CN107418973A - A kind of construction method of genotype VII NDV low virulent strain - Google Patents

A kind of construction method of genotype VII NDV low virulent strain Download PDF

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CN107418973A
CN107418973A CN201710388557.0A CN201710388557A CN107418973A CN 107418973 A CN107418973 A CN 107418973A CN 201710388557 A CN201710388557 A CN 201710388557A CN 107418973 A CN107418973 A CN 107418973A
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李明义
孙化露
金红岩
李思菲
于泽坤
毕云英
刘阳
单学强
栾志舫
马礼照
李朝阳
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Qingdao Sinder Pharmaceutical Co ltd
Shandong Sinder Technology Co ltd
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Abstract

The present invention provides a kind of construction method of genotype VII NDV low virulent strain, is that the amino acid of 237 of the P albumen of VII type NDVs and 240 are sported into threonine and isoleucine respectively.Vaccine prepared by the genotype VII NDV low virulent strain of the method structure of the present invention, which can induce, produces higher neutralizing antibody, can resist the infringement of strong virus force genotype VII NDV, have broad application prospects.

Description

A kind of construction method of genotype VII NDV low virulent strain
Technical field
The invention belongs to vaccine virus strain constructing technology field, and in particular to a kind of weak poison of genotype VII NDV The construction method of strain.
Technical background
Ewcastle disease is a kind of acute deadly infectious disease as caused by NDV, to the very harmful of aviculture, once A classes disease is classified as by International Office of Epizootics (OIE).ND is found in Indonesia Java first in nineteen twenty-six, and the same year is found in English The Newcastle city of state, hereafter rapidly to propagating all over the world.Worldwide there occurs four times to be very popular by ND at present, and And host range constantly expands.
29 plants of NDVs that Liu Xiu Buddhist etc. is isolated from Zhejiang And Jiangsu area to 1985-2003 carry out sequence analysis, As a result 17 plants are shown and belongs to genotype VII, wherein 10 plants of separation idiopathy gaggles.Liu Hualei etc. was isolated from China to 2005 83 plants of NDVs of Continental Area carry out analysis and shown, wherein 64 plants belong to genotype VII.Lien etc. is to 2003-2006 20 plants of NDVs that Taiwan is isolated between year are genotype VII.In addition the South Korea with stage China periphery and day This popular NDV is also mostly genotype VII.The part NDV strains that we separate in recent years to this laboratory are also sequenced Analysis, as a result shows, more than 70% strain is genotype VII.It is therefore seen that genotype VII NDV is in Chinese Poultry It is widely current in group, is current absolute predominance genotype.
Prevention and control on Vehicles Collected from Market to NDV mainly use vaccine strain Lasota, clone30 and II systems of IV systems epidemic disease The strain of the gene II types such as seedling strain B1, although they can provide certain immunoprotection, can not prevent ewcastle disease completely The propagation of virus, the prevalence of diffusion, particularly genotype VII NDV cause huge economic loss to aquaculture. Because the genotype VII NDV of prevalence is all strong poison, therefore most of strains are not suitable as vaccine strain use, unless It is Natural Avirulent Strain or the exquisite weak strain of genetic engineering means people.It is the key for producing vaccine to obtain low virulent strain.
The content of the invention
It is an object of the invention to provide a kind of construction method of genotype VII NDV low virulent strain, so as to structure Build the genotype VII NDV low virulent strain for obtaining less toxic power
The method of structure VII type NDV low virulent strains provided by the invention, it is by the P eggs of VII type NDVs The white amino acid of 237 and 240 sports threonine (T) and isoleucine (I) respectively.
The method of above-mentioned acquisition VII type NDV low virulent strains, a kind of its concrete operation method is by reversely losing Operating method is passed to build VII type NDV low virulent strains, it includes the steps：
The cell line of construction expression t7 rna polymerase,
Described cell line, it is to transfect DF1 cells after the gene for expressing t7 rna polymerase is connected with carrier PCI-neo Obtain；
The three auxiliary recombinant plasmids and a full base needed during genotype VII NDV NDV rescues are provided Because of a group recombinant vector, three auxiliary recombinant plasmids include NDV NP albumen, P albumen and L GFPs respectively；Wherein P albumen The amino acid of 237 and 240 is respectively T and I；
Three auxiliary recombinant plasmids and a full-length genome recombinant vector are transferred to the cell line of expression t7 rna polymerase In, culture obtains genotype VII NDV low virulent strain.
Further, described construction method, be by 237 of the P albumen of genotype VII NDV and When the amino acid of 240 sports T and I respectively, F protein is also subjected to hypotoxicity mutation.
Vaccine prepared by the genotype VII NDV low virulent strain of the method structure of the present invention can induce generation higher Neutralizing antibody, the infringement of strong virus force genotype VII NDV can be resisted, had broad application prospects.
Brief description of the drawings
Fig. 1：Ewcastle disease F genetic fragment PCR qualification figures；
Fig. 2：The phylogenetic tree of NDV strains (WF strains, SL strains) based on F genetic fragments；
Fig. 3：9 genetic fragment PCR qualification result figures of ewcastle disease；
Fig. 4：Full-length genome segmentation structure PCR qualification result figures；
Fig. 5：NP, P, L helper plasmid digestion qualification figure.
Embodiment
For the present invention by studying two pnca gene VII types NDV being clinically separated, it is medium virulence on the weak side to find one plant Strain (WF strains), one plant is velogen strain (SL strains)；By the gene order-checking to them, and the genotype VII announced with NCBI NDV P genes are largely compared, it is found that WF strains are different from other strains in the 237th of P albumen and the 240th amino acids, Speculate that it may be relevant with the virulence of ewcastle disease.P genes according to the sequence pair SL strains of the P albumen of WF strains are transformed, as a result Obtain a plant height degree and cause weak genotype VII newcastle disease vaccine candidate strain.The life of WF strains and improved SL strains is studied respectively Thing characteristic, and make respectively inactivated vaccine be immunized animal after, good immune protective effect can be provided；So as to facilitate this hair It is bright.
The present invention is described in detail with reference to embodiment.
The screening and sequence analysis of the genotype VII NDV street strains of embodiment 1
Two plants of doubtful NDV strains are separated to from clinical onset chicken, inoculated into chick embryo collects allantoic fluid, determines HA potency.Root Primer is designed according to the NCBI NDV announced F gene orders, identifies ewcastle disease strain.Sense primer：NDV F‐F： ATGGGCTCCAAACCTTCTACCAG；Anti-sense primer：NDV F‐R：AAACTGCTGCATCTTCCCAACCG.
The allantoic fluid 250uL collected is taken to extract RNA, reverse transcription according to a conventional method.Expand by primer of NDV F-F/NDV F-R Increase the Partial Fragment of F genes, clip size about 500bp.After nucleic acid electrophoresis, positive band is cut, is reclaimed, connection carrier T sequencing. The sequence between F genes 47nt-420nt is taken to draw phylogenetic trees, the genotype of Analyze ＆ separate strain.For analysis of experiments Strain GenBank accession number is：1‐2(AY935499)、1‐2(AY935500)、18719‐03(GQ288385)、B1(NC_ 002617)、HB92(AY225110)、Herts(AY741404)、Italien(EU293914)、JS‐07(FJ766527)、La sota(AF077761)、NA‐1(DQ659677)、paramyxovirus‐1(AJ880277)、ZJ1(AF431744)、 Mukteswar(EF201805)。
As a result show, two plants of isolated strains are NDV, and the amino acid sequence of F gene cracking sites is112R-R- Q-K-R-F117, meet the feature of velogen strain, further gene evolution, which is analyzed, shows that they are genotype VII strain.HA potency Measure display, the potency of two strain virus chick embryo allantoic liquids are stablized 2 respectively9With 27‐28.The NDV strains of separation are respectively designated as WF Strain and SL strains.Wherein genotype VII NDV low virulent strain WF strains, it was preserved in Wuhan, China, force on March 7th, 2017 The China typical culture collection center of Chinese university, deposit number are CCTCC NO:V201708.
The ewcastle disease SL strains of embodiment 2 and WF strains MDT, ICPI measure
MDT refers to that minimum lethal dose can make the average time of chicken embryo death, and MDT is strong virus force NDV strains less than 60h； MDT values are medium virulence strain between 60h-90h；MDT is low virulent strain more than 90h.With sterile saline by fresh receipts The toxic allantoic fluid obtained is made 10 times and is serially diluted, and takes 10‐6、10‐7、10‐8With 10‐94 dilution factors, 10 age in days chickens are inoculated with respectively Embryo, each dilution factor are inoculated with 5 pieces, per embryo allantois intracavitary administration 0.1mL.Every morning and afternoon respectively according to embryo once, record each The death time of chicken embryo, Continuous Observation 7 days.(the results are shown in Table 1) MDT calculation formula are：
ICPI refers to 1 Japanese instar chickling ICPI.Take 1 age in days SPF chick 15, each intracerebral injection 10‐1Dilution Fresh toxic allantoic fluid 0.05mL.It is another to take 5 each 0.05mL conducts of sterile saline in kind injecting same dose Control.After inoculation, daily in the time observation being accordingly inoculated with, the situation of chick is recorded, point normal (flexibly, action is without altogether for activity Ji detuning phenomena), morbidity (including benumb, sleeping ground does not rise, but do not include only showing blunt chicken) it is and dead.
Observation 8 days, normal, morbidity, the sum of dead chicken are calculated, (be normally 0, fall ill for 1, extremely according to different weights Die 2) to add up gross score.
ICPI is the average value (the results are shown in Table 1) of accumulative total score divided by the cumulative total of normal, morbidity and dead animal.
Table 1：WF strains and SL strains MDT, ICPI measure
The above results show that SL strains meet the feature of velogen strain, consistent with the velogen strain characteristic of F gene cracking sites；And Although the F genes cracking site of WF strains also complies with the feature of velogen strain, but the result of table 1 shows that WF strains belong to medium strain on the weak side, Prove that the virulence of ewcastle disease is not only relevant with F protein, thus it is speculated that may be also relevant with other albumen such as P albumen etc..
Embodiment 3：Ewcastle disease WF strains, the measure of SL strain whole genome sequences
In order to determine whether that p albumen also generates variation, according to the NDV gene orders of NCBI announcements, 9 pairs of primers are designed, There is partial nucleic acid sequence overlapped between adjacent amplified fragments.Primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd, sequence Row are shown in Table 2.
Table 2 expands the primer sequence of NDV SL strain full-length genomes
Conventional method extracts viral RNA, reverse transcription, and above-mentioned primer enters performing PCR amplification, and connection PMD 19-T carriers are sequenced, The sequence of survey compares in NCBI websites, is spliced with Lasergene softwares, obtains WF strains and SL strain complete genome sequences.
As a result show, WF strains and SL pnca gene group total length 15192bp, compared with the strains such as Lasota, in 1646-1647 There are 6 unnecessary bases between bit base, meet the characteristic of genotype VII ewcastle disease strain.
Further the P genes of the P genes of WF strains and SL strains and the NCBI other genotype VII NDVs announced are carried out Compare and find, (its amino acid sequence is seqID NO to WF strain P albumen:1) the 237th and the 240th amino acids and other strains Variant, the amino acid that other strains include the 237th and 240, SL strain P albumen is K/R and K, and WF strains are respectively T and I.
Embodiment 4：The structure of genotype VII new castle disease virus weakening strain
Whether the variation that WF strain P albumen is verified by reverse genetics manipulation technology is exactly to cause original of the WF strains as low virulent strain Because of place.Reverse genetics manipulation technology generally refers to the Genomic cDNA clone by building virus, in culture cell or/and susceptible Again " bring back to life " virus in host, the methods of being inserted or being lacked by gene modification virus genome sequence, disease is carried out with this The development of the functional genome research and new generation vaccine of poison.The NDV of the present embodiment rescue process is by the full-length genome of structure CDNA clone (transcription vector) and helper plasmid (expression vector) cotransfection cells respectively containing NP, P and L gene, are carried in helper plasmid It is provided with the presence of closing enzyme, cDNA clone carries out transcription and the expression of each gene, is finally assembling to the virion of infectious, so The NDV of rescue is obtained come a large amount of amplicon virus by inoculated into chick embryo afterwards.The NDV genomes saved out are ultimately derived from cDNA clone.
Specific step is described below.
1) structure of the cell line of t7 rna polymerase is expressed
The single clone of picking e. coli bl21, test tube shake bacterium, take 400uL bacterium solutions, and 13000rpm/min is centrifuged 10 minutes, Remove supernatant；Centrifuged after being resuspended with ultra-pure water, remove supernatant；It is resuspended with 100uL ultra-pure water, boils 10min, ice bath 10min；From The heart, supernatant is taken as DNA profiling.The BL21 bacterial strain t7 rna polymerase gene orders announced according to NCBI, design primer：T7‐F： 5'CTGCTCGAGCCACCATGAACACGATTAACATCGCTAAGAACGAC 3', T7-R：5' CTGTCTAGATTACGCGAACGCGAAGTCCGACTCTAAGATGT, sense primer I containing Xho restriction enzyme site, anti-sense primer contain Xba I restriction enzyme sites.Enter performing PCR amplification, amplified fragments size about 2.6Kb using the DNA of preparation as template.
Take 5uL PCR primers to carry out nucleic acid electrophoresis identification, if band is correct, remaining PCR primer is tried with nucleic acid purification Agent box is purified.Purified product carries out the reaction of Xba I/Xho I double digestions together with carrier for expression of eukaryon PCI-neo.Recovery Positive band, it is attached with T4DNA Ligase.Conversion, a small amount of extractions of plasmid, digestion identification etc. are carried out according to a conventional method. Positive plasmid serves the sequencing of Hai Shenggong bioengineering Co., Ltd, obtains recombinant plasmid PCI-T7.
With the DMEM medium culture DF1 cells containing 10% hyclone, 60mm Dish culture dishes are uniformly inoculated in, are treated Transfected when cell length is to 70%-90%.2ug PCI-T7 recombinant plasmids are taken, by the requirement of calcium phosphate transfection kit specification Transfect DF1 cells.4h changes the fresh growth medium without G418 into after transfection, changes the G418's containing 400ng/mL into after 24h Growth medium.First passage changes the growth medium of the G418 containing 600ng/mL into, and passage later gradually steps up the dense of G418 Degree, until G418 concentration reaches 1mg/mL.In the generation of cell continuous passage 20, extract RNA, reverse transcription after being handled with DNA enzymatic, with drawing Thing T7-F/T7-R is expanded, and identifies the expression of t7 rna polymerase.
Identified, the cell line of structure, which can be stablized, to pass on, and transfects the sustainable table of t7 rna polymerase gene of acquisition Reach.The cell line of the stable expression t7 rna polymerase of structure is named as DF1-T7.
2) structure of SL strains full length cDNA clone carrier
According to the SL strain whole genome sequences measured, with its restriction enzyme site of the software analysis of Oligo 7., by full genome component Into 7 sections, segmentation is connected on the carrier PBRT by transformation.Wherein, by the 710th in P genes and the mutation transformation of 719 bit bases It is attached again afterwards, the full length cDNA clone carrier of structure is named as PBRT-NDV-P, after sequencing is correct, saves backup.In structure On the basis of the PBRT-NDV-P carriers built, the basic amino acid cracking site that F genes in the 3rd section are represented to velogen strain characteristic is mutated For the sequence of low pathogenicity feature, the full length cDNA clone carrier of structure is named as PBRT-NDV, after sequencing is correct, preserved standby With.Primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd, and primer sequence is shown in Table 3.Wherein CL2-T1/CL2-T2/CL2-T3/ CL2-T4 is F gene mutation primers, and CL2-2F/CL2-T5/CL2-T6/CL2-2R is P gene mutation primers.
Table 3：NDV full-length genome vector construction primer sequences
3) structure of helper plasmid
Restriction enzyme site is introduced in the upstream and downstream of SL strain NP, P and L genes respectively, digestion after PCR amplifications, is connected through identical interior The treated PCI-neo carriers of enzyme cutting, preserve after sequencing, are respectively designated as PCI-NP, PCI-P and PCI-L.The primer is by upper Hai Shenggong bioengineering Co., Ltd synthesizes, and primer sequence is shown in Table 4.
Table 4：NDV helper plasmid structure primer sequences
4) virus rescue
DF1-T7 cells are inoculated in when growing to 70%-80% individual layers in the orifice plates of 35mm six, will transcribe plasmid PBRT-NDV- P and helper plasmid PCI-NP, PCI-P and PCI-L are respectively with 5ug, 2.5ug, 1.25ug, 1.25ug ratio, cotransfection DF1- T7 cell lines, using calcium phosphate transfection kit, operation is carried out by kit specification.Meanwhile plasmid PBRT-NDV will be transcribed Identical amount and method cotransfection DF1-T7 cell lines are pressed with above-mentioned helper plasmid.After transfection 3-5 days, harvest culture supernatant connects The SPF chick embryo allantoic cavities of kind 9-11 ages in days, are cultivated 3-5 days, take chick embryo allantoic liquid to survey HA potency.Harvest HA result of the test positive urines Cyst fluid, -70 DEG C freeze after packing.
The strain of the only P genetic modifications of rescue is named as rSL-P strains, genotype VII NDV rSL-P strains, its Wuhan, China, the China typical culture collection center of Wuhan University are preserved on March 20th, 2017, deposit number is CCTCC NO:V201710；
The strain that the P and F genes of rescue are transformed jointly is named as rSL strains；Genotype VII NDV rSL strains, its Wuhan, China, the China typical culture collection center of Wuhan University are preserved on March 20th, 2017, deposit number is CCTCC NO:V201709；
Transfection liquid harvests allantoic fluid after connecing embryo 4 days, blood clotting (HA) experiment is positive, and chicken embryo potency is 26‐28Between.Harvest Allantoic fluid 1:1000‐1:10000 dilutions, continue to connect embryo, embryo are received after 0.1mL/ pieces, 4-5 days；Continue to be passaged to for the 15th generation.Respectively Take rSL-P strains and rSL strains are primary, the 5th generation, the sequence of the 10th generation and extraction RNA, PCR the amplification mutation of the 15th generation virus allantoic fluid Site (P genes and P/F genes) is simultaneously sequenced, and qualification result is consistent with expected result.Show virus rescue success, and pass on steady Fixed, back mutation does not occur again for the site of transformation.
The chicken embryo median lethal time (MDT) of embodiment 5, new Revive virus (rSL-P strains and rSL strains)
The toxic allantoic fluid newly harvested work is serially diluted for 10 times with sterile saline, takes 10‐7、10‐8、10‐93 dilute Degree of releasing, instar chicken embryo on the 10th is inoculated with respectively, each dilution factor is inoculated with 5 chicken embryos, per embryo allantois intracavitary administration 0.1mL, is connecing daily The same time of kind shines egg 1 time, records the death time of each chicken embryo, and determines HA activity.Continuous Observation 7 days, inoculated into chick embryo All dead highest dilution is minimum lethal dose.
As a result show, the MDT values of rSL-P strains are 108h, and the MDT values of rSL strains are 128h, and concrete outcome is shown in Table 5.
Table 5：The measure of rSL strain chicken embryo median lethal times (MDT)
Embodiment 6, rSL-P strains, the measure (ICPI) of rSL strain ICPIs
Take 1 age in days SPF chick 10, each intracerebral injection 10‐1The fresh toxic allantoic fluid 0.05mL of dilution.It is another take 5 with Each 0.05mL of physiological saline of same method injection dilution virus.
After inoculation, observed daily in corresponding inoculation time, record the situation of chick, point normal, morbidity and death.Observation 8 Day, normal, morbidity, the sum of dead chicken are calculated, (is normally 0, falls ill for 1,2) death is adds up total score according to different weights Number.ICPI is the average value of accumulative total score divided by the cumulative total of normal, morbidity and dead chicken.
As a result show, the ICPI values of rSL-P strains are that the ICPI of 0.9, rSL strains is 0.35, and concrete outcome is shown in Table 6.
Table 6：RSL-P strains, rSL strains ICPI measure
Embodiment 7, rSL-P strains, the measure (IVPI) of rSL strain vein pathogenic index
Take 6 week old SPF chickens 10, each intravenous inoculation 10‐1The toxic allantoic fluid 0.1mL of dilution, separately take 5 inoculation physiology salts Water compares.Observed daily in the corresponding time of inoculation, record is inoculated with chicken situation, point normal, morbidity, paralysis and death.
After observation 10 days, add up normal, morbidity, paralysis and dead animal number, (be normally 0, falling ill is according to different weights 1, benumb as 2,3) death is adds up gross score.IVPI is accumulative total score divided by the cumulative total of normal, morbidity and dead animal.
As a result show, rSL-P strains, the IVPI values of rSL strains are 0, and concrete outcome is shown in Table 7.
With reference to the measurement result analysis of two plants of rescue strain MDT, ICPI and IVPI values, the virulence of rSL-P strains is compared with wild strain The virulence of SL strains has declined to a great extent, and illustrates that two sites of P genes affect the virulence of SL strains really, and passes through P genes and F The rSL strain virulence that gene association transformation obtains declines completely, the characteristic of low virulence strain is complied fully with, as vaccine candidate strain energy More ensure the security that vaccine uses.
Table 7：RSL-P strains, rSL strains IVPI measure
Stability test after embodiment 8, WF strains, rSL-P strains and the inactivation of rSL strain virus and inactivation
Find that WF strains and rSL-P strains, rSL strains are guaranteed on biological safety, have by a series of above-mentioned checkings Potentiality as vaccine candidate strain.
By the fresh viral allantoic fluid (EID50 10 of the WF strains, rSL-P strains, rSL strains of harvest9.5/ 0.1mL) in, add eventually Concentration is 0.1% formalin, 37 DEG C of inactivation 24h, is placed in 4 DEG C of preservations.After inactivation 0h, 24h, 48h, 7 days, 15 My god, 1 month measure HA potency, determine the stability of inactivation of viruses, the results are shown in Table 8.The another stoste taken after inactivation, is inoculated with 10 ages in days 10 pieces of SPF chicken embryos, 0.1mL/ pieces, 37 DEG C of culture 120h, receive embryo and survey potency, examine inactivation completeness.
As a result show, viral relatively stable after inactivation, after placing a period of time, HA potency is not remarkably decreased.And Inactivation is more thorough, and the antigen stock inoculated into chick embryo after inactivation is without death, HA potency total negatives.
Table 8：Antigen HA potency after inactivation
Embodiment 9, WF strains, rSL-P strains, the preparation of rSL strain oil adjuvant killed vaccines and safety testing, steriling test
Virus stock solution used after inactivation, prepares oil adjuvant killed vaccine according to a conventional method.Every group of inactivated vaccine is respectively with 7 ages in days SPF chickens 10, vaccine 1.0mL is subcutaneously injected in every neck, while respectively sets control 5, raises under the same conditions, continuous to see Examine 14, the feeding of record test chicken, drinking-water and clinical setting.As a result show, vaccine assimilation effect is good after being immunized, and immune chicken does not have Any adverse reaction locally and systemically is occurred, the state of chicken is completely normal.
The WF strains, rSL-P strains, rSL strain oil-adjuvant vaccines of preparation are taken, by version in 2010《Chinese veterinary pharmacopoeia》Annex carries out nothing Bacterium is examined, and as a result meets standard, without germ contamination.
Embodiment 10, WF strains, rSL-P strains, the Efficacy evaluation of rSL strain oil adjuvant killed vaccines
21 age in days SPF chickens 50, are randomly divided into five groups, and one group is negative control group, and one group is WF strain vaccine immune groups, and one Group is rSL-P strain vaccine immune groups, and one group is rSL strain vaccine immune groups, and another group is Lasota strain immune groups.Immune group every Vaccine 0.2mL is subcutaneously injected in neck, and the PBS of same dose, immune group and control group isolated rearing is immunized in control group.Respectively at exempting from Take a blood sample within 21 days afterwards, 28 days, separate serum, determine antibody.It is malicious by force with the genotype VII street strain being clinically separated after blood sampling in 28 days Poison, every chicken muscle injection 10 are attacked in WH strains6EID50, isolator raising, is observed 14 days, records the morbidity of chicken and dead feelings daily Condition；Attack after poison the 3rd day, the throat swab and cloacal swab for gathering four groups of immune group chickens connect embryo, detect toxin expelling situation.
As a result show, can produce higher level antibody after immune group is immune 21 days, antibody level is higher within 28 days, as a result sees Shown in table 9.After attacking poison, there is not any death in immune group, and negative control group chicken is all dead after 5 days.Toxin expelling situation shows Show, toxin expelling do not occur in immune WF strains, rSL-P strains, the animal of rSL strain vaccines, and Lasota strains still have toxin expelling phenomenon, are shown in Table 10 Shown in table 11.
Table 9：Antibody titer measure after immune
Table 10：Immune efficacy experimental result
Table 11:Attack the 3rd day virus purification result after poison
In summary, the low virulent strain constructed by the present invention, by being found after they are made into vaccine immunity animal, can be lured Higher neutralizing antibody is given birth in artificial delivery, can resist the infringement of strong virus force genotype VII NDV, have broad application prospects.
SEQUENCE LISTING
<110>Shandong Sinder Technology Co., Ltd.
<120>A kind of construction method of genotype VII NDV low virulent strain
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 395
<212> PRT
<213> 1
<400> 1
Met Ala Thr Phe Thr Asp Ala Glu Ile Asp Asp Ile Phe Glu Thr Ser
1 5 10 15
Gly Thr Val Ile Asp Ser Ile Ile Thr Ala Gln Gly Lys Ser Ala Glu
20 25 30
Thr Val Gly Arg Ser Ala Ile Pro Gln Gly Lys Thr Lys Gly Leu Ser
35 40 45
Thr Ala Trp Glu Lys His Gly Ser Val Gln Pro His Ala Ser Gln Asp
50 55 60
Ala Pro Asp Gln Pro Asp Arg Thr Glu Lys Gln Pro Ser Thr Pro Gly
65 70 75 80
Gln Ala Thr Pro His Asn Asn Pro Pro Ile Thr Ser Thr Glu Pro Pro
85 90 95
Pro Thr Gln Ala Ala Ser Glu Thr Ser Asp Thr Gln Leu Lys Thr Gly
100 105 110
Ala Ser Asn Ser Leu Leu Ser Met Leu Asp Lys Leu Ser Asn Lys Ser
115 120 125
Ser Asn Ala Lys Lys Gly Pro Trp Ser Gly Pro Gln Glu Gly His His
130 135 140
Gln Ser Pro Ala Gln Gln His Gly Asp Gln Pro Ser His Gly Ser Asn
145 150 155 160
Gln Gly Arg Pro Gln His Gln Ala Lys Ala Val Pro Gly Asn Arg Gly
165 170 175
Ile Asp Glu Asn Thr Ala Tyr His Gly Gln Arg Lys Glu Ser Gln Pro
180 185 190
Ser Ala Gly Ala Thr Pro His Ala Pro Gln Ser Gly Gln Ser Gln Asp
195 200 205
Asn Ile Pro Val Pro Val Asp Arg Val Gln Leu Pro Ala Asp Phe Ala
210 215 220
Gln Ala Met Met Ser Met Met Glu Ala Leu Ser Gln Thr Val Ser Ile
225 230 235 240
Val Asp His Gln Leu Asp Leu Val Leu Lys Gln Thr Ser Ser Ile Pro
245 250 255
Met Met Arg Ser Glu Ile Gln Gln Leu Lys Thr Ser Val Ala Ile Met
260 265 270
Glu Ala Asn Leu Gly Met Met Lys Ile Leu Asp Pro Gly Cys Ala Asn
275 280 285
Val Ser Ser Leu Ser Asp Leu Arg Ala Val Ala Arg Ser His Pro Val
290 295 300
Leu Val Ser Gly Pro Gly Asp Pro Ser Pro Tyr Val Thr Gln Glu Gly
305 310 315 320
Glu Met Thr Leu Asn Lys Leu Ser Gln Pro Val Gln His Pro Ser Glu
325 330 335
Leu Ile Lys Ser Ala Thr Ala Ser Gly Pro Asp Met Gly Val Glu Lys
340 345 350
Asp Thr Val Arg Ala Leu Ile Thr Ser Arg Pro Met His Pro Ser Ser
355 360 365
Ser Ala Lys Leu Leu Ser Lys Leu Asp Ala Ala Lys Ser Ile Glu Glu
370 375 380
Ile Arg Lys Ile Lys Arg Leu Ala Leu Asn Gly
385 390 395
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atggctactt ttacagatgc ggagatagat gacatatttg agaccagcgg gactgtcatt 60
gatagcataa ttacggccca gggcaaatca gctgagaccg tcggaagaag cgcgatcccg 120
cagggcaaga ccaaaggtct aagcacagca tgggagaagc acgggagtgt ccagccacac 180
gccagtcagg acgcccctga ccaaccagac agaacagaaa aacagccatc cacacctggg 240
caggcgactc cacacaacaa cccgccgatc acatccactg aaccgccccc cactcaggcc 300
gcaagcgaga ccagcgacac acagctcaaa accggagcaa gcaactccct tctgtccatg 360
ctcgacaaat tgagcaataa atcgtctaat gctaaaaagg gcccatggtc gggtccccaa 420
gaagggcatc accaatctcc ggcccaacaa cacggggacc agccgagcca tggaagcaac 480
cagggaagac cacagcacca ggccaaagcc gtccctggaa accggggcat agacgagaac 540
acagcatatc atggacaacg gaaggagtca caaccatcag ctggtgcaac ccctcatgcg 600
ccccagtcag ggcagagcca agacaatatt cctgtacctg tggatcgtgt ccagctacct 660
gccgactttg cgcaggcgat gatgtctatg atggaggcat tatctcagac ggtaagtata 720
gttgatcatc agctggacct agtcttgaaa cagacatcct ctattcctat gatgcgatct 780
gaaatccaac agctcaagac atctgttgcg atcatggaag ctaacttagg catgatgaaa 840
attctggacc ctggttgtgc caacgtttca tccttaagtg atctccgggc agtagcccga 900
tcccacccag tcctagtttc aggccccgga gacccatctc cttacgtgac acaagaaggt 960
gaaatgacgc tcaataaact ctcacaacca gtgcagcacc cttctgaatt gattaagtct 1020
gccaccgcaa gcgggcctga catgggagtg gagaaggaca ctgtccgcgc attaatcacc 1080
tcacgcccga tgcatccaag ctcctcggct aagctcctga gcaagctaga tgcagccaag 1140
tcaattgaag agatcaggaa gattaaacgc cttgcgctga atggttga 1188

Claims (5)

  1. A kind of 1. method of structure VII type NDV low virulent strains, it is characterised in that described method is by VII type new cities Epidemic disease poison 237 of P albumen and the amino acid of 240 sport threonine T and isoleucine I respectively.
  2. 2. the method as described in claim 1, it is characterised in that described method, be come structure by reverse genetic manipulation method Build VII type NDV low virulent strains.
  3. 3. method as claimed in claim 2, it is characterised in that described method includes the steps：
    The cell line of construction expression t7 rna polymerase,
    The three auxiliary recombinant plasmids needed during the rescue of genotype VII NDV and a full-length genome restructuring are provided Carrier, three auxiliary recombinant plasmids include NP albumen, P albumen and the L GFPs of NDV respectively；Wherein P albumen The amino acid of 237 and 240 is respectively T and I；
    Three auxiliary recombinant plasmids and a full-length genome recombinant vector are transferred in the cell line of expression t7 rna polymerase, Culture obtains genotype VII NDV low virulent strain.
  4. 4. method as claimed in claim 3, it is characterised in that described cell line, be by the gene of expression t7 rna polymerase DF1 cell constructions are transfected after being connected with carrier PCI-neo.
  5. 5. the method as described in claim 1, it is characterised in that described method, be by genotype VII NDV When the amino acid of 237 of P albumen and 240 sports T and I respectively, F protein is also subjected to hypotoxicity mutation.
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