CN102559612A - 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 proteic recombinant Newcastle disease virus LaSota of Buddhist nun handkerchief encephalitis G vaccine strain

Technical field

The present invention provides the proteic recombinant Newcastle disease virus LaSota of a kind of expression Buddhist nun handkerchief encephalitis G vaccine strain.Particularly, the present invention provides the application of expressing Buddhist nun handkerchief encephalitis G proteic recombinant Newcastle disease virus LaSota vaccine strain rLa-NiVG (its preserving number is CGMCC No.5402) and being used for preventing the vaccine of the sick encephalitis of Buddhist nun's handkerchief in preparation.

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 generic has only Heng Dela 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 breaks out in Malaysia a year mid-June in late September, 1998 to 1999 first, sends out case load up to 265 examples, and dead 105 examples, mortality ratio are 385%.For spreading of control 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 takes place in states such as India, Bangladesh again successively, 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.

Though China should disease not broken out at present, it has constituted grave danger to China.Flying fox extensively distributes in south east asia, and it is more to distribute at China southern area, and domestic existing scholar has detected exist [4] of Nipah virus antibody in the middle of the bat of China's southern area.Because a plurality of national outbursts of China periphery should disease, the pressure that this disease of continuous intensification that is accompanied by trade contacts is imported China into also constantly increases.And China's quantity of raising pigs is very big, and raising pigs becomes one of mainstay property industry of China's agricultural already, therefore should disease in case outburst will certainly cause serious harm to pig industry of China and the people's orthobiosis and life security, 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 mediation virus combines to start virus with cell receptor a course of infection.G albumen is the main immune protective antigen of Nipah virus, has good immunogenicity, is to induce body to produce the primary structure albumen of neutralizing antibody.This research is carrier with NDV LaSota low virulent strain; Utilize the reverse genetic manipulation technology successfully to make up the proteic recombinant Newcastle disease virus live vector vaccine-rLa-NiVG of expression Nipah virus G; And in mouse and pig body, the immune effect of recombiant vaccine is estimated; This vaccine of experiment proof can produce induces the immunoreation of body good humoral; Be the outstanding candidate vaccine of prevention and control Buddhist nun handkerchief encephalitis, in the potential hazard of China's reply Nipah virus, shown good prospects for application, have the important strategic meaning.

Summary of the invention

The present invention provides the proteic recombinant Newcastle disease virus LaSota of a kind of expression Buddhist nun handkerchief encephalitis G vaccine strain.Particularly, the present invention provides the application of expressing Buddhist nun handkerchief encephalitis G proteic recombinant Newcastle disease virus LaSota vaccine strain rLa-NiVG (its preserving number is CGMCC No.5402) and being used for preventing the vaccine of the sick encephalitis of Buddhist nun's handkerchief in preparation.

Particularly; The present invention has successfully made up expression Nipah virus (Nipah virus; NiV) the proteic recombinant Newcastle disease virus LaSota of G vaccine strain rLa-NiVG (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 NDV LaSota low virulent strain reverse genetic operating system to make up the proteic recombinant Newcastle disease virus rLa-NiVG of expression Nipah virus G.Confirm the proteic expression of Nipah virus G 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 EUSA (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 shows that rLa-NiVG can correctly express Nipah virus G albumen.With said 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 to Nipah virus; The pig immunity test shows that rLa-NiVG can induce pig to produce high-caliber Nipah virus neutralizing antibody.This achievement in research does not all have report at present both at home and abroad, has very strong novelty.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 crucial strategic importance to China's reply Nipah virus potential threat.

The present invention provides and prepares the method for expressing the proteic recombinant Newcastle disease virus LaSota of Nipah virus G vaccine strain, and said method comprises the steps:

(1) make up reorganization and transcribe plasmid, this transcribes the genome cDNA sequence that plasmid comprises the said newcastle disease LaSota vaccine strain that wherein inserts Nipah virus G gene, and plasmid called after pBRN-NiVG is transcribed in said reorganization;

(2) make up the helper plasmid system that transcribes, this helper plasmid system comprises the helper plasmid pBSL of cDNA sequence of big polymerase protein (L) of helper plasmid pBSP and the said newcastle disease LaSota vaccine strain of encoding of cDNA sequence of phosphorprotein (P) of helper plasmid pBSNP, the said newcastle disease LaSota vaccine strain of encoding of cDNA sequence of the nucleoprotein (NP) of the said newcastle disease LaSota vaccine strain of encode;

(3) the said reorganization of step (1) is transcribed the host cell of transcribing helper plasmid pBSNP, pBSP, the said newcastle disease LaSota of pBSL cotransfection vaccine strain copy permission of plasmid pBRN-NiVG and step (2), cultivate the host cell after the transfection; With

(4) results supernatant filters back inoculated into chick embryo allantoic cavity rescue recombinant virus.

More specifically; Preparing the method for expressing the proteic recombinant Newcastle disease virus LaSota of Nipah virus G vaccine strain is described below: the Nipah virus G gene cDNA with synthetic is a template; Utilize PCR method to insert new castle disease virus specific gene initial sum terminator sequence and Pme I restriction enzyme site respectively at G gene two ends; Order-checking is identified errorless, and the G gene is inserted into LaSota full-length gene group plasmid, and (this plasmid is by this prepared in laboratory, and its preparation method can be referring to the patent of having obtained the authorization: application number: 200510097997.8; Denomination of invention is " newcastle disease LaSota vaccine strain reverse genetic operating system and application thereof "; The contriver: go on foot CHIGO, ageing is blue, the 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 this recombinant plasmid in a large number, with helper plasmid pBSNP, pBSP, the common transfection BHK-21 cell of pBSL (structure of said helper plasmid is referring to reference [5]) (infecting the recombinant poxvirus of expressing the T7 polysaccharase in advance), inoculate 10 age in days SPF chick embryo allantoic cavities with the cells transfected supernatant after 72 hours simultaneously; 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 proteic eukaryon expression plasmid PCAGG-NiVF of while transfection expression Nipah virus F, and the specific cell that can observe the mediation of Nipah virus G and F albumen co expression after 24 hours merges; Do one with mouse-anti NiVG protein monoclonal antibody in addition and resist, under fluorescent microscope, can observe specific intensive fluorescent signal with the expression of indirect immunofluorescence detection Buddhist nun's handkerchief G albumen on rLa-NiVG infected B HK-21 cell.The cytogamy test proves all that with indirect immunofluorescence recombinant virus rLa-NiVG can correctly express Nipah virus G albumen.The used newcastle disease LaSota vaccine strain AV1615 of the present invention (Chinese veterinary microorganism culture presevation administrative center; CVCC) also by this prepared in laboratory; Its preparation method can be referring to the patent of having obtained the authorization: application number: 200510097997.8, denomination of invention is " newcastle disease LaSota vaccine strain reverse genetic operating system and application thereof ", the contriver: the step CHIGO; Ageing is blue, the application time: on September 2nd, 2005.

The present invention also provides the proteic recombinant Newcastle disease virus LaSota of said expression Nipah virus G vaccine strain to be used to prepare the application of the vaccine that prevents the sick encephalitis of Buddhist nun's handkerchief; Preferably, the proteic recombinant Newcastle disease virus LaSota of said expression Nipah virus G vaccine strain is rLa-NiVG.Said application comprises that also the proteic recombinant Newcastle disease virus LaSota of said expression Nipah virus G vaccine strain and the proteic recombinant Newcastle disease virus LaSota of expression Nipah virus F vaccine strain carry out combined immunization.

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

The sick encephalitis of Buddhist nun's handkerchief that the vaccine that is used for preventing the sick encephalitis of Buddhist nun's handkerchief of the present invention can be used to prevent Mammals, said 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 the Mammals; Said method comprises to the vaccine of the proteic recombinant Newcastle disease virus LaSota of comprising of said administration significant quantity of expression of the present invention Buddhist nun handkerchief encephalitis G vaccine strain, is preferably rLa-NiVG.

The accompanying drawing summary

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

The structure synoptic 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 are represented Log10 chicken embryo median infective dose, and transverse axis is represented inoculation time;

Changes of weight behind the 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 are represented the multiple of serum dilution;

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

Embodiment

Below come further to illustrate the present invention through embodiment.But should be appreciated that said embodiment is illustrational purpose, and be not intended to limit scope of the present invention and spirit.

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

1 material and method

1.1: material

1.1.1: strain and plasmid: NDV LaSota attenuated vaccine strain (GenBank accession number AY845400.2; By this prepared in laboratory; Its preparation method can be referring to the patent of having obtained the authorization: application number: 200510097997.8, denomination of invention is " newcastle disease LaSota vaccine strain reverse genetic operating system and application thereof ", the contriver: the step CHIGO; Ageing is blue; Application time: on September 2nd, 2005), express the Nipah virus proteic recombinant baculovirus rBac-NiVG of G [6] and 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, and ℃ preservation of the viral liquid-70 after the titration is subsequent use.LaSota full-length gene group is transcribed plasmid pBRN-FL and helper plasmid pBSNP, pBSP, pBSL preserves [5] (reference 5 has specified the preparation method of above-mentioned plasmid) 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 " newcastle disease LaSota vaccine strain reverse genetic operating system and application thereof "; Contriver: go on foot CHIGO; Ageing is blue, September 2 2005 application time.Express Nipah virus G and the proteic eukaryon expression plasmid pCAGG-NiVG of F, pCAGG-NiVF [8] preserves (reference 8 has specified the preparation method of above-mentioned plasmid) by this laboratory.The reorganization VSV genome cDNA cloned plasmids pVSV Δ G*GFP and auxiliary expression plasmid pBS-G, pBS-L, pBS-N and the pBS-P that are used to prepare the reorganization vesicular stomatitis pseudovirion (VSV Δ G*GFP-NiVG/F) of chimeric Nipah virus G of cyst membrane and the proteic expressing green fluorescent protein of F provide [9] (reference 9 has specified the preparation method of above-mentioned plasmid) 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 is used.The BHK-21 cell is grown and kept liquid is the DMEM (available from Gibco) that contains 5% foetal calf serum; Sf9 insect cell nutrient solution is Sf-900II (available from a Gibco) serum-free medium.

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

1.1.4: laboratory animal: 6 ages in week, female BALB/c mouse was available from Beijing dimension tonneau China laboratory animal company.Age, weanling pig was available from veterinary institute laboratory animal base, Harbin and by breeding of veterinary institute laboratory animal base, Harbin and raising all around.

1.2: the structure of expressing the viral genome cDNA clone of NiV G gene

With PCAGG-NiVG is template; (its gene order is SEQ ID No.1 at Nipah virus G gene through PCR primer (seeing table 1); Coded protein sequence is SEQ ID No.2) ORFs 5 ' end introduces the transcription termination sequence GE (TTAAGAAAAAA) and the transcriptional initiation sequence GS (ACGGGTAGAA) of Pme I restriction enzyme site recognition sequence (GTTTAAAC) and NDV (NDV) 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 genome cDNA clone called after pBRN-FL-NiVG that is built into after Pme I enzyme is cut.

The used primer of table 1 construction expression NiV G full length gene genomic clone plasmid

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

1.3: the rescue of recombinant virus

With BHK-21 cell inoculation six orifice plates (available from Corning); Treat that cell growth reaches 80% when converging; Infect vTF7-3 (MOI=0.01) in preceding 1 hour in transfection and (express the recombinant poxvirus vTF7-3 of T7 polysaccharase; Bernard doctor Moss by America NI H provides [7]), infect the back that finishes adopt 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; Restrain cell 2.5 minutes with the PBS buffering liquid that contains 10%DMSO; Add complete DMEM nutrient solution (available from Gibco) afterwards, change Opi-MEM substratum (available from Gibco) after 12 hours, and add pancreatin (the 1 μ g/mL that TPCK handles; (available from Sigma)) continue to hatch results culture supernatant after 72 hours,, 0.22 μ M aperture filter (available from Millipore) inoculates 9-11 age in days SPF chick embryo allantoic cavity (available from Harbin veterinary institute state poultry seed resource center) after filtering; Inoculate and get chick embryo allantoic liquid 50 μ L after 72 hours and carry out NDV blood clotting (HA) and blood clotting and suppress (HI) test.The chick embryo allantoic liquid that results HA and HI test-results are positive ,-70 ℃ of preservations are frozen subsequent use after the packing.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 0.01 infection BHK-21 cell with MOI respectively; Infect and discard nutrient solution after 24 hours; With twice of Xian PBS; Use fixedly 30min of 3% Paraformaldehyde 96 (available from Sigma) room temperature afterwards; Wash 3 times with PBST (PBS that contains 0.05%Tween-20); Mouse anti NiV G monoclonal antibody G3E9 (this prepared in laboratory, 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]) and the non-immune BalB/c mice serum (taking mouse vein blood also to separate) that the cell of rLa-NiVG infection are added 1: 50 times of dilution respectively with adopting the ball rear vein beard to puncture blood-collecting method behind the mouse anesthesia; The LaSota vaccine strain infects the anti-NDV serum of chicken [5], mouse-anti NiV G monoclonal antibody G3E9 (see before and state) and the non-immune serum (see before and state) that the hole adds 1: 50 times of dilution respectively, room temperature effect 2 hours; PBST rinse three times, the anti-mouse IgG of FITC labelled goat (or anti-chicken IgY) antibody (available from Sigma) the room temperature lucifuge that adds 1: 200 times of dilution is more respectively hatched 1h, washes 5 times with PBST, places observations under the fluorescent microscope (Leica DMIRES2).

1.4.2: cytogamy is active to be detected

To express the proteic eukaryon expression plasmid PCAGG-NiVF of Nipah virus F and grow 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 MOI, and be LaSota and infect contrast, infect adding DMEM complete culture solution after a hour, be positioned over the 5%CO2 environment, cultivate observation of cell fusion situation after 48 hours for 37 ℃ with recombinant virus rLa-NiVG.

1.5 the chicken embryonic development kinetics of recombinant virus

RLa-NIVG is inoculated SPF chicken embryo (available from Harbin veterinary institute state poultry seed resource center), 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 safety experiment to mouse

With rLa-NiVG with maximal dose (10 ⁶EIDS0/0.1ml) while collunarium 30ul, intramuscular injection 100ul immunity 10 6 weeks female Balb/c mouse in age (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) are set up LaSota vaccine strain immune group (dosage is identical with rLa-NiVG with approach) and PBS control group simultaneously.Immunity back set time every day weighing mouse body weight also writes down 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 behind first and booster immunization, get 5 mouse respectively for every group and take euthansia, 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 EUSA (ELISA) and virus neutralization tests (VNA).

The ELISA experimentation is summarized as follows: with the Sf9 cell lysate of expressing the proteic recombinate shape virus infection of Nipah virus G as envelope antigen; In order to detect behind the first and booster immunization of rLa-NiVG the concentration of IgG in the mice serum, the concrete operations step is referring to document [11].Use the mouse IgG (Southern Biotech) of concentration known to do gradient dilution simultaneously and encapsulate elisa plate production standard curve.Linear relationship calculation formula between OD value of obtaining according to typical curve and the standard antigen concentration that encapsulates is calculated the content of antigen-specific IgG in the serum according to the gained formula, shows with the nanogram numerical table 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].It is subsequent use that the BHK-21 cell grows 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 contains 200IU with equal-volume approximately mixes, and every part of serum is done three repetitions.1 hour postoperative infection BHK-21 cell is made in 37 ℃ of senses, places 5%CO2, cultivates after 24 hours that (Leica DMIRES2) observes and counting 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, behind the initial immunity around booster immunization, immunization route is identical with initial immunity with dosage.The separation of serum of taking a blood sample weekly after the immunity, and measure serum neutralization tire (experimental procedure is the same).

2 results

2.1 expressing the proteic recombinant Newcastle disease virus of NiV G makes up and virus rescue

On newcastle disease 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, be built into and express the proteic recombinant full-lenght cDNA clone of NiV G rLa-NiVG.

With pBRN-FL-NiVG and express NP, P, the common transfection BHK-21 of the proteic helper plasmid of L cell, 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 results HA and HI test is positive after 72 hours.RT-PCR and sequencing result show that 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 China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) (BeiJing, China on October 26th, 2011; Institute of Microorganism, Academia Sinica, 100101), preserving number is CGMCC No.5402.

2.2 recombinant virus rLa-NiVG can correctly express Nipah virus G albumen

For confirming that G albumen can access correct expression in the recombinant virus, infect the BHK-21 cell with rLa-NiVG, LaSota vaccine strain respectively, be that an anti-indirect immunofluorescence that carries out detects with mouse-anti NiV G protein monoclonal antibody G3E9, the anti-NDV serum of chicken.The result is as shown in Figure 2: rLa-NiVG infects the BHK-21 cell can detect specificity green fluorescent signal with G3E9, and LaSota infected group and do not infect control group and all be negative, and shows that recombinant virus can correctly express 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 take place 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 NDV 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 (express and verify [8]) transfection BHK-21 cell; And then infect with recombinant virus rLa-NiVG; Microscopically promptly can be observed tangible cytogamy (Fig. 3 is shown in arrow) after 48 hours, shows that rLa-NiVG can correctly express the G albumen with BA.

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

Insert the chicken growth of the embryo characteristic that Nipah virus G gene does not change recombinant virus rLa-NiVG in order to verify, 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, the 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 with 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; Three groups of mouse all survive in observation period, and the mental status is good, and the mouse body weight all 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 the good humoral immunoreation 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 can detect tangible Nipah virus specific IgG antibodies and neutralizing antibody (P＜0.05); Two kinds of antibody horizontals significantly rise (P＜0.05) than initial immunity behind the booster immunization, and the result shows that Nipah virus G albumen recombinant Newcastle disease virus LaSota vaccine strain can induce the good humoral immunoreation in mouse.

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

Pig is that Nipah virus is propagated most important intermediate host to Nipah virus height susceptible, also is 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 judging the vaccine success or not.We with 2ml recombinant virus SPF chick embryo allantoic liquid through intramuscular injection immunity weanling pig in 4 age in week.4 week of initial immunity, the back was with identical immunizing dose and approach booster immunization.Take a blood sample and separation of serum for the first time and behind the 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 that from the result of Fig. 8 initial immunity can produce neutralizing antibody after two weeks, the 3rd, antibody horizontal rises to some extent all around, but does not have significant difference.The serum neutralizing antibody significantly rises (P＜0.01) after than initial immunity behind the booster immunization, and the antibody extended period is grown (lasting till for 21 weeks at least).Neutralization test result shows that rLa-NiVG can induce generation high level and persistent neutralizing antibody in the pig body.

3 discuss

The present invention utilizes NDV LaSota vaccine strain reverse genetic operating system; With Nipah virus G gene as one independently transcriptional units insert the LaSota genome; Success makes up and rescue obtains expressing the proteic recombinant Newcastle disease virus rLa-NiVG of Nipah virus G; Test-results proof rLa-NiVG can correctly express Nipah virus G albumen, and recombinant virus has kept the high chicken embryonic development titre consistent with LaSota and to mammiferous tight security.NDV LaSota vaccine strain has following advantage as carrier: NDV heredity is relatively stable, and a serotype is only arranged, and reorganization and virulence take place between strain, and to return strong possibility minimum.NDV is a passing infection on Mammals, and security is very high; Reproduction process is accomplished in cytoplasm, from RNA to RNA, and the possibility that does not have the DNA stage and integrate with cellular genome; The NDV less toxic vaccine is a kind of natural adjuvant to Mammals, and good innate immunity and specificity humoral and cellular immunization produce more comprehensively, the equilibrated immunoprotection 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 did not take place in China at present as yet, and operated Nipah virus and need under Biosafety IV level condition, carry out, 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 that possesses a passing infection ability and expressing green fluorescent protein simulates the NiV live virus and 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 gets into 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 half the highest serum extension rate.This method has safety, stable, quick, the characteristics that susceptibility is high, and practical application effect is good.

Humans such as Guillaume are expressed Nipah virus G or the proteic recombinant poxvirus immunity hamster of F 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 people such as Weingartl make up, the proteic reorganization canary pox virus of F ALVAC-G, ALVAC-F 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.The proteic recombinant Newcastle disease virus rLa-NiVG of the expression Nipah virus G initial immunity of this research and establishment can induce pig to produce neutralizing antibody (the average extension rate of serum 1: 200) rapidly after two weeks, booster immunization post neutralization antibody horizontal is than initial immunity (the average extension rate of serum 1: 4000 that rises significantly; P＜0.01).Kaku people's such as [16] research has been compared the serum dilution of doing neutralization test with Nipah virus G and the chimeric vesicular stomatitis pseudovirion of F albumen cyst membrane and Buddhist nun's handkerchief live virus and has been tired, and 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 in the attack of lethal dose Nipah virus, protection to be provided for pig.

Though Buddhist nun's handkerchief encephalitis does not take place in China at present; But the Nipah virus encephalitis does not stop in the popular of China surrounding countries (especially country in Southeast Asia) and outburst all the time; 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 natural hole such as the upper respiratory tract, oral cavity and drain, and therefore becomes the contagium to the most threatening property of the mankind.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 basically, but not the direct result who 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 proteic recombinant Newcastle disease virus LaSota of the expression Nipah virus G vaccine strain 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 the good humoral immunoreation 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 property as a kind of, and prospective outstanding Nipah virus candidate vaccine has crucial 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 NDV has been seen in research report [17], have in addition got into phase ii clinical trial [18].The Nipah virus G albumen that rLa-NiVG provided by the invention is expressed; Can under the condition of Nipah virus F albumen co expression, (express the proteic recombinant Newcastle disease virus LaSota of F vaccine strain) like co-infected; 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 the influence of carrier.Because rLa-NiVG or kept the mammalian safety of the height identical, so remain high safety for the normal health tissue with its vector virus.

Should be appreciated that; Although with reference to its exemplary embodiment; The present invention is shown particularly and describe, 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 (10)

1. express the proteic recombinant Newcastle disease virus LaSota of Buddhist nun handkerchief encephalitis G vaccine strain for one kind.

2. recombinant Newcastle disease virus LaSota vaccine strain according to claim 1, wherein the proteic aminoacid sequence of Buddhist nun's handkerchief encephalitis G is SEQ ID No.2.

3. recombinant Newcastle disease virus LaSota vaccine strain according to claim 1, wherein the proteic gene order of Buddhist nun's handkerchief encephalitis G is SEQ ID No.1.

4. recombinant Newcastle disease virus LaSota vaccine strain according to claim 1, wherein said to be used to express the proteic newcastle disease LaSota of G vaccine strain be AV1615.

5. recombinant Newcastle disease virus LaSota vaccine strain according to claim 1, its called after rLa-NiVG, preserving number are CGMCC No.5402.

6. vaccine that is used to prevent the sick encephalitis of Buddhist nun's handkerchief, it comprises the proteic recombinant Newcastle disease virus LaSota of the described expression of the claim 1 Buddhist nun handkerchief encephalitis G vaccine strain of significant quantity.

7. vaccine according to claim 6, it comprises the described recombinant Newcastle disease virus LaSota of the claim 5 vaccine strain rLa-NiVG of significant quantity.

8. the application that the proteic recombinant Newcastle disease virus LaSota of each described expression Buddhist nun handkerchief encephalitis G vaccine strain is used to prepare the vaccine that is used to prevent the sick encephalitis of Buddhist nun's handkerchief among the claim 1-5.

9. application according to claim 8, it comprises that also the proteic recombinant Newcastle disease virus LaSota of said expression Nipah virus G vaccine strain and the proteic recombinant Newcastle disease virus LaSota of expression Nipah virus F vaccine strain carry out combined immunization.

10. the method for the proteic recombinant Newcastle disease virus LaSota of construction expression Buddhist nun handkerchief encephalitis G vaccine strain, said method comprises the steps:

(1) make up reorganization and transcribe plasmid, this transcribes the genome cDNA sequence that plasmid comprises the said newcastle disease LaSota vaccine strain that wherein inserts Nipah virus G gene, and plasmid called after pBRN-NiVG is transcribed in said reorganization;

(2) make up the helper plasmid system that transcribes, this helper plasmid system comprises the helper plasmid pBSL of cDNA sequence of big polymerase protein (L) of helper plasmid pBSP and the said newcastle disease LaSota vaccine strain of encoding of cDNA sequence of phosphorprotein (P) of helper plasmid pBSNP, the said newcastle disease LaSota vaccine strain of encoding of cDNA sequence of the nucleoprotein (NP) of the said newcastle disease LaSota vaccine strain of encode;

(3) the said reorganization of step (1) is transcribed the host cell of transcribing helper plasmid pBSNP, pBSP, the said newcastle disease LaSota of pBSL cotransfection vaccine strain copy permission of plasmid pBRN-NiVG and step (2), cultivate the host cell after the transfection; With

(4) results supernatant filters back inoculated into chick embryo allantoic cavity rescue recombinant virus.

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