CN102559612B - Recombinant newcastle disease virus LaSota vaccine strain for expressing Nipah virus encephalitis G-protein - Google Patents

Abstract

The invention provides a recombinant newcastle disease virus LaSota vaccine strain for expressing Nipah virus encephalitis G-protein and a preparation method and application thereof. Particularly, the invention provides the recombinant newcastle disease virus LaSota vaccine strain rLa-NiVG for expressing the Nipah virus encephalitis G-protein, of which the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.5402, and an application thereof in the preparation of the vaccine used for preventing the Nipah virus encephalitis.

Description

Express the recombinant Newcastle disease virus LaSota vaccine strain of Buddhist nun's handkerchief encephalitis G albumen

Technical field

The invention provides recombinant Newcastle disease virus LaSota vaccine strain of a kind of Buddhist nun's of expression handkerchief encephalitis G albumen and its preparation method and application.Particularly, the invention provides the recombinant Newcastle disease virus LaSota vaccine strain rLa-NiVG (its preserving number is CGMCC No.5402) that expresses Buddhist nun's handkerchief encephalitis G albumen and for the preparation of the application in the vaccine of the sick encephalitis of prevention Buddhist nun handkerchief.

Background technology

The sick encephalitis of Buddhist nun's handkerchief is the strong infectious diseases common to human beings and animals of a kind of height, and its cause of disease Nipah virus belongs to Paramyxoviridae, and prosperous Nipah virus belongs to, and what belong to together only has Hendra virus [1].The Nipah virus host range is very extensive, and wherein pig is important susceptible animal.The natural reservoir (of bird flu viruses) of Nipah virus is flying fox [2].This disease is year mid-June in late September, 1998 to 1999 in Malaysian the first explosion, and the morbidity number of cases is up to 265 examples, and dead 105 examples, mortality ratio are 385%.In order to control spreading of disease, more than 100 ten thousand pigs have been massacred by Malaysian government, account for this state's amount of raising pigs 40%.After this, this disease occurs successively in states such as India, Bangladesh again, and this sick lethality rate also presents ascendant trend (67%-92%) [3].Nipah virus has caused serious harm for these national animal husbandry developments and people ' s health.

Although China should disease not broken out at present, it has consisted of grave danger to China.Flying fox extensively distributes in south east asia, distributes in south China area more, and domestic existing scholar has detected exist [4] of Nipah virus antibody in the middle of the bat in south China area.This disease is broken out in a plurality of countries due to China's periphery, and this disease of continuous intensification that is accompanied by trade contacts is imported the also constantly increase of pressure of China into.And China's quantity of raising pigs is very big, and raising pigs becomes one of the mainstay of China's agricultural industry already, therefore should disease in case outburst will certainly be to the pig industry of China and the people's orthobiosis and life security harm, the prevention and control situation allows of no optimist.Therefore, deposit effectively the vaccine of prevention and control Nipah virus have very important strategic importance.

Nipah virus G albumen is a kind of II type transmembrane glycoprotein that is positioned at the virus particle surface, thereby its major function is to mediate the viral Viral infection process that starts of being combined with cell receptor.G albumen is the main immune protective antigen of Nipah virus, has good immunogenicity, is to induce body to produce the major structural protein of neutralizing antibody.This research is take Avian pneumo-encephalitis virus LaSota low virulent strain as carrier, utilize the reverse genetic manipulation technology successfully to build the recombinant Newcastle disease virus live vector vaccine-rLa-NiVG that expresses Nipah virus G albumen, and in mouse and pig body, the immune effect of recombiant vaccine is estimated, experimental results show that this vaccine can produce the humoral immune reaction of inducing body good, it is the outstanding candidate vaccine of prevention and control Buddhist nun handkerchief encephalitis, show good application prospect in the potential hazard of China's reply Nipah virus, had important strategic importance.

Summary of the invention

The invention provides recombinant Newcastle disease virus LaSota vaccine strain of a kind of Buddhist nun's of expression handkerchief encephalitis G albumen and its preparation method and application.Particularly, the invention provides the recombinant Newcastle disease virus LaSota vaccine strain rLa-NiVG (its preserving number is CGMCC No.5402) that expresses Buddhist nun's handkerchief encephalitis G albumen and for the preparation of the application in the vaccine of the sick encephalitis of prevention Buddhist nun handkerchief.

Particularly, the present invention has successfully built expression Nipah virus (Nipah virus, NiV) the recombinant Newcastle disease virus LaSota vaccine strain rLa-NiVG of G albumen (this vaccine strain is preserved in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms on October 26th, 2011), and on mouse and weanling pig system evaluation the humoral immune reaction that produces of recombinant virus.

The present invention adopts Avian pneumo-encephalitis virus LaSota low virulent strain reverse genetic operating system to build the recombinant Newcastle disease virus rLa-NiVG that expresses Nipah virus G albumen.Confirm Nipah virus G protein expression with indirect immunofluorescence and specific cell fusion experiment; With the recombinant virus immune mouse, do neutralization test (VNA) to detect the humoral immunity level of recombinant virus with enzyme linked immunosorbent assay (ELISA) and the pseudo-C-type virus C particle of the chimeric Buddhist nun's handkerchief of vesicular stomatitis virus cyst membrane encephalitis G/F albumen.The result demonstration, rLa-NiVG can correction Nipah virus G albumen.With described recombinant virus immunity Balb/c mouse, ELISA shows that the immune group mouse can produce the specific IgG of higher level; Virus neutralization tests shows that immune mouse can produce the neutralizing antibody for Nipah virus; The pig immunity test shows, rLa-NiVG can induce pig to produce high-caliber Nipah virus neutralizing antibody.This achievement in research both at home and abroad all without report, has very strong novelty at present.RLa-NiVG is as a kind of forward-looking, and novelty, and the candidate vaccine of the prevention and control Nipah virus of highly effective and safe have very important strategic importance to China's reply Nipah virus potential threat.

The invention provides preparation and express the method for the recombinant Newcastle disease virus LaSota vaccine strain of Nipah virus G albumen, described method comprises the steps:

(1) build restructuring and transcribe plasmid, this transcribes the genome cDNA sequence that plasmid comprises the described ND LaSota vaccine strain of wherein inserting Nipah virus G gene, and plasmid called after pBRN-NiVG is transcribed in described restructuring;

(2) build the helper plasmid system of transcribing, this helper plasmid system comprises the helper plasmid pBSL of cDNA sequence of the large polymerase protein (L) of the helper plasmid pBSP of cDNA sequence of phosphorprotein (P) of helper plasmid pBSNP, the described ND LaSota vaccine strain of encoding of cDNA sequence of nucleoprotein (NP) of the described ND LaSota vaccine strain of encode and the described ND LaSota vaccine strain of encoding;

(3) the described restructuring of step (1) is transcribed the host cell of transcribing helper plasmid pBSNP, pBSP, the described ND LaSota vaccine strain of pBSL cotransfection copy permission of plasmid pBRN-NiVG and step (2), the host cell after the cultivation transfection; With

(4) results supernatant liquor, inoculated into chick embryo allantoic cavity rescue recombinant virus after filtering.

more specifically, the method that the recombinant Newcastle disease virus LaSota vaccine strain of Nipah virus G albumen is expressed in preparation is described below: take the Nipah virus G gene cDNA of synthetic as template, utilize PCR method respectively at G gene two ends insertion new castle disease virus specific gene initial sum terminator sequence and Pme I restriction enzyme site, order-checking is identified errorless, the G gene is inserted into LaSota full-length gene group plasmid, and (this plasmid is by this laboratory preparation, its preparation method can be referring to the patent of having obtained the authorization: application number: 200510097997.8, denomination of invention is " ND LaSota vaccine strain reverse genetic operating system and application thereof ", contriver: go on foot CHIGO, ageing is blue, application time: the Pme I site between P (phosphorprotein) on September 2nd, 2005) and M (stromatin) gene, be built into total length recombinant plasmid pBRN-NiVG, utilize plasmid extraction kit to prepare in a large number this recombinant plasmid, simultaneously with helper plasmid pBSNP, pBSP, the common transfection BHK-21 cell of pBSL (structure of described helper plasmid is referring to reference [5]) (infecting in advance the recombinant poxvirus of expressing the T7 polysaccharase), cell conditioned medium with transfection after 72 hours is inoculated 10 age in days SPF chick embryo allantoic cavities, inoculate that the allantoic fluid of getting inoculated into chick embryo after 72 hours is hemagglutination test HA and newcastle disease hemagglutination-inhibition test HI all is positive, tentatively show the virus rescue success.The recombinant virus rLa-NiVG that obtains with rescue infects the BHK-21 cell, and the eukaryon expression plasmid PCAGG-NiVF of while transfection expression Nipah virus F albumen, and the specific cell that can observe the mediation of Nipah virus G and F albumen co expression after 24 hours merges; Separately make primary antibodie with the expression of indirect immunofluorescene assay Buddhist nun handkerchief G albumen on the BHK-21 cell that rLa-NiVG infects with mouse-anti NiVG protein monoclonal antibody, can observe specific strong fluorescent signal under fluorescent microscope.The cytogamy test proves all that with indirect immunofluorescence recombinant virus rLa-NiVG can correction Nipah virus G albumen.The present invention ND LaSota vaccine strain AV1615 used (Chinese veterinary microorganism culture presevation administrative center, CVCC) also by this laboratory preparation, its preparation method can be referring to the patent of having obtained the authorization: application number: 200510097997.8, denomination of invention is " ND LaSota vaccine strain reverse genetic operating system and application thereof ", contriver: go on foot CHIGO, ageing is blue, the application time: on September 2nd, 2005.

The present invention also provides the application of the recombinant Newcastle disease virus LaSota vaccine strain of described expression Nipah virus G albumen for the preparation of the vaccine of the sick encephalitis of prevention Buddhist nun handkerchief, preferably, the recombinant Newcastle disease virus LaSota vaccine strain of described expression Nipah virus G albumen is rLa-NiVG.Described application comprises that also the recombinant Newcastle disease virus LaSota vaccine strain of described expression Nipah virus G albumen and the recombinant Newcastle disease virus LaSota vaccine strain of expressing Nipah virus F albumen carry out combined immunization.

The present invention also is provided for preventing the vaccine of the sick encephalitis of Buddhist nun's handkerchief, and described vaccine comprises the recombinant Newcastle disease virus LaSota vaccine strain of the expression Buddhist nun handkerchief encephalitis G albumen of the present invention of significant quantity.Preferably, described vaccine comprises the recombinant Newcastle disease virus LaSota vaccine strain rLa-NiVG of significant quantity.In addition, described vaccine can also comprise medicinal adjuvant or vehicle etc.

Vaccine for the sick encephalitis of prevention Buddhist nun handkerchief of the present invention can be used for the sick encephalitis of Buddhist nun's handkerchief of prevention Mammals, and described Mammals includes, but not limited to pig, dog, cat, mouse and horse etc.

The present invention also is provided for preventing the method for the sick encephalitis of Buddhist nun's handkerchief in Mammals, described method comprises to the vaccine of the recombinant Newcastle disease virus LaSota vaccine strain of comprising of described administration significant quantity of expression of the present invention Buddhist nun handkerchief encephalitis G albumen, is preferably rLa-NiVG.

The accompanying drawing summary

With reference to following accompanying drawing, further describe the present invention, in described accompanying drawing:

The structure schematic diagram of Fig. 1 rLa-NiVG;

The indirect immunofluorescence of Fig. 2 rLa-NiVG cells infected;

The synplasm that Fig. 3 Nipah virus G, F albumen coexpression form;

The chicken embryonic development curve of Fig. 4 recombinant virus, the longitudinal axis represent Log10 chicken embryo median infective dose, and transverse axis represents inoculation time;

Body weight change after mouse after the immunity of Fig. 5 maximal dose recombinant virus;

Specific IgG level (ng/ml) in Fig. 6 A rLa-NiVG immune serum; Fig. 6 B rLa-NiVG immune mouse neutralizing antibody level, ordinate zou represent the multiple of serum dilution;

Fig. 7 recombinant virus induces pig to produce anti-Nipah virus neutralizing antibody.

Embodiment

Come by the following examples further to illustrate the present invention.But should be appreciated that, described embodiment is illustrational purpose, and is not intended to limit the scope of the invention and spirit.

Need to prove, it should be appreciated by those skilled in the art that reagent used in following embodiment, enzyme etc. except specifying, be other reagent of analytical pure level or the enzyme that can be purchased from reagent company.

1 materials and methods

1.1: material

1.1.1: strain and plasmid: Avian pneumo-encephalitis virus LaSota attenuated vaccine strain (GenBank accession number AY845400.2, by this laboratory preparation, its preparation method can be referring to the patent of having obtained the authorization: application number: 200510097997.8, denomination of invention is " ND LaSota vaccine strain reverse genetic operating system and application thereof ", contriver: go on foot CHIGO, ageing is blue, application time: on September 2nd, 2005), express the recombinant baculovirus rBac-NiVG[6 of Nipah virus G albumen] preserve (its detailed construction process is referring to reference [6]) by this laboratory, the recombinant poxvirus vTF7-3 of expression T7 polysaccharase is provided by doctor Bernard.Moss of America NI H, its preparation is all carried out [7] by document with titration process, virus liquid after titration-70 ℃ save backup.LaSota full-length gene group is transcribed plasmid pBRN-FL and helper plasmid pBSNP, pBSP, pBSL preserves [5] (reference 5 describes the preparation method of above-mentioned plasmid in detail) by this laboratory, the detailed description in document [5] of the detailed process of the reverse genetic manipulation of LaSota.The reverse genetic operating system of LaSota vaccine strain can be referring to the patent of having obtained the authorization in addition: application number: 200510097997.8, denomination of invention is " ND LaSota vaccine strain reverse genetic operating system and application thereof ", contriver: go on foot CHIGO, ageing is blue, September 2 2005 application time.Express the eukaryon expression plasmid pCAGG-NiVG of Nipah virus G and F albumen, pCAGG-NiVF[8] preserve (reference 8 describes the preparation method of above-mentioned plasmid in detail) by this laboratory.Provide [9] (reference 9 describes the preparation method of above-mentioned plasmid in detail) for the preparation of the restructuring VSV Genomic cDNA clone plasmid pVSV Δ G*GFP of the restructuring vesicular stomatitis pseudovirion (VSV Δ G*GFP-NiVG/F) of the chimeric Nipah virus G of cyst membrane and F protein expression green fluorescent protein and auxiliary expression plasmid pBS-G, pBS-L, pBS-N and pBS-P by Michael doctor Whitt of U.S. University of Tennessee.

1.1.2: cell and serum: clone BHK-21, Sf9 insect cell (available from American type culture collection ATCC) is also preserved in this laboratory and uses.BHK-21 Growth of Cells and maintenance medium are the DMEM (available from Gibco) that contains 5% foetal calf serum; Sf9 insect cell nutrient solution is Sf-900II (available from Gibco) serum-free medium.

1.1.3 monoclonal antibody: Nipah virus G protein-specific monoclonal antibody G3E9 is prepared and is preserved by this laboratory, and the preparation method sees document [10] in detail;

1.1.4: laboratory animal: 6 age in week female BALB/c mouse available from Beijing dimension tonneau China laboratory animal company.Surrounding weanling pig in age breeds and raises available from veterinary institute laboratory animal base, Harbin and by veterinary institute laboratory animal base, Harbin.

1.2: the structure of expressing the full-length cDNA clone of NiV G gene

Take PCAGG-NiVG as template, (its gene order is SEQ ID No.1 at Nipah virus G gene by PCR primer (seeing Table 1), coded protein sequence is SEQ ID No.2) open reading frame 5 ' end introduces transcription termination sequence GE (TTAAGAAAAAA) and the transcriptional initiation sequence GS (ACGGGTAGAA) of Pme I restriction enzyme site recognition sequence (GTTTAAAC) and NDV (Avian pneumo-encephalitis virus) self-polymerization enzyme L identification, at 3 ' end introducing Pme I restriction enzyme site; The PCR product is after sequence verification, and end inserts pBRN-FL-PmeI (that is, the pBRN-FL plasmid that process PmeI enzyme is cut), the recombinant full-lenght Genomic cDNA clone called after pBRN-FL-NiVG that is built into after Pme I enzyme is cut.

Table 1 construction expression NiV G full length gene genomic clone plasmid primer used

Annotate: runic represents Pme I restriction enzyme site sequence; Lowercase represents new castle disease virus specific gene initial sum terminator sequence; Italic represents the Kozak sequence; Underscore represents G gene specific complementary sequence.

1.3: the rescue of recombinant virus

With BHK-21 cell inoculation six orifice plates (available from Corning), reach 80% when converging until Growth of Cells, infect vTF7-3 (MOI=0.01) in front 1 hour in transfection and (express the recombinant poxvirus vTF7-3 of T7 polysaccharase, Bernard doctor Moss by America NI H provides [7]), infect complete rear employing calcium phosphate transfection method with the full-length gene group plasmid pBRN-FL-NIVG that builds and helper plasmid pBS-N, pBS-P and pBS-L respectively with the ratio cotransfection BHK-21 cell of 5 μ g, 2.5 μ g, 1.25 μ g, 1.25 μ g.after transfection 8-12 hour, discard the transfection supernatant, with the PBS buffering liquid gram cell that contains 10%DMSO 2.5 minutes, add afterwards complete DMEM nutrient solution (available from Gibco), change Opi-MEM substratum (available from Gibco) after 12 hours, and pancreatin (the 1 μ g/mL that adds TPCK to process, (available from Sigma)) continue to hatch and gather in the crops the culture supernatant after 72 hours, inoculation 9-11 age in days SPF chick embryo allantoic cavity (available from veterinary institute state poultry seed resource center, Harbin) after 0.22 μ M aperture filter (available from Millipore) filters, inoculate and get chick embryo allantoic liquid 50 μ L after 72 hours and carry out Avian pneumo-encephalitis virus blood clotting (HA) and blood clotting and suppress (HI) test.The chick embryo allantoic liquid that results HA and HI test-results are positive, after packing ,-70 ℃ of preservations are frozen standby.The recombinant virus called after rLa-NiVG that saves out.

1.4: the evaluation of recombinant virus

1.4.1: indirect immunofluorescence (IFA)

rLa-NiVG and LaSota vaccine strain are respectively take MOI as 0.01 infection BHK-21 cell, infect and discard nutrient solution after 24 hours, with twice of Xian PBS, use afterwards fixedly 30min of 3% paraformaldehyde (available from Sigma) room temperature, wash 3 times with PBST (PBS that contains 0.05%Tween-20), the anti-NiV G of mouse monoclonal antibody G3E9 (this laboratory preparation that the cell of rLa-NiVG infection is added respectively 1: 50 times of dilution, as previously mentioned, the preparation method is referring to document [10] in detail), the anti-NDV positive serum of chicken [5] (source is referring to reference [5]), with nonimmune BalB/c mice serum (take mouse vein blood and separate adopting the ball rear vein beard to puncture blood-collecting method after mouse anesthesia), the LaSota vaccine strain infects the anti-NDV serum of chicken [5], mouse-anti NiV G monoclonal antibody G3E9 (see above and state) and the nonimmune mice serum (see above and state) that the hole adds respectively 1: 50 times of dilution, room temperature effect 2 hours, PBST rinse three times, add respectively again mountain sheep anti-mouse igg (or anti-chicken IgY) antibody (available from Sigma) the room temperature lucifuge of the FITC mark of 1: 200 times of dilution to hatch 1h, wash 5 times with PBST, be placed in observations under fluorescent microscope (Leica DMIRES2).

1.4.2: cytogamy is active to be detected

The eukaryon expression plasmid PCAGG-NiVF that expresses Nipah virus F albumen is grown to the individual layer BHK-21 cell of 80%-90% with Lipofectamine 2000 lipofectamine (available from Invitrogen) transfection.Discard nutrient solution after 4 hours, be about 0.1 cells infected with recombinant virus rLa-NiVG with MOI, and be LaSota and infect contrast, infects and add the DMEM complete culture solution after one hour, be positioned over the 5%CO2 environment, cultivate observation of cell fusion situation after 48 hours for 37 ℃.

1.5 the chicken embryonic development kinetics of recombinant virus

RLa-NIVG is inoculated SPF chicken embryo (available from veterinary institute state poultry seed resource center, Harbin), respectively 24,48, took a sample in 72,96 hours, and measure the chicken embryo median infective dose EID50 of each time point sample.

1.6rLa-NiVG the safety experiment to mouse

With rLa-NiVG maximal dose (10 ⁶EIDS0/0.1ml) 10 6 all female Balb/c mouse in age (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) of collunarium 30ul, intramuscular injection 100ul immunity simultaneously, set up LaSota vaccine strain immune group (dosage is identical with rLa-NiVG with approach) and PBS control group simultaneously.The rear set time every day weighing Mouse Weight of immunity also records incidence, continues for two weeks.

1.7: the humoral immunization evaluation of the malicious immune Balb/c mouse of recombinating.

Recombinant Newcastle disease virus rLa-NiVG is by 10 ⁶EID50/ intramuscular injection immunity 10 female Balb/c mouse (available from Beijing Vital River Experimental Animals Technology Co., Ltd.), and set up PBS immunity control group.Initial immunity booster immunization after 4 weeks, immunizing dose is identical with initial immunity with approach.Two weeks after first and booster immunization, get respectively 5 mouse for every group and take euthanasia, gather blood separation serum and measure humoral immunity level with ELISA and neutralization test.

1.7.1 the humoral immunity level of immune mouse is estimated in enzyme linked immunosorbent assay (ELISA) and virus neutralization tests (VNA).

The ELISA experimentation is summarized as follows: with the Sf9 cell lysate of the recombinate shape virus infection of expressing Nipah virus G albumen as envelope antigen, in order to detect after the first and booster immunization of rLa-NiVG the concentration of IgG in mice serum, concrete operation step is referring to document [11].Use simultaneously the mouse IgG (Southern Biotech) of concentration known to do the coated elisa plate production standard curve of gradient dilution.Linear relationship calculation formula between the OD value of obtaining according to typical curve and coated standard antigen concentration is calculated the content of antigen-specific IgG in serum according to the gained formula, represent with the nanogram number that contains in every milliliter of blood.

1.7.2 neutralization test (VNA)

Utilize the replication defect type vesicular stomatitis virus pseudovirion VSV Δ G*GFP-NiVG/F of the chimeric Nipah virus G of cyst membrane and F albumen and expressing green fluorescent protein to carry out neutralization test (VNA) [9,12].The BHK-21 Growth of Cells is standby in 96 well culture plate to 80% density.Serum to be checked is done the twice serial dilution, each extent of dilution 25 μ l, and the pseudo-C-type virus C VSV Δ G*GFP-NiVG/F suspension that approximately contains 200IU with equal-volume mixes, and every part of serum is done three repetitions.1 hour postoperative infection BHK-21 cell is made in 37 ℃ of senses, is placed in 5%CO2, cultivates after 24 hours that (Leica DMIRES2) observes and count the GFP positive cell under fluorescent microscope for 37 ℃.

1.8rLa-NiVG the NAT of immune swine

Get 2ml rLa-NiVG SPF chick embryo allantoic liquid through intramuscular injection immunity weanling pig in 4 age in week, surrounding booster immunization after initial immunity, immunization route is identical with initial immunity with dosage.The separation of serum of taking a blood sample weekly after immunity, and measure serum neutralization tire (experimental procedure is the same).

2 results

2.1 expressing the recombinant Newcastle disease virus of NiV G albumen builds and virus rescue

On ND LaSota vaccine strain reverse genetic operating system basis [5], insert in the genomic Pme I of LaSota site 5 ' end introduced the gene of NDV polysaccharase specific recognition initial/the Nipah virus G gene of terminator sequence (GS/GE) is as a transcriptional units independently, is built into the recombinant full-lenght cDNA clone rLa-NiVG that expresses NiV G albumen.

With pBRN-FL-NiVG and express the common transfection BHK-21 of the helper plasmid cell of NP, P, L albumen, results transfectional cell supernatant and be inoculated in 9-11 age in days SPF chicken embryo after 72 hours.The chick embryo allantoic liquid that after 72 hours, results HA and HI test is positive.RT-PCR and sequencing result demonstration, the G gene correctly is inserted into rLaSota genome Pme I site.The recombinant virus called after rLa-NiVG that rescue obtains, be preserved in (BeiJing, China, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on October 26th, 2011, Institute of Microorganism, Academia Sinica, 100101), preserving number is CGMCC No.5402.

2.2 recombinant virus rLa-NiVG can correction Nipah virus G albumen

For confirming that in recombinant virus, G albumen can access correction, infect the BHK-21 cell with rLa-NiVG, LaSota vaccine strain respectively, carry out indirect immunofluorescene assay take mouse-anti NiV G protein monoclonal antibody G3E9, the anti-NDV serum of chicken as primary antibodie.Result is as shown in Figure 2: rLa-NiVG infects the BHK-21 cell can detect specificity green fluorescence signal with G3E9, and LaSota infected group and do not infect control group and all be negative shows that recombinant virus can correction NiV G albumen.

2.3rLa-NiVG cause cytogamy with the PCAGG-NiVF coexpression

Nipah virus attachment proteins G and fusion protein F co expression can cause fusion phenomenon to occur in cell surface, and either party all can not produce cytogamy single expression.And the cytogamy that Nipah virus G and F albumen cause has specificity, and Nipah virus G albumen and Avian pneumo-encephalitis virus F albumen or Nipah virus F albumen and newcastle disease virus HN albumen coexpression all can not cause cytogamy [13].Therefore we are with Nipah virus F albumen eukaryon expression plasmid PCAGG-NiVF ([8] are verified in expression) transfection BHK-21 cell, and then infect with recombinant virus rLa-NiVG, after 48 hours, microscopically namely can be observed obvious cytogamy (Fig. 3 as shown by arrows), shows the G albumen that rLa-NiVG can correction has biologic activity.

2.4 insert the chicken embryonic development characteristic that Nipah virus G gene does not change recombinant virus rLa-NiVG

Insert in order to verify the chicken growth of the embryo characteristic that Nipah virus G gene does not change recombinant virus rLa-NiVG, we inoculate SPF chicken embryo with rLa-NiVG, and at the EID50 of different time points sampling and each time point sample of titration.Referring to Fig. 4, result shows that the chicken embryonic development curve of rLa-NiVG is consistent with LaSota, shows that recombinant virus has kept the high titre chicken embryonic development characteristic similar to parent's strain.

2.5 recombinant virus rLa-NiVG has good security to mouse.

RLa-NiVG with maximal dose simultaneously through collunarium and intramuscular injection path immune mouse, compare with control group, in observation period, three groups of mouse all survive, the mental status is good, the Mice Body weight average increases to some extent, but no significant difference (referring to Fig. 5) shows that recombinant virus is the same with its parent's strain mouse is had good security.

2.6rLa-NiVG can produce good humoral immune reaction by inducing mouse

Referring to Fig. 6, recombinant virus rLa-NiVG can produce good ELISA antibody and neutralizing antibody by inducing mouse.Initial immunity is after two weeks, compare with control group and obvious Nipah virus specific IgG antibodies and neutralizing antibody (P＜0.05) can be detected, after booster immunization, two kinds of antibody horizontals significantly rise (P＜0.05) than initial immunity, and result shows that Nipah virus G Protein reconstitution Avian pneumo-encephalitis virus LaSota vaccine strain can induce good humoral immune reaction in mouse.

2.7rLa-NiVG can induce pig to produce high-level anti-Nipah virus neutralizing antibody.

Pig to Nipah virus height susceptible, is that Nipah virus is propagated most important intermediate host, is also the contagium most threatening to the mankind, thereby is the important step of Nipah virus prevention and control.Can the Nipah virus vaccine induce neutralizing antibody in the pig body be the major criterion of judgement vaccine success or not.We use 2ml recombinant virus SPF chick embryo allantoic liquid through intramuscular injection immunity weanling pig in 4 age in week.4 weeks of initial immunity are rear with identical immunizing dose and approach booster immunization.Take a blood sample and separation of serum for the first time and after booster immunization, utilize the replication defect type vesicular stomatitis virus pseudovirion VSV Δ G*GFP-NiVG/F simulation Nipah virus particle of the chimeric Nipah virus G of cyst membrane and F albumen and expressing green fluorescent protein to carry out neutralization test.Can find out from the result of Fig. 8, initial immunity can produce neutralizing antibody after two weeks, and the 3rd, the surrounding antibody horizontal rises to some extent, but without significant difference.After booster immunization, the serum neutralizing antibody significantly rises (P＜0.01) after than initial immunity, and the antibody extended period is grown (lasting till at least for 21 weeks).The neutralization test result shows that rLa-NiVG can induce generation high level and lasting neutralizing antibody in the pig body.

3 discuss

The present invention utilizes Avian pneumo-encephalitis virus LaSota vaccine strain reverse genetic operating system, with Nipah virus G gene as one independently transcriptional units insert the LaSota genome, success builds and saves the recombinant Newcastle disease virus rLa-NiVG that obtains expressing Nipah virus G albumen, experiment results proved rLa-NiVG can correction Nipah virus G albumen, and recombinant virus has kept the high chicken embryonic development titre consistent with LaSota and to mammiferous tight security.Avian pneumo-encephalitis virus LaSota vaccine strain has following advantage as carrier: Avian pneumo-encephalitis virus heredity is relatively stable, and a serotype is only arranged, and restructuring and virulence occur between strain, and to return strong possibility minimum.NDV is a passing infection on Mammals, and security is very high; Reproduction process is completed in cytoplasm, from RNA to RNA, and the possibility that does not have the DNA stage and integrate with cellular genome; The NDV attenuated vaccine is a kind of natural adjuvant to Mammals, and good innate immunity and specificity humoral and cellular immunization produce more comprehensively, the immunoprotection of balance thereby can induce; The weak poison of NDV has the chicken embryonic development characteristic of high titre, and production cost is very cheap.

The Nipah virus encephalitis not yet occured in China at present, and the operation Nipah virus need to carry out under Biosafety IV level condition, thereby this research can not utilize Buddhist nun's handkerchief live virus itself to carry out neutralization test.Therefore we have prepared the chimeric Nipah virus G of cyst membrane and F albumen, and replication defect type (VSV genomic deletion itself G gene) the pseudo-C-type virus C VSV of the vesicular stomatitis virus Δ G*-NiVG/F simulation NiV live virus that possesses a passing infection ability and expressing green fluorescent protein carries out neutralization test [8].The Nipah virus that is neutralized-vesicular stomatitis pseudovirion can not be invaded cell, thereby can not produce green fluorescence; Otherwise the pseudovirion that is not neutralized enters cell via Nipah virus G and protein mediated absorption and the fusion of F, thereby make and invaded cell and produce green fluorescence, so the serum neutralization is tired to be expressed as and can be made fluorocyte reduce the highest serum extension rate of half.This method has safety, stable, quick, the characteristics that susceptibility is high, and practical application effect is good.

The humans such as Guillaume are expressed the recombinant poxvirus immunity hamster of Nipah virus G or F albumen and are prepared hyper-immune serum, can make its attack to the lethal dose Nipah virus produce protection [14] with hyper-immune serum passive immunization hamster.The expression Nipah virus G that the people such as Weingartl build, restructuring canary pox virus ALVAC-G, the ALVAC-F of F albumen separately and the combined immunization pig all can produce protection [15] to the attack of lethal dose Nipah virus.Neutralizing antibody is resisted and is removed in the Nipah virus process at body and plays a crucial role.Can induce pig to produce rapidly neutralizing antibody (the average extension rate of serum 1: 200) after two weeks of recombinant Newcastle disease virus rLa-NiVG initial immunity of the expression Nipah virus G albumen of this research and establishment, after booster immunization, the neutralizing antibody level is than initial immunity (the average extension rate of serum 1: 4000 that significantly rises; P＜0.01).Kaku[16] etc. the people research and comparison do neutralization test with Nipah virus G and the chimeric vesicular stomatitis pseudovirion of F albumen cyst membrane and Buddhist nun's handkerchief live virus the serum dilution tire, finds that serum that two kinds of methods draw neutralizes to tire to have positive correlation.Therefore, we rLa-NiVG that has every reason to believe induces the high-level neutralizing antibody of generation to provide protection in the attack of lethal dose Nipah virus for pig.

Although Buddhist nun's handkerchief encephalitis does not occur in China at present, but the Nipah virus encephalitis does not stop all the time at the popular of China surrounding countries (especially country in Southeast Asia) and outburst, along with the foundation of ASEAN Free Trade Area and the further raising of China market degree of opening, the danger that Nipah virus imports China into also increases day by day, and the prevention and control situation allows of no optimist.Pig is to Nipah virus height susceptible, in case infect, although mortality ratio is not high, atypical symptoms, virus levels of replication in vivo are very high, and can pass through the natural hole excretion such as the upper respiratory tract, oral cavity, therefore becomes the contagium to human beings tool menace.Broke out in 1999 in Buddhist nun's handkerchief encephalitis epidemic situation of Malaysia, Singapore and directly cause 283 people to infect, 109 people are dead, and its source of infection be pig substantially, but not the direct result that contacts with flying fox.Therefore, pig is the important step of Buddhist nun's handkerchief encephalitis prevention and control.In case Buddhist nun's handkerchief encephalitis epidemic situation occurs, the epidemic place susceptible animal is monitored, when slaughtering, set up the particularly immunity band of pig of peripheral susceptible animal, reduce susceptibility, significant.

The recombinant Newcastle disease virus LaSota vaccine strain of the expression Nipah virus G albumen of this laboratory research and development report that at home and abroad still belongs to the first time at present has very strong novelty.RLa-NiVG not only can produce good humoral immune reaction in the Mammals model, the more important thing is that it can make the terrain pig produce high-caliber Nipah virus neutralizing antibody.RLa-NiVG has a deposit as a kind of, and prospective outstanding Nipah virus candidate vaccine has very important strategic importance to the potential threat of China's reply Nipah virus.

Except being used as prevention Nipah virus encephalitis, this vaccine strain also can be used for oncotherapy.The vaccine carrier of doing oncotherapy with Avian pneumo-encephalitis virus has been seen in research report [17], and what have enters phase ii clinical trial [18] even.The Nipah virus G albumen that rLa-NiVG provided by the invention is expressed, can (express the recombinant Newcastle disease virus LaSota vaccine strain of F albumen as common infection) under the condition of Nipah virus F albumen co expression, form the specific synplasm of Nipah virus, thereby greatly strengthen the ability of recombinant Newcastle disease virus dissolving and destruction tumor tissues, and the newcastle epidemic disease antibody that exists before avoiding is to Support effect.Because rLa-NiVG or kept the mammalian safety of the height identical with its vector virus, so remain high safety for the normal health tissue.

Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and describes, but will be understood by those skilled in the art that, under the condition that does not deviate from by the defined the spirit and scope of the present invention of accompanying claim, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.

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Claims (3)

1. recombinant Newcastle disease virus LaSota vaccine strain of expressing Buddhist nun's handkerchief encephalitis G albumen, its preserving number is CGMCC No.5402.

2. vaccine that is used for the sick encephalitis of prevention Buddhist nun handkerchief, it comprises the recombinant Newcastle disease virus LaSota vaccine strain of the expression Buddhist nun handkerchief encephalitis G albumen claimed in claim 1 of significant quantity.

3. the recombinant Newcastle disease virus LaSota vaccine strain of expression claimed in claim 1 Buddhist nun handkerchief encephalitis G albumen is for the preparation of the application in the vaccine of the sick encephalitis of prevention Buddhist nun handkerchief.

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