CN1772909A - Reverse genetic operation system of New castle disease LaSota vaccine strain and its applciation - Google Patents

Abstract

The present invention is reverse genetic operation system of Newcastle disease Lasota low virulent vaccine strain and its application. The system includes one transcription plasmid including the genome cDNA sequence of the low virulent vaccine strain; one or several transcription aiding plasmids including the cDNA sequence coding the nucleoprotein of the low virulent vaccine strain, the cDNA sequence coding the phosphoprotein of the low virulent vaccine strain and the cDNA sequence coding the large polymerase protein of the low virulent vaccine strain; and the host cell of the Newcastle disease Lasota low virulent vaccine strain. Wild viral strain is obtained by means of the reverse genetic operation system. The present invention lays firm foundation for further development of Newcastle disease virus live carrier vaccine and Newcastle disease virus related research.

Description

Newcastle disease LaSota vaccine strain reverse genetic operating system and application thereof

Technical field

The present invention relates to viral genetic manipulation field, more specifically, the present invention relates to a kind of newcastle disease LaSota vaccine strain reverse genetic operating system and application thereof.

Background technology

(Newcastle disease virus NDV) is non-segmented negative sub-thread minus-stranded rna virus to Avian pneumo-encephalitis virus, as the important member and the model virus of Paramyxoviridae, obtains further investigation.Reorganization NDV has outstanding advantage as the live-virus vaccine carrier: the NDV attenuated vaccine that comprises the LaSota strain is used for the poultry epidemic prevention for a long time always, and its safety and effectiveness is fully proved; 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; Reproduction process is finished in cytoplasm, from RNA to RNA, and the possibility that does not exist DNA stage and cellular genome to integrate; The NDV attenuated vaccine can be induced the formation of general humoral immunization, local mucous membrane immunity and cellular immunization simultaneously, forms more comprehensive, certain immunoprotection; Can or inject multiple mode and give seedling by drinking-water, spraying, collunarium, eye droppings, extremely easy to use; NDV has the chicken embryonic development characteristic of high titre, and production cost is very cheap ^{(1,7,8,11,12,13)}NDV is the chickenpest cause of disease of hyperinfection and height lethality, and China is used for the attenuated vaccine of the anti-system of newcastle disease every year at least more than 10,000,000,000 plumage parts.The economic implications that NDV uses as the live-virus vaccine carrier is very huge.

The reverse genetic manipulation of minus-stranded rna virus (Reverse genetic) is a process of making new virus by operation viral genome cDNA, its primary process is: 1. assemble complete viral genome (or recombinant type genome) cDNA clone, 5 ' end is accurately sewed after the T7 promotor, 3 ' terminal accurately sewing before nuclease sequence and T7 transcription termination signal that the oneself shears, constitute genome cDNA and transcribe template; 2. transcribe template with starting the necessary expression plasmid (T7 promotor) of transcribing correlation function structural protein such as nucleoprotein (NP), phosphoric acid albumen (P) and polymerase protein (L) of virus replication, the virus replication permissive cell of cotransfection integrative gene expression T7 polysaccharase with genome cDNA; 3. gather in the crops culture supernatant after 24-72 hour, filter that the back continues that sensitive cells goes down to posterity or the inoculated into chick embryo allantoic cavity is rescued and obtained (rescue) virus.Genome cDNA is suddenlyd change, after disappearance or foreign gene insert and modify, can obtain the minus-stranded rna virus of corresponding sudden change or reorganization by reverse genetic operating system (reverse genetic system, RGS system) ^{(1,2,3,4,5,6)}

NDV genome total length 15186 Nucleotide, the same with other paramyxovirus, comprise nucleoprotein (NP), phosphorprotein (P), stromatin (M), fusion rotein (F), lectin neuraminic acid zymoprotein (HN) and big six of polymerase proteins (L) are independently transcribed coding unit (Figure 1A).This research is cloned into GFP albumen between P and the M, studies the expression appropriate location of foreign protein in NDV.NDV is the same with other minus-stranded rna virus, and its minimum infectious unit is the ribonucleoprotein mixture, and the RNA of no albumen parcel itself there is no infectivity.The geneome RNA of NDV is by forming the nucleoprotein complex body with NP, P, L albumen, and the first run of startup RNA is transcribed and the translation of viral protein is synthetic, produce infectious progeny virus ^(7,10)According to this principle, European scholars in 1999 have taken the lead in setting up the reverse genetic operating system (reversegenetic system, RGS system) of first highly pathogenic NDV ⁽²⁾Have at least 4 laboratories to utilize the RGS technology of NDV carrying out the keen competition Journal of Sex Research aspect basis and the applied research in Europe, the United States at present.2001-2002; Palese.P. wait the reorganization NDV B1 strain of the virus of construction expression H1 subtype influenza in succession HA immunogen gene and express the reorganization NDVB1 strain of H7 subtype influenza virus HA immunogen gene, immunity test shows that these two kinds of NDV live vector vaccines can induce protective immunological reaction mouse and bird respectively.But because a little less than B1 itself highly causes; relatively poor in the intravital replication of immunization chicken; thereby the induction of immunity chicken form effective immunoprotection ability also relatively a little less than; test shows; the NDV B1 strain of expressing H7 hypotype HA gene only is respectively 60% and 40% to the survival protection of NDV and H7 hypotype highly pathogenicity avian influenza lethal hit, and can not stop virus duplicating and discharging in vivo ⁽¹²⁾

Studies show that the NDV genome inserts external source reporter gene or immunogen gene in different loci, going down to posterity through cell or the continuous high generation of chicken embryo still keeps the heredity and the expression stability of height ^(11,12,13)The NDV LaSota attenuated vaccine strain that we select that China cultivates voluntarily, production practice widespread use, immune effect are good has been set up corresponding reverse genetic operating system, successfully rescues and has obtained expression GFP recombinant virus.Rescue and obtain virus and have good chicken embryonic development characteristic, immune newborn SPF chick can be induced high-caliber HI antibody response.This research has been established solid basis for further carrying out the viral relevant rudimentary of development of Avian pneumo-encephalitis virus live vector vaccine and NDV.

Summary of the invention

An object of the present invention is to provide a kind of reverse genetic operating system of new castle disease LaSota attenuated vaccine strain, this system comprises:

(1) transcribe plasmid, this transcribes the genome cDNA sequence that plasmid comprises described new castle disease LaSota attenuated vaccine strain;

(2) one or more helper plasmids of transcribing, this helper plasmid comprise the cDNA sequence of the big polymerase protein (L) of the cDNA sequence of the nucleoprotein (NP) of the described new castle disease LaSota attenuated vaccine strain of encoding, the cDNA sequence of phosphoric acid albumen (P) of the described new castle disease LaSota attenuated vaccine strain of coding and the described new castle disease LaSota attenuated vaccine strain of encoding;

(3) host cell of the virus replication of described new castle disease LaSota attenuated vaccine strain permission, BHK-21.

In a preferred embodiment, preferred described new castle disease LaSota attenuated vaccine strain is the newcastle disease LaSota vaccine strain AV1615 available from Chinese veterinary microorganism culture presevation administrative center (CVCC).

In an embodiment of above-mentioned reverse genetic operating system, being included in described genome cDNA sequence of transcribing in the plasmid is positioned at after the T7 promotor, and before the sequence and T7 transcription terminator of the nuclease that the coding oneself shears, constitute genome cDNA and transcribe template.In a preferred embodiment, the nuclease of described oneself's shearing is hepatitis D virus ribozyme (Rib).

In another embodiment of above-mentioned reverse genetic operating system, the cDNA sequence that is included in the big polymerase protein (L) of the cDNA sequence of phosphoric acid albumen (P) of described cDNA sequence of transcribing the nucleoprotein (NP) of the described new castle disease LaSota attenuated vaccine strain of coding in the helper plasmid, the described new castle disease LaSota attenuated vaccine strain of coding and the described new castle disease LaSota attenuated vaccine strain of encoding all is positioned at after the T7 promotor.

In a preferred embodiment, the described plasmid of transcribing is the plasmid pBRNFL-GFP3 that comprises external source label gene GFP.

In a preferred embodiment, the described helper plasmid of transcribing is plasmid pBSNP, pBSP and pBSL.

In a preferred embodiment, described host cell is BHK-21.

Another object of the present invention provides the application of above-mentioned reverse genetic operating system in the preparation minus-stranded rna virus.In a preferred embodiment, described genome cDNA sequence of transcribing the new castle disease LaSota attenuated vaccine strain in the plasmid is suddenlyd change, is lacked or inserted by foreign gene, to prepare the minus-stranded rna virus of corresponding sudden change, disappearance or reorganization.

A further object of the invention provides the method for utilizing above-mentioned reverse genetic operating system to prepare minus-stranded rna virus, may further comprise the steps:

(1) with described plasmid and described host cell of transcribing the described virus replication permission of helper plasmid cotransfection, the host cell after the cultivation transfection of transcribing;

(2) results supernatant liquor filters that the back continues that sensitive cells goes down to posterity or the inoculated into chick embryo allantoic cavity is rescued and obtained virus.

Description of drawings

Fig. 1. assemble total length NDV cDNA from the overlapping cDNA fragment of subgene group that high-fidelity RT-PCR produces.The cDNA fragment is connected at total restriction site, and in transcribing plasmid pBR322, assemble, in transcribing plasmid pBR322, RBZ and T7 terminator sequence are cloned between EcoRI and the salI site (seeing specification sheets for details) in advance.(A) first and last Nucleotide of the whole full-length gene group of demonstration parental generation NDV.(B) cDNA that shows the NDV that contains the GFP gene at the top clones, and the sea line under genetic map shows the position of single cDNA.

Fig. 2. produce the Nucleotide of introducing the modifying enzyme site by RT-PCR and change, and by using the order-checking of PRISM test kit (Perkin-Elmer) and Applied Biosystems ABI310 automatic sequencer.What add frame is a nucleotide substitution (sporting G by A) of introducing in pBRN1-10 by PCR mutagenesis.

The immunofluorescence analysis of Fig. 3 .rNDV protein expression.Converge the BHK-21 cell with the rLaSota-GFP infection.(A) MOI is the cell that infected in 1 o'clock, (B) negative control.With the cell fixation that infects, saturatingization and with chicken anti--the NDV antiserum(antisera) surveys, follow with FITC-link coupled rabbit to resist-the chicken igg antibody incubation.Observation of cell under Leica DMIRES2 fluorescent microscope.Show and infect back 28 hours data.

Fig. 4. with the green fluorescence of rLasota-GFP infected B HK-21 cell.The left side is to infect 10 ⁹The BHK-21 cell of pFU/ml rLasota-GFP.The right is normal BHK-21 cell.

The stably express of Fig. 5 .EGFP in the recombinant virus of different generations.With rLasota-GFP infected chicken embryo primary cell (CEF) (available from the Harbin veterinary institute), fluorescence microscope behind the 24h: (A) first-generation, (B) third generation, (C) the 5th generation, (D) the 7th generation, (E) the 9th generation.

Fig. 6. the growth curve of rNDV/LaSota virus in containing the egg of embryo.With 1 * 10 ⁴EID ₅₀RNDV/LaSota inoculation embryo egg (is inoculated back 24,48 and 72 hours) constantly in difference and is collected allantoic fluid.Pass through EID ₅₀Measure the virus titer of embryo egg.Numerical value is available from two independently experiments, and each experiment is carried out in triplicate.The Log titre derives from average virus titer.

The plasmid map of Fig. 7 .pBTRT.

The dna sequence dna of Fig. 8 .pBTRT plasmid.First italicized item: T7 promotor; Band underscore part: ribozyme sequence; The italicized item of second band underscore: T7 terminator.

The plasmid map of Fig. 9 .pBRN-FL-GFP3.

The dna sequence dna of Figure 10 .pBRN-FL-GFP3 plasmid.First italicized item: T7 promotor; Band underscore part: ribozyme sequence; The italicized item of second band underscore: T7 terminator.

Embodiment

Hereinafter describe reference example in detail the present invention, described embodiment only is intended as illustrative explanation the present invention, rather than intention limits the scope of the invention.Scope of the present invention is specifically limited by accompanying Claim.

The structure of the reverse genetic operating system of embodiment 1 new castle disease LaSota attenuated vaccine strain

Cell and virus

BHK-21 cell (newborn hamster nephrocyte ATCC CCL-10), the DMEM (Eagle ' s substratum of Dulbecco ' s improvement) of substratum for containing 10% foetal calf serum (Hyclone) and 1 μ g/m1 G418; NDV Lasota vaccine strain AV1615 (available from Chinese veterinary microorganism culture presevation administrative center (CVCC)).-70 ℃ of inoculation 9-10 age in days SPF chick embryo allantoic cavity amplification backs are frozen standby; The anti-NDV height of the chicken property exempted from serum is by this research department's preparation (Chu, H.P., G.Snell, D.J.Alexander and G.C.Schild.1982.Avian Pathol 11:227-234); SPF chicken embryo and SPF chicken provide by Harbin veterinary institute SPF Experimental Animal Center.

The structure of transcription vector

Geneome RNA transcription vector pBTRT be a skeleton and at the insertion of EcoRI/salI site T7 promotor (T7promotor), fourth hepatovirus ribozyme (Rib) and T7 transcription termination signal (T7terminal) with low copy cloning vector pBR322 (Invitrogen), is made up voluntarily by this laboratory.The dna fragmentation that is cloned between T7 promotor and the ribozyme can be transcribed under the effect of t7 rna polymerase, and because the autocatalysis function of Rib can guarantee that 3 of transcription product ' end is accurately consistent with clone's dna segment.

The structure of NDV LaSota pnca gene group full-length cDNA

For setting up the reverse genetic operating system of NDV newcastle disease Lasota vaccine strain, must at first make up the full length cDNA clone of corresponding gene group, transcribe template as the genome strand RNA, ten cDNA cloned sequences that cover whole genome have been made up for this reason, utilize the restriction enzyme site of lap between each segment, assemble the global cDNA clone who has obtained 15186nt at low copy plasmid transcription vector plasmid pBTRT, the sequencing result has logined GenBank, accession number is AY845400, and GFP gene (GenBank AAZ16159.1) is cloned into P, between the M.In full-length cDNA fragment 5 ' terminal prefix t7 rna polymerase promotor, having no progeny at the cDNA sheet is connected with hepatitis δ ribozyme (GenBank X04451) and the T7 transcription termination signal with self-catalysis.The plasmid called after pBRN-FL-GFP that structure is finished.For avoiding methylating of Xba site, be C with F protein-coding region the 6178th bit base in the genome cDNA by T same sense mutation by the pcr gene group, and as the viral molecule marker of rescue.The same with other investigators, at T7 polymerase promoter two unnecessary G of 5 ' terminal introducing in genome cDNA, this has the virus rescue that helps the paramyxovirus reverse genetic manipulation simultaneously for we.Specific as follows:

NDV Lasota vaccine strain virus egg inoculation allantoic fluid extracts geneome RNA through conventional method (animal virology, second edition); Whole genome is divided into 10 fragments of terminal portions eclipsed (F1-F10) and carries out RT-PCR amplification, and the cDNA fragment cloning is to pBluescript (Clontech) SmaI site and in full accord through sequential analysis conclusive evidence and virus genome RNA sequence; The sequencing result has logined GenBank, and accession number is AY845400.For introducing special molecular genetic label, select Lasota vaccine strain genome cDNA 6172bp place to have monomethylated XbaI site, sequence is TCTAGATCA, utilize the PCR means that it is sported TCTAGACCA, make it discerned by methylase, thereby can discern by being limited property restriction endonuclease XbaI; The restriction site that utilizes the adjacent segment lap to exist connects into the complete NDV genome cDNA (Figure 1A) of assembling, and with the GFP gene clone to P, PmeI site between the M, and on pre-installed gene stop and gene homing sequence (GE/GS) (TTAAGAAAAAA/T/ACGGGTAGAA), and be cloned on the transcription vector pBTRT, be built into and contain GFP gene viruses genome and transcribe plasmid pBRN-FL-GFP3 (Figure 1B); And then open reading frame (ORF) cDNA that expresses nucleoprotein (NP), phosphoric acid albumen (P) and big polymerase protein (L) gene is cloned in pBluescriptII (+/-) plasmid T7 promotor downstream respectively, constitute respectively and transcribe helper plasmid pBSNP, pBSP and pBSL.

Rescue from full length cDNA clone and to obtain infectious NDV (virus rescue)

For the infectious NDV of rescue from clone's cDNA, at first with pBRN-FL-GFP3 and express NDV NP, the proteic helper plasmid cotransfection of P, L BHK-21 cell.The fusion protein F 0 of NDV must be cracked into F1 and F2 just has infectivity, for the Lasota low virulent strain, the BHK-21 cell can not be secreted the required proteolytic enzyme of cracking F0 albumen, therefore in substratum, add the corresponding protein enzyme, so should change serum free medium into and add TPCK (tosylphenylalanine chloromethyl ketone .Sigma) (1 μ g/ml) this moment, continue to cultivate 2-3 days, results transfectional cell supernatant is inoculated in the SPF chicken embryo of 9-11 age in days.Gather in the crops chick embryo allantoic liquid after 4 days, blood clotting (HA) the test-results positive, the HA valency of Different Chicken embryo is between 2 ^8-11NDV immune serum blood clotting suppresses (HI) analysis of experiments and presents positive findings equally.Results virus-positive allantoic fluid is as rescuing the F1 generation of obtaining viral rLasota-GFP.Further RT-PCR and The sequencing results show, it is C that F1 generation is rescued the 6178 site bases that obtain viral genome cDNA, but not the C of former LaSota parent plant, with expection conform to fully (Fig. 2).The result shows, by anti-Genetic Manipulative Technology, utilizes NDV LaSota vaccine strain genome cDNA clone successfully to rescue to obtain and has infective progeny virus rLaSota-GFP.More specifically, experimental procedure is as follows:

The BHK-21 cell inoculation is grown when reaching the 50-80% individual layer in 35mm six orifice plates, transcribe plasmid and helper plasmid pBRNFL-GFP3, pBSNP, pBSP and pBSL respectively with 5 μ g, 2.5 μ g, 1.25 μ g, 1.25 μ g, cotransfection BHK-21 cell, adopt CaP04 transfection reagent box (Invitrogene), operation is undertaken by the test kit specification sheets.After the transfection 8-12 hour, discard transfection mixture, with the PBS liquid shock cell that contains 10%DMSO 2.5 minutes, add complete DMEM night incubation, changed serum free medium in second day into, and add after TPCK (1 μ g/ml) continues to hatch 2-3 days, results culture supernatant, 0.22um aperture filter filter 9-11 days SPF embryo allantoic cavity of back inoculation; Postvaccinal SPF embryo continues to cultivate, 3-5 days, get blood clotting (HA) and blood clotting inhibition (HI) test (Thayer SG that chick embryo allantoic liquid 50 μ l carry out Avian pneumo-encephalitis virus routinely, Nersessian BN, RivetzB, Fletcher OJ.Comparison of serological tests for antibodies againstNewcastle disease virus and infectious bronchitis virus using ImmunoCombsolid-phase immunoassay, a commercial enzyme-linked immunosorbent assay, and the hemagglutination-inhibition assay.Avian Dis.1987 Jul-Sep; 31 (3): 459-63.).The positive allantoic fluid of results HA and HI test-results ,-70 ℃ are frozen, and according to a conventional method respectively at 9-10 day instar chicken embryo and every milliliter of EID of chick embryo fibroblast titration ₅₀And PFU viral level ⁽¹⁴⁾Called after rLaSota-GFP.

Embodiment 2 indirect immunofluorescence assay (IFA) test

The weak zooblast of NDV LaSota vaccine strain energy one passing infection vitro culture.For proof rLaSota-GFP virus is duplicated and virus antigen is expressed in that BHK-21 is intracellular, the rLaSota-GFP allantotoxicon is the individual layer BHK-21 cell that 1 virus quantity infects about 70-80% with MOI, infect back 24 hour cells and early stage CPE (cytopathy) phenomenon occurs, exempting from SPF chicken positive serum with the NDV height immediately serves as to detect antibody to carry out indirect IF staining, observe strong positive reaction (Fig. 3 A) as a result under the virus infected cell fluorescent microscope, SPF chicken control serum fluorescent dye then negative (Fig. 3 B).More specifically, experimental procedure is as follows:

Egg inoculation allantois virus liquid rLaSota-GFP dilutes with the DMEM suitable multiple, be about 5 by MOI, 50 μ l volumes infect the BHK-21 that grows in 24 orifice plates, 37 ℃, wash three times with DMEM after hatching 1h, adding complete DMEM then continues to cultivate, behind the 24h with 95% ethanol fixed cell 5min, after PBST (phosphate buffered saline buffer that contains 0.05% polysorbas20) washes behind the cell and to seal 1 hour with the SPF chicken serum, anti-with anti-NDV hyper-immune serum of chicken and SPF serum as one, with 1: the 100 anti-NDV height of the dilution chicken property exempted from serum was that an anti-SPF chicken negative serum with identical extension rate is contrast, after acting on after 30 minutes the PBST washing, two anti-(Sigma) that add 1: 160 dilution fluorescein (FITC) mark, effect 30min, PBST washing back fluorescent microscope (Leica DMIRES2) observations.

The proteic expression of embodiment 3 GFP

Allantotoxicon rLaSota-GFP is that 1 virus quantity infects about 80% individual layer BHK-21 cell with MOI, infects back 24 hour cells and early stage CPE phenomenon occurs, (Leica DMIRES2) observations under fluorescent microscope occurs after the pathology.Have green fluorescence to occur in the positive, wild-type NDV is as negative control, result negative (Fig. 4).

For proof rLaSota-EGFP virus is duplicated and is inserted external source reporter gene GFP and going down to posterity and still can keep stably express in the process in that BHK-21 is intracellular, rLaSota-EGFP chick embryo allantoic cavity inoculation continuous passage 9 times, each generation chick embryo allantois virus liquid 10 times of continuous gradient dilution 100ul volumes is respectively inoculated 24 orifice plates cultivation CEO for cell (CEF), fluorescent microscope behind the 24h (LeicaDMIRES2) direct viewing is (see figure 5) as a result, according to the cell quantity of expressing the strong positive green fluorescent protein under the mirror, determine that every milliliter of plaque forming unit of each generation rLaSota-EGFP (PFU) is between 10 ⁹-10 ¹⁰Between (table 1).The result proves, rLaSota-EGFP virus is duplicated and inserted external source reporter gene GFP and going down to posterity and still can keep stably express in the process in that BHK-21 is intracellular.

The stably express of the GFP of rNDV-EGFP during table 1 goes down to posterity

Go down to posterity	The HA titre	PFU/ml	GFP expresses
				F1 F2 F3 F4 F5 F6 F7 F8 F9	2 9 2 8 2 10 2 11 2 10 2 10 2 8 2 10 2 12	8×10 9 2×10 9 4×10 10 2×10 10 8×10 9 3×10 10 4×10 9 1×10 10 2×10 9	+ + + + + + + + +

Embodiment 4 rNDV are at chicken growth of the embryo characteristic and pathogenic property

For determining that reverse genetic manipulation rescues the chicken embryonic development characteristic that obtains rLaSota-GFP and pathogenic to the chicken embryo thereof, rLaSota-GFP virus F1 for allantois virus liquid by 1 * 10 ⁴EID ₅₀Inoculation SPF chick embryo allantoic cavity.1 * 10 ⁴EID ₅₀Inoculate the SPF chicken embryo that do not cause death fully in back 96 hours.Inoculate back 24 hours, 48 hours, 72 hours and 96 hours results allantoic fluids, HA titre mean value is respectively 2 ^7.5, 2 ^10.3, 2 ^11.3With 2 ^11.6, and every milliliter of allantoic fluid EID ₅₀Then be respectively 10 ^-8.54, 10 ^-8.635, 10 ^-10.085With 10 ^-9.43(Fig. 6).Being respectively according to OIE standard test rLaSota and rLaSota-GFP intracerbral pathogenicity index (ICPI) is 0.37 and 0.35.The result shows that reverse genetic manipulation is rescued and obtains viral rLaSota-GFP and still keep high titre growth and the low lethal biological characteristics of NDV LaSota vaccine parent plant at the chicken embryo.

The immune effect of embodiment 5 induced animal protection antibodies

Rescue and obtain the immunogenicity of rLaSota, chicken embryo amplification allantotoxicon 2 * 10 for measuring reverse genetic manipulation the SPF chicken ⁶EID ₅₀Dosage comes prosperous SPF chicken (Harbin veterinary institute SPF Experimental Animal Center provides) through add eye droppings approach artificial immunization 12 plumages seven ages in days white through collunarium, and other establishes non-immune group control group 8 plumages; Immune group and non-immune control group are raised respectively in air negative pressure and are filtered in the shield retaining.3 weeks were detected the special HI antibody of NDV routinely with hind wing venous blood collection separation of serum.In addition, back 21 days of immunity with strong malicious F48E9 (available from the Harbin veterinary institute) with 10 ³EID ₅₀Dosage adds the eye droppings approach through collunarium to be attacked, and observes for two weeks, statistics morbidity and death condition.

Experimental result: in rLaSota allantois virus liquid F1 generation, is with 2 * 10 ⁶EID ₅₀Dosage adds eye droppings approach artificial immunization seven ages in days white through collunarium and comes prosperous SPF chicken, and 3 weeks were observed in the immunity back, during all chick of immune group do not have any unusual, feed intake and growing and non-immune control group no significant difference.3 weeks of immunity back are detected the special HI antibody horizontal of serum N DV, all between 2 ⁷-2 ⁹, average out to 2 ^8.2(table 2).Immune group any morbidity or death do not occur after attacking poison, and protection (10/10) fully is with the then all morbidities dead (0/4) (table 3) of the non-immune control group of age in days.The result shows that immunized chicks of rLaSota-GFP can be induced high-caliber protection antibody reaction, has good immunogenicity, and keeps the good security of low pathogenicity LaSota vaccine strain.

The recombinant poxvirus of stably express T7 polysaccharase is adopted in this research ^(9,10), infect the BHK-21 cell and in transfectional cell, transcribe full-length gene group strand RNA, express required NP, P and the L albumen of startup virus replication simultaneously, obtained the efficient of very high virus rescue.In addition, adding proteolytic enzyme in the substratum of transfectional cell also is to rescue the prerequisite that obtains virus.In chick embryo allantoic liquid, there is special proteolytic enzyme; can be cracked into F1 and F2 by special F0 with low virulent strain; discharge the highly hydrophobic fusion function zone of F1N end; be that the assembling virus particle obtains further infection ability; and there is not the corresponding protein enzyme in the BHK-21 cell that is used for transfection; therefore we add acetylize trypsin TPCK) substitute chick embryo allantoic liquid, infectious to promote that virus particle obtains.On the contrary, do not adding under the situation that pancreatin carries out transfection, we fail to rescue and obtain virus.

The immunogenicity of table 2. reorganization NDV

^aDosage, 2 * 10 ⁶EID ₅₀. ^bAfter the inoculation. nucleotide sequence .NDV LaSota vaccine strain genome full length cDNA sequence (15186nt) GeneBank accession number: AY845400.

Inoculation a?	Chicken quantity	Inoculate age	Serum HI to NDV tires: the 19th day b
				rNDV ? ? ? ? ? ? ? ? ?	1 2 3 4 5 6 7 8 9 10	7 7 7 7 7 7 7 7 7 7	2 8 2 8 2 8 2 8 2 7 2 7 2 9 2 9 2 9 2 9
Mean value	-	-	2 8.2

Malicious protection test is attacked in table 3. reorganization LaSota NDV immunity

Group	Inoculum *	Dosage	Blood clotting suppresses valency **	Survival rate ***
					The test group control group	rLaSota-GFP PBS	2×10 6EID 50 -	2 7.3 0	10/10 0/4

^*7 age in days SPF chick are through collunarium eye droppings inoculation rLaSota (1 * 10 ⁶EID ₅₀/ only).

^*Venous blood collection under the wing is measured the HI titre, calculating mean value.

^{* *}In immunity three weeks of back, attack poison (1 * 10 with strong poisons collunarium of NDV and intramuscular injection ³EID ₅₀/ only)

This research can reach the growth titre similar to parent LaSota vaccine strain by the expression GFP attenuated vaccine of reverse genetic operating system rescue in inoculated into chick embryo, and rescues and obtain virus with 10 ³EID ₅₀Inoculation 9-10 age in days SPF chicken embryo in the dosage allantoic cavity, 96 hours chicken embryos that do not cause death; 24,48, the 72 and 96 hours allantoic fluid HA valencys in inoculation back are respectively 2 ^7.5, 2 ^10.3, 2 ^11.3With 2 ^11.6, every milliliter of allantoic fluid EID ₅₀Then be respectively 10 ^-8.54, 10 ^-8.635, 10 ^-10.085With 10 ^-9.43Immunity 7 age in days SPF chick can be induced high-caliber protection antibody reaction, and back 19 days antibody serum HI antibody titerss of immunity are between 2 ^8-9, good immunogenicity when fully showing high safety, the good chicken embryonic development adaptability that the vaccine strain of this reverse genetic operating system of the present invention rescue should possess as the live vector vaccine and containing foreign gene.Reorganization NDV live vector production of vaccine is with low cost, extremely easy to use, can pass through drinking-water, spraying, collunarium, eye droppings to seedling; Because respiratory tract, digestive tube are NDV and the former natural infection route of entry of other multiple important eqpidemic diseases, reorganization NDV is alive, and malicious vaccine can be induced high-caliber mucosal immunoreaction, and more certain effective immunoprotection is provided; The NDV attenuated vaccine immunity almost is the requisite immune programme for children of all newborn chick in China's aviculture, naturally becomes the most attractive reorganization live-virus vaccine carrier.

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Claims (12)

1. the reverse genetic operating system of a new castle disease LaSota attenuated vaccine strain, this system comprises:

(1) transcribe plasmid, this transcribes the genome cDNA sequence that plasmid comprises described new castle disease LaSota attenuated vaccine strain;

(2) one or more helper plasmids of transcribing, this helper plasmid comprise the cDNA sequence of the big polymerase protein (L) of the cDNA sequence of the nucleoprotein (NP) of the described new castle disease LaSota attenuated vaccine strain of encoding, the cDNA sequence of phosphoric acid albumen (P) of the described new castle disease LaSota attenuated vaccine strain of coding and the described new castle disease LaSota attenuated vaccine strain of encoding;

(3) host cell of the virus replication of described new castle disease LaSota attenuated vaccine strain permission, the clone of preferred stably express T7 polysaccharase provides T7 polysaccharase as the vaccinia virus (VV-T7) by stably express T7 polysaccharase.

2. according to the reverse genetic operating system of claim 1, comprising after described genome cDNA sequence of transcribing in the plasmid is positioned at the T7 promotor, and before the sequence and T7 transcription terminator of the nuclease that the coding oneself shears, constitute genome cDNA and transcribe template.

3. according to the reverse genetic operating system of claim 2, the nuclease that wherein said oneself shears is a fourth hepatovirus ribozyme (Rib).

4. according to the reverse genetic operating system of claim 1, all be positioned at the T7 promotor comprising cDNA sequence at the big polymerase protein (L) of the cDNA sequence of the phosphoric acid albumen (P) of described cDNA sequence of transcribing the nucleoprotein (NP) of the described new castle disease LaSota attenuated vaccine strain of coding in the helper plasmid, the described new castle disease LaSota attenuated vaccine strain of coding and the described new castle disease LaSota attenuated vaccine strain of encoding after.

5. according to any one reverse genetic operating system among the claim 1-4, wherein said new castle disease LaSota attenuated vaccine strain is AV1615.

6. according to the reverse genetic operating system of claim 5, the wherein said plasmid of transcribing is the plasmid pBRNFL-GFP3 that comprises external source label gene GFP, and foreign gene GFP inserts the NDV viral genome by manually-injected PmeI site between P and M gene.

7. according to the reverse genetic operating system of claim 5, the wherein said helper plasmid of transcribing is plasmid pBSNP, pBSP and pBSL.

8. according to any one reverse genetic operating system among the claim 1-4, wherein said host cell is BHK-21.

9. claim 1 or 5 the reverse genetic operating system application in the preparation minus-stranded rna virus.

10. according to the application of claim 9, wherein said genome cDNA sequence of transcribing the new castle disease LaSota attenuated vaccine strain in the plasmid is suddenlyd change, is lacked or inserted by foreign gene, to prepare the minus-stranded rna virus of corresponding sudden change, disappearance or reorganization.

11. a method of utilizing the reverse genetic operating system of any one among the claim 1-4 to prepare minus-stranded rna virus may further comprise the steps:

(1) with described plasmid and described host cell of transcribing the described virus replication permission of helper plasmid cotransfection, the host cell after the cultivation transfection of transcribing;

(2) results supernatant liquor filters that the back continues that sensitive cells goes down to posterity or the inoculated into chick embryo allantoic cavity is rescued and obtained virus.

12. NDV LaSota vaccine strain progeny virus rLaSota-GFP according to the preparation of the method for claim 11.

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