CN106047823A - Bivalent vaccine strain for recombining and expressing NCV HN gene by virtue of IBV H120 strain based on reverse genetic technique - Google Patents

Bivalent vaccine strain for recombining and expressing NCV HN gene by virtue of IBV H120 strain based on reverse genetic technique Download PDF

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CN106047823A
CN106047823A CN201610502356.4A CN201610502356A CN106047823A CN 106047823 A CN106047823 A CN 106047823A CN 201610502356 A CN201610502356 A CN 201610502356A CN 106047823 A CN106047823 A CN 106047823A
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lasota
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infectious bronchitis
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王红宁
杨鑫
周泷
周英顺
门帅
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Sichuan University
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Abstract

The invention discloses a R-H120-Lasota (HN) bivalent vaccine saving strain obtained by replacing a 5a gene (Figure 1) of H120 strain with a HN gene of poultry Newcastle disease virus based on a constructed poultry infectious bronchitis virus H120 reverse genetic system. The recombinant vaccine strain can simultaneously well protect IBV standard toxin counteracting strains and Newcastle disease standard toxin counteracting strains and can be used for preventing the poultry infectious bronchitis virus and the poultry Newcastle disease virus.

Description

A kind of based on Reverse Genetics by IBV H120 strain recombinant expressed NDV HN gene Bigeminy vaccine strain
Technical field
The present invention relates to animal medicine bioengineering field, be that a kind of avian infectious bronchitis virus H120 is recombinant expressed Fowl Avian pneumo-encephalitis virus Lasota strain HN gene vaccine.
Background technology
The infectious bronchitis of fowl (IB) that avian infectious bronchitis virus (IBV) cause, is the weight of harm aviculture One of big infectious disease, with respiratory symptom, laying hen egg drop reduction 30-50%, Egg Quality decline, nephropathy and gastrointestinal disease Become principal character.At this disease sickness rate 100% of China, mortality rate 10-30%, the domestic economic loss caused every year is more than 1,000,000,000 Unit.Poisonous carrier is to utilize recombinant DNA technology to be inserted through inside the viral genome of transformation by exogenous gene, and postoperative infection is thin Born of the same parents, make exogenous gene at intracellular effective expression.Viral vector is used for epidemic disease as a kind of instrument being usually used in molecular biology The directions such as Seedling research.The maturation of Reverse Genetics, has expedited the emergence of out many RNA viral vector, and Zhao H etc. is strong at NDV respectively Strain, low virulent strain 4 different genes between insert exogenous gene, find that the duplicating efficiency of virus and titre be not by substantially Impact, it was demonstrated that NDV is feasible as vaccine carrier.2001 is recombinant expressed in Avian pneumo-encephalitis virus by reverse genetic Bird flu hemagluttinin proteins, the Revive virus of the restructuring constructed by discovery can induce the humoral antibody to bird flu virus is strong to exempt from Epidemic disease reaction has complete protective effect to mouse lethal dose bird flu virus.Huang Z H etc. are successively with restructuring NDV Infectious bursal disease virus VP2 is expressed in Lasota strain, attacks NDV virulent strain and infectious bursa of Fabricius virus highly virulent strain Hit and all can play the best protection.Within 2007, Joshua M and PANS utilizes Avian pneumo-encephalitis virus sick as vector expression SARS Poison vaccine constructed by S gene is inoculated in the SARS virus that laboratory animal carries out doubling dosage, finds in virus multiple in pulmonary The content of hayday processed will be well below comparison.Within 2008, Man-Seong Park application ND viral vector expresses bird flu H5, H9 hemagglutinin gene is configured to vaccine, finds the protective effect not only to newcastle with lethal dosage, simultaneously to fowl Influenza virus has good protective effect simultaneously.2007 breathe out beasts grinds Ge etc. by reverse genetic means by AIV H5N1 HA base Because being inserted in NDV Lasota, forming Combined vaccine can be to homology or the Avian pneumo-encephalitis virus of allos, high pathogenic avian influenza There is immanoprotection action completely.
Summary of the invention
The avian infectious bronchitis virus H120 reverse genetics system that the present invention builds according to the success of this laboratory is base Plinth, replaces H120 strain 5a gene by fowl NDV HN chimeric gene, it is thus achieved that R-H120-Lasota(HN) rescue strain.
Accompanying drawing explanation
Fig. 1: H120 recombinant expressed NDV HN gene construction strategy figure.
Detailed description of the invention
1, avian infectious bronchitis virus H120 strain reverse genetics system used by the present invention is Sichuan University's animal epidemic disease Sick prevention and control build with food safety Key Laboratory of Sichuan Province.The passage cell BHK21 used is that Sichuan University's animal epidemic is prevented Control preserves with food safety Key Laboratory of Sichuan Province.PCR high-fidelity enzyme, restricted enzyme BsaI, BsmB I, NciI, T4 Ligase is that pMD-19T reclaims test kit purchased from Dalian treasured biology company limited glue and purchases purchased from NEB company, PCR high-fidelity enzyme It is that Invitrogen Corp. completes from TIANGEN Co., Ltd, design of primers and sequencing.
2, design primer: the fowl NDV HN chimeric gene that design primer amplification is transformed, in order to replace avian infectious 5a gene in bronchitis virus H120 strain reverse genetics system, construction strategy is shown in Fig. 1.
3, RNA extracts: using Trizol method to extract its Lasota strain full-length genome RNA, its process is: take 200 μ L Avian infectious bronchitis virus liquid adds 750 μ L TRIzol Reagent (Invitrogen, Carlsbad, CA)；Mixed Even, to place 5 min, add 200 μ L chloroforms for 15-30 DEG C, acutely vibration 15 s, room temperature is placed 3 min, 4 DEG C of 12000 rpm and is centrifuged 10 min, take upper strata aqueous phase, proceed in new centrifuge tube, are slowly added to 1 times of volume 70% isopropanol, mixing, place 10-20 Min, 4 DEG C of 12000 rpm are centrifuged 30 s, abandon waste liquid；4 DEG C of 12000 rpm is centrifuged 2 min, removes residual liquid.Add 30 μ L RNase-free water, room temperature is placed 2 min, 4 DEG C of 12000 rpm and is centrifuged 2 min, collects centrifuged supernatant, take 5 μ L in Electrophoresis is carried out on the agarose gel of 1.0%.Other saves backup in-70 DEG C.The synthesis of cDNA, uses precious biological reverse transcription examination Agent box primeScripts, illustrates to carry out according to test kit, its reaction system Buffer 2 μ L, oligdT 0.5 μ L, Radome 0.5 μ L, RNA template, 37 degree Celsius of 15 min, 85 DEG C of 5 s.Fowl NDV HN chimeric gene PCR obtains, with CDNA is template, adds KOD plus polymerase (Toyobo, Japan).Polymerase, upstream and downstream primer, dNTP are carried out Amplified reaction.The reaction system of PCR is: 10 × buffer 5 μ L, 4 × dNTP mix(2.5 mmol/L) 4 μ L, upstream and downstream are drawn The each 2 μ L of thing (20 μm ol/L), Mgcl23 μ L, cDNA 2 μ L, ddH2O 31.75 μ L, 0.25 μ L.Response parameter is: 94 DEG C 5 min, 25 circulations, 94 DEG C of 30 s, 60 DEG C, 30 s, 72 DEG C of 2 last 72 DEG C of 10 min of min.PCR primer exists 1.0% agarose gel electrophoresis is observed, and amplified band size is consistent with expected result.
PCR primer purification, agarose gel electrophoresis, cuts purpose band, carries out pure by using glue to reclaim test kit Change and reclaim, it is thus achieved that the fowl NDV HN chimeric gene PCR fragment of purification.
The clone of PCR fragment, by as follows to the fowl newcastle disease virus HN PCR fragment of purification and pMD-19T coupled reaction, PCR primer 4.5 μ L, pMD-19T carrier 0.5 μ L solution I 5 μ L, mixing, 16 DEG C overnight, will connect product and convert DH5a competent cell, on the LB flat board containing ampicillin after 37 DEG C of cultivation 16 h, at the LB containing ampicillin The single bacterium colony of picking on flat board, inoculates in 5 mL fluid mediums, 37 DEG C, cultivates, and then chooses positive bacterium solution 2-3 and serves Hai Shenggong checks order, if comparison analysis result sequence is consistent with GenBank, shows to clone successfully.Alkaline lysis extracting matter Grain DNA, by sub-for identified positive colony mass propgation, alkaline lysis method of extracting plasmid.
The enzyme action of purpose cDNA fragment reclaims: the plasmid being loaded with HN fragment is carried out BsmB I enzyme action
Enzyme action system is BsmB I:NEB buffer 35 μ L, BSA(10mg/Ml) 0.5 μ L, BsmB I 2 μ L, plasmid 20 μ L, ddH2O 22.5 μ L, total system is 50 μ L.55 DEG C of incubation 8 h.
4, the external structure of full-length cDNA
Use the avian infectious bronchitis virus H120 strain reverse genetics system set up, reconnect improved total length cDNA。
5, the in vitro transcription of cell adaptation H120 strain cDNA and the acquisition of recovered virus
In vitro transcription test kit is transcribed: take about 200 ng purified connection product as template, use mMESSAGE MMACHINE T7 Ultra Kit carries out in vitro transcription, and reaction system is as follows:
(1) full-length cDNA in vitro transcription system: 10 × T7 Reaction Buffer 2 μ L, T7 2 × NTP/ARCA 10 μ L, GTP 3 μ L, Template DNA5 μ L, T7 Enzyme Mix 2 μ L, 20 μ L
(2) N-3 ' cDNA in vitro transcription system: 10 × T7 Reaction Buffer 2 μ L, T7 2 × NTP/ARCA 10 μ L, GTP 3 μ L, Template DNA 5 μ L, T7 Enzyme Mix 2 μ L
After 37 DEG C of incubation 2 h, add 1 μ L TURBO DNase, incubation 15 min.-70 DEG C of preservations.
6, electric shock transfection
(1) when cultivating bhk cell to monolayer, use 0.25% trypsinization, add 8 mL DMEM complete mediums, suspend thin Born of the same parents.4 DEG C of 400 g is centrifuged 3 min.Abandon supernatant, add 10 mL and do not contain FBS, the DMEM re-suspended cell of mycillin, centrifugal.
(2) re-suspended cell is in without FBS, the DMEM of mycillin, makes cell concentration be about 1 × 107/mL。
(3) cell is put 10 min in ice bath, take 0.8 mL and proceed in 0.4 mL electric shock cup, by 1 μ g transcription and cell Careful mixing.
(4) carrying out electroporation, use Gene Pulser Xcell system, shock parameters is: voltage 400 v, pulse duration 25 ms, pulse number 3, pulse spacing 0.5 s.
(5) after electric shock terminates, electric shock cup is put 5 min in ice bath, proceed to, in culture bottle, add cultivation after being diluted by cell Liquid dilutes 20 times, cultivates and observes for 37 DEG C.
(6) cell culture is passed Embryo Gallus domesticus and gather in the crops its chick embryo allantoic liquid.And chick embryo allantoic liquid is stored in-70 DEG C of refrigerators.
7, the qualification of reverse genetic rescue strain R-H120-Lasota (HN)
(1) silent mutation is identified
Respectively to the Cell sap of 48 h after transfection with Cell sap is accessed the chick embryo allantoic liquid RT-PCR obtained in Embryo Gallus domesticus identify, Primer amplified region covers the silent mutation site introduced, a length of 584 bp of target fragment, and primer sequence is:
IBV-H-R-F:5 '-AATATAAGACAGAGCACAAG-3 '
IBV-H-R-R:5 '-CTGTCATACAAAGCAGCACTACA-3 '
(2) HN gene identification
5-ATGGACCGCGCCGTTAGCCA-3
5-CGTCTCCTAGCCAGACCTGGCTTCTCTA-3, target length 1800 bp
(3) IFA identifies
BHK21 cell is incubated at 96 porocyte culture plates, puts 37 DEG C, 5% CO2 calorstat is cultivated 12-24 h, treat that cell is long To the monolayer of 80%-90%, discard culture fluid, wash 2 times with DMEM stock solution.100TCID will be diluted50IBV add thin In the corresponding plate hole of born of the same parents' culture plate, every hole 50 μ L, set the normal cell of not virus inoculation as blank simultaneously.Virus is allowed to inhale Attached 1 h, shakes up 1 time every 20 min therebetween, then all plate holes adds cell maintenance medium to 200 μ L, be placed in 37 DEG C, 5% CO2Incubator is cultivated 6 h.Discarding the cell maintenance medium in Tissue Culture Plate, every hole adds 70% acetone of-20 DEG C of pre-coolings 100 L, fix 15 min, discard fixative, dry for 4 DEG C, and this Tissue Culture Plate can use or put in-20 DEG C of refrigerators immediately Preserve, standby.Close: being washed 3 times by the Tissue Culture Plate secured with PBS, every hole adds 1% BSA 100 μ L, and room temperature is put Put 30 min, then wash 3 times with PBS.Add that 50 μ L have diluted one resists in cultivating plate hole, and cell culture incubator is hatched suitable Working as the time, PBS vibration washing 3 times, each 5 min, be subsequently adding that 50 μ L have diluted two resist in cultivating plate hole, at incubator Interior lucifuge hatches appropriate time, PBS vibration washing 3 times, each 5 min.Microscopy: show specific yellow green in the cytoplasm of infection Immunofluorescence can determine that as the positive, does not show that specific yellow green immunofluorescence is then judged to feminine gender.
8, reverse genetic rescue strain R-H120-Lasota (HN) genetic biology characteristic
(1) rescue strain R-H120-Lasota (HN) growth curve in Embryo Gallus domesticus be there was no significant difference with parent strain H120.
(2) extract continuously the rescue of 10 generations strain RNA, RT-PCR expand smoothly rescue strain R-H120-Lasota (HN) and Parent's strain N gene, amplifies the specificity purpose fragment of about 490 bp size smoothly.And amplify R-H120-smoothly NDV-HN/ (5a) strain HN gene, shows that saving strain R-H120-Lasota (HN) can stablize heredity.
(3) rescue strain R-H120-Lasota (HN) HA measures
Rescue strain R-H120-Lasota (HN) with positive control H120 strain through clostridieum welchii process after can coagulation chicken red Cell, titre is 211, and undressed R-H120-Lasota (HN) and positive control Lasota energy coagulation chicken red blood cell, blood Solidifying valency is 211。
(4) rescue strain EID50 measures
R-H120-Lasota (HN) rescue strain inoculation SPF Embryo Gallus domesticus shows the classical symptom infecting IBV, hypoevolutism, short Little, roll up.Calculating by Reed-Muench method, the EID50 of H120 strain is EID50=10-7/ 0.2mL, R-H120-Lasota (HN) strain EID50 is 10-6.8EID50/0.2mL.Strain EID50 is different without significant difference with parent's strain in rescue.
(5) rescue strain pathological change (CPE) in passage cell CK cell
After R-H120-Lasota (HN) strain inoculation CK cell, it is possible to make CK cell produce obvious cytopathy (CPE), de- Fall, dead.

Claims (3)

1. an avian infectious bronchitis virus H120 recombinant expressed fowl Avian pneumo-encephalitis virus Lasota strain HN gene vaccine strain Constructing technology, it is characterised in that: use Reverse Genetics, by avian infectious bronchitis virus H120 strain to fowl newcastle Virus Lasota strain HN expresses, and builds the Combined vaccine strain R-with avian infectious bronchitis virus H120 as carrier H120-Lasota(HN).
R-H120-Lasota(HN the most according to claim 1) vaccine strain, it is characterised in that: according to No see'M strategy Design primer, the upstream and downstream primer at amplification HN gene introduces BsmB I restricted enzyme, and initiates at HN gene
Codon ATG is previously incorporated GeneEnd, Genestar, korzea " AGAAAAAATACGGGTAGAACACTAGTCCGCCA CCA ",
Replace the 5a reading frame of H120 strain with the HN gene expanded and obtain rescue strain R-by Reverse Genetics H120-Lasota(HN).
The most according to claim 1, the R-H120-Lasota(HN that rescue obtains) vaccine strain, its biological property is: institute The R-H120-Lasota(HN obtained) stably can pass in Embryo Gallus domesticus；Can stably express fowl newcastle HN albumen；IFA energy Immunofluorescence enough detected；Rescue strain R-H120-Lasota (HN) with through clostridieum welchii process after can coagulation chicken red carefully Born of the same parents, titre is 211；EID50 is determined as 10-6.8EID50/0.2 mL；After R-H120-Lasota (HN) strain inoculation CK cell, CK cell can be made to produce obvious cytopathy (CPE)；IBV standard counteracting toxic substances strain and newcastle standard counteracting toxic substances strain are had There is good protective effect, can be used for preventing avian infectious bronchitis virus and fowl Avian pneumo-encephalitis virus.
CN201610502356.4A 2016-07-01 2016-07-01 Bivalent vaccine strain for recombining and expressing NCV HN gene by virtue of IBV H120 strain based on reverse genetic technique Pending CN106047823A (en)

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CN108009401A (en) * 2017-11-29 2018-05-08 内蒙古大学 A kind of method for screening finger-print genetic marker
CN109207492A (en) * 2018-09-26 2019-01-15 四川大学 A kind of recombination, recombinant expression plasmid, recombinant Lactobacillus and its application comprising IBV multi-epitope EpiC and NDV F gene
CN110184287A (en) * 2019-05-24 2019-08-30 华南农业大学 A kind of method and its application of preparation and reorganization virus
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107190022A (en) * 2017-06-28 2017-09-22 浙江大学 A kind of method of rapid build avian infectious bronchitis virus reverse genetic strain
CN107190022B (en) * 2017-06-28 2020-10-30 浙江大学 Method for quickly constructing reverse genetic strain of avian infectious bronchitis virus
CN108009401A (en) * 2017-11-29 2018-05-08 内蒙古大学 A kind of method for screening finger-print genetic marker
CN108009401B (en) * 2017-11-29 2021-11-02 内蒙古大学 Method for screening fingerprint genetic markers
CN109207492A (en) * 2018-09-26 2019-01-15 四川大学 A kind of recombination, recombinant expression plasmid, recombinant Lactobacillus and its application comprising IBV multi-epitope EpiC and NDV F gene
CN109207492B (en) * 2018-09-26 2021-12-28 四川大学 Recombinant gene containing IBV multi-epitope EpiC and NDV F genes, recombinant expression plasmid, recombinant lactobacillus and application thereof
CN110184287A (en) * 2019-05-24 2019-08-30 华南农业大学 A kind of method and its application of preparation and reorganization virus
CN110184287B (en) * 2019-05-24 2024-01-30 华南农业大学 Method for preparing recombinant virus and application thereof
CN112852758A (en) * 2021-02-07 2021-05-28 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Recombinant Newcastle disease virus for expressing avian infectious bronchitis virus S protein and preparation method and application thereof

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