (for the test kit that adds cap purchased from NEB, article No. is S1405L and S1406S, by specification operation), P32 mark utilizes the vaccinia virus capping enzyme (M2080S) of NEB company to complete, in process, add the inorganic pyrophosphatase (NEB, M0296L) of 0.004 unit to increase the output of m7G*pppA-RNA190 and the G*pppA-RNA190 of P32 mark.Product is carried out to purifying with sephadex column, then use phenol chloroform extracting and purifying, and precipitate with ethanol.

The protein liquid of wild-type Methyl transporters enzyme fragment that respectively prepared by detecting step 3 and the methyl transferase activity of the protein liquid of saltant type Methyl transporters enzyme fragment.

The N-7 methylation analysis system of adenylic acid (AMP) ribose (20 μ l): containing 4pmol G*pppA-RNA ₁₉₀the unlabelled S-adenosylmethionine of 50 μ M (purchased 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): containing 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 above-mentioned system, Methyl transporters enzyme fragment all adds with the form of the protein liquid of wild-type Methyl transporters enzyme fragment or the protein liquid of saltant type Methyl transporters enzyme fragment, and the quality of Methyl transporters enzyme fragment is in the total protein quality in protein liquid.

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

While 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.

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

Four, the rescue of encephalitis b virus E218A

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

2, take the linearizing fragment of step 1 is 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 quantitative ,-80 ℃ of transcription RNA packing postposition after purifying are frozen stand-by.

3, with liposome Lipofectamine 2000(purchased from Invitrogen company) transcription RNA transfection individual layer BHK-21 cell that step 2 is obtained, method is as follows: first 50 μ l OPTI-MEM substratum and 4 μ l liposomes are mixed, room temperature is placed after 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 BHK-21 cell in 6 orifice plates), then add 450 μ l OPTI-MEM substratum, 37 ℃, 5%CO ₂under condition, hatch 6h, remove supernatant and add the DMEM substratum containing 2%FBS, 37 ℃, 5%CO ₂under condition, hatch to occurring cytopathy, centrifugal collection culture supernatant, is re-seeded into culture supernatant BHK-21 cell and cultivates, and centrifugal collection supernatant after pathology appears in cell, is encephalitis b virus E218A seed liquor, frozen in-80 ℃.

4, the RNA of encephalitis b virus E218A detects

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

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

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

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

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

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

1, with transcription RNA, infect individual layer BHK-21 cell, 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 ₂under condition, cultivate 8-12h, be antigen sheet, antigen sheet is placed in to fixedly 30-60min of-20 ℃ of acetone, after drying, be put in-20 ℃ of refrigerator sealings stand-by.

2, adopt encephalitis b virus envelope E protein to resist (purchased from abcam company, catalog number is ab41671) more, by indirect immunofluorescence, the viral differential protein in BHK-21 cell is detected.Method is as follows: antibody, by suitable proportion dilution, is hatched to 1-2h with the BHK-21 cell in 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; On virus antigen sheet, add the sheep anti-mouse igg antibody with the FITC mark of 800 times of dilutions of PBS damping fluid, put 37 ℃ of effect 60min, then virus antigen slide is put into PBS damping fluid vibration washing 3 times, each 10min, room temperature is dried, observations under fluorescent microscope.

IIFly the results are shown in Figure 3.In 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, 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 detected as follows:

By titre, be 1 * 10 ⁸the encephalitis b virus E218A seed liquor of PFU/mL is carried out 10 times of gradient dilutions with the DMEM substratum containing 2%FBS, and (extent of dilution is followed successively by 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6with 10 ^-7).By titre, be 4 * 10 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 containing 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 to the individual layer BHK-21 cell that is laid on 6 orifice plates, 37 ℃, 5%CO with 500 μ l/ holes respectively ₂standing 1-2h under condition, inhales and abandons culture supernatant, adds agar lid (the DMEM substratum that contains 2%FBS and 1% agar), 37 ℃, 5%CO in each hole ₂under condition, cultivate 3d, then with the fixing 1h of 4% formaldehyde room temperature, abandon agar lid, with Viola crystallina room temperature dyeing 10min, observe plaque form, and calculate plaque forming unit (PFU).

Fig. 4 is shown in by photo.In 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, 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, the plaque diameter of encephalitis b virus E218A is significantly less than 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 detected as follows:

Encephalitis b virus seed liquor is inoculated to BHK-21 cell, Vero cell or the C6/36 cell in 24 orifice plates, 37 ℃, 5%CO with the inoculum size of MOI=1 ₂standing 1h under 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 ₂under condition, cultivate, within the 24th, 48 and 72 hours, collect culture supernatant after inoculation, with plaque titration measuring virus titer, (method, with embodiment 3, all adopts BHK-21 cell to carry out Plaque assay; 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.In 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 is all weaker than wild-type encephalitis b virus (virulent strain).In BHK-21 cell, Vero cell and C6/36 cell, encephalitis b virus E218A reaches and copies peak respectively at 48h, 72h after infecting and 72h.Result shows, encephalitis b virus E218A can effectively copy in different clone, 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 detected as follows:

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

2, viral supernatant previous step being 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, the primer pair that adopts J-BamHI – F and J-XbaI-R to form carries out pcr amplification, and pcr amplification product is checked order.In per generation,, the sequencing result of virus supernatant was all consistent, all if the sequence 2 of sequence table is from as shown in 5 ' end 5568-9153 position Nucleotide.Result shows, after Vero passage, 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 BHK-21 cell to carry out Plaque assay).Fig. 6 is shown in by photo.Go down to posterity through some generations on 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

By encephalitis b virus with 8 * 10 ³pFU, 8 * 10 ²pFU, 8 * 10 ¹the dosage encephalic inoculation BALB/c mouse in 3 week age (purchased 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), mean survival time (survival time of the mouse also surviving for the 15th day to inoculation is designated as 15 days) and the mld (LD of encephalitis b virus to mouse that within 15 days, add up afterwards this dosage group mouse ₅₀).Carry out repeating for three times experiment, results averaged.

The results are shown in Table 1.

The neurovirulence feature result of table 1 encephalitis b virus

The above results shows, 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, by encephalitis b virus with 4 * 10 ⁷the dosage intraperitoneal inoculation BALB/c mouse in 3 week age (purchased from Military Medical Science Institute's Experimental Animal Center) of PFU, observes mouse invasion and death condition in 15 days.Carry out repeating for three times experiment, repeat to test 8 mouse of every kind of virus inoculation, results averaged at every turn.

The results are shown in Figure 7.In 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, the neural invasiveness of mouse of encephalitis b virus E218A is significantly smaller than parent's wild-type encephalitis b virus (virulent strain), has obvious attenuation feature.

2, by encephalitis b virus with 4 * 10 ⁷the dosage intraperitoneal inoculation BALB/c mouse in 3 week age (purchased from Military Medical Science Institute's Experimental Animal Center) of PFU, puts to death mouse to pluck eyeball mode in inoculation after 1,3,5,7 day respectively, puts to death 4 at every turn, gets blood and cerebral tissue.Blood is the centrifugal serum of collecting after 4 ℃ of standing 3h, and 56 ℃ of deactivation 30min measure virus titer in serum (method, with embodiment 3, adopts BHK-21 cell to carry out Plaque assay) by plaque method.After cerebral tissue grinds and homogenizes, suspension in the centrifugal 10min collection of 10000rpm, measures virus titer (method, with embodiment 3, adopts BHK-21 cell to carry out Plaque assay) by plaque method equally.Carry out repeating for three times experiment, repeat to test 16 mouse of every kind of virus inoculation, results averaged at every turn.

The results are shown in Figure 8.In Fig. 8, WT represents wild-type encephalitis b virus (virulent strain), and E218A represents encephalitis b virus E218A.After wild-type encephalitis b virus (virulent strain) infecting mouse 24h, the virus of about 640PFU/ml in serum, detected, be reduced under 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) 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 detection threshold, the virus titer in wild-type encephalitis b virus (virulent strain) infected group mouse brain tissue is 1.4 * 10 the value of the 5th day ⁷pFU/ml, and continued to be increased to 3.6 * 10 at the 7th day ⁷pFU/ml.Above result shows, encephalitis b virus E218A in mouse neural invasiveness be significantly smaller than wild-type encephalitis b virus (virulent strain).

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

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

1, experimental group: 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 5, then culture plate is hatched to 1h at 37 ℃ of incubators, suction is abandoned supernatant and is washed three times with PBS damping fluid, then the IFN-α A/D (Sigma) that adds respectively various dose (10,100 or 500U/ml), continue to be placed in 37 ℃ of incubators and incubate 48h, after collecting cell supernatant, with the virus titer in Plaque determination method mensuration supernatant liquor, (method is with embodiment 3, adopt 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).Arranging does not add IFN-α A/D only to add the blank group of encephalitis b virus.Arranging does not add encephalitis b virus only to add each negative control group of corresponding dosage IFN-α A/D.

Inhibiting rate=(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 virus titer/experimental group cell conditioned medium of inhibition multiple=blank group cell conditioned medium.

The results are shown in Figure 9.In 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 all significantly higher than the inhibiting rate of wild-type encephalitis b virus (virulent strain) and suppresses multiple the inhibiting rate of encephalitis b virus E218A (in born of the same parents) and inhibition multiple (born of the same parents are outer), has significant difference.E218A is more responsive to Interferon, rabbit for this explanation encephalitis b virus.

2, BHK-21 cell is inoculated in to 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, with PBS damping fluid, wash three times, then add the IFN-α A/D (Sigma) of 10U/ml to process, continue to be placed in 37 ℃ of incubators and hatch 48h.After collecting cell supernatant, by Plaque determination method, measure the virus titer (method, with embodiment 3, adopts BHK-21 cell to carry out Plaque assay) in supernatant liquor, by the method for MTS, measure cell viability.

The results are shown in Figure 10.In 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 all significantly higher than the inhibiting rate of wild-type encephalitis b virus (virulent strain) and suppresses multiple the inhibiting rate of encephalitis b virus E218A (in born of the same parents) and inhibition multiple (born of the same parents are outer), 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 detected as follows:

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

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

Respectively at 14d and 28d after immunity, cut tail and get blood.The centrifugal serum of collecting after 4 ℃ of standing 3h, 56 ℃ of deactivation 30min ,-20 ℃ are frozen stand-by.

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

With 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, then measures the IgG antibody titer in serum by indirect immunofluorescence assay.Antigen in indirect immunofluorescence assay is encephalitis b virus SA14-14-2 strain.Concrete grammar is as follows: by above-mentioned diluent respectively with antigen sheet (i.e. absorption has the slide glass of BHK-21 cell) in 37 ℃, 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 fluorescent microscope will can be observed special green fluorescence and be judged to be the positive under field of microscope.

IgG antibody titers can be viewed as positive maximum dilution multiple.Experimental mice the results are shown in Figure 11B.Immunity 14d after, experimental mice serum in IgG antibody all have remarkable rising, antibody titer can reach 1:1280, the antibody titer of negative control group is less than 1:20.In the time of after immunity 28d, the IgG antibody titer of experimental mice can reach 1:2560.The above results shows, with 10 ⁴after 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 immune serum

Adopt PRNT to measure the NAT in serum.Concrete grammar is as follows: with 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 to (encephalitis b virus that contains about 100PFU in 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 supernatant, adopt BHK-21 cell to carry out Plaque assay), according to Reed-Muench method, calculate 50% NAT.Calculation formula is as follows:

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

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

Experimental mice the results are shown in Figure 11A.Immune function brings out mouse and produces to tire and be greater than the neutralizing antibody of 1:20, more than wherein the highest NAT can reach 1:100.In addition, high-caliber neutralizing antibody can maintain more than 4 weeks.Above-mentioned data show, 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

After immunity, 28d puts to death the disconnected neck of mouse, in 70% alcohol immersion in a moment, opens abdominal cavity, gets spleen.With syringe nook closing member, at 200 order Stainless Steel Cloths, push gently spleen, make unicellularly in woven wire flows into plate, (15ml to be housed containing 10%FBS RPMI-1640 substratum).Single cell suspension after 400 order woven wires filter, by 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.In the most backward precipitation, add 5ml to contain 10%FBS RPMI-1640 substratum, hanged cell, counting cells concentration, puts ice bath standby.

With 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, in 37 ℃, cultivates 4 days.

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

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

Result demonstration, 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 can produce stronger cellular immune level with this recombinant virus immune mouse.

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

Experimental group (10): by 20 of encephalitis b virus E218A subcutaneous abdomen inoculations BALB/c mouse in 4 week age, 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.

Result shows sees Figure 12, 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 in the situation of mouse with lethal dose abdominal cavity, and this recombinant virus can be mouse protection is completely provided, and further shows that this recombinant virus has good immune protective.

Claims

1. a protein, the protein that protein shown in the sequence of sequence table 3 is obtained after N-terminal the 218th amino acids residue sports nonpolar amino acid by polare Aminosaeren, described polare Aminosaeren is L-glutamic acid E, and described nonpolar amino acid is L-Ala A.

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

3. gene as claimed in claim 2, is characterized in that: the sequence 2 that described gene is sequence table is from the DNA molecular shown in 5 ' end 7677-10391 position Nucleotide.

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

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

6. the application of recombinant virus in preparing Vaccinum Encephalitis B described in claim 4 or 5.

7. a Vaccinum Encephalitis B, its activeconstituents is the recombinant virus described in claim 4 or 5.

8. a method that reduces the methyl transferase activity of encephalitis b virus, it is the encoding sequence that is nonpolar amino acid from the nucleotide coding sequence of N-terminal the 218th amino acids residue by the sequence encoding mutant of polare Aminosaeren by protein shown in the sequence of sequence table 3, described polare Aminosaeren is L-glutamic acid E, and described nonpolar amino acid is L-Ala A.