CN104962580A - Recombinant plant rhabdovirus vector and construction method thereof - Google Patents

Abstract

The invention discloses a recombinant plant rhabdovirus vector and a construction method thereof. The recombinant plant rhabdovirus vector comprises a modified plant rhabdovirus genome, a transcription unit which is induced newly and a heteroduplex nucleotide sequence, wherein the transcription unit is inserted into the plant rhabdovirus genome to express the heteroduplex nucleotide sequence, the heteroduplex nucleotide sequence is replaced into a glycoprotein transcription unit of a recombinant plant rhabdovirus; the recombinant plant rhabdovirus has copying and infecting capacity, and one antigenic polypeptide or other protein is coded by the heteroduplex nucleotide sequence. The virus expression vector is the first one which can express a negative-sense RNA virus vector of foreign protein on plants, and the recombinant plant rhabdovirus vector has the advantages that the expression quantity is high, the expression stability is good, a relative long foreign gene segment can be inserted, and inserted foreign gene segment is not prone to loosing; the recombinant plant rhabdovirus vector can be used for expressing of multiple kinds of foreign protein and can also be used for preparing animal vaccines, and wide application values and application prospects are achieved.

Description

A kind of recombinant plant Rhabdovirus vector and construction process thereof

Technical field

The present invention relates to plant genetic engineering and viral molecular biology field, particularly, the present invention relates to a kind of plant and bear adopted RNA rhabdovirus---the construction process of sonchus yellow net virus recombinant virus expression vector and the method for expression alien gene.

Background technology

Along with the development of plant genetic engineering, plant is utilized to produce as bio-reactor the concern that foreign protein is more and more subject to people, research in recent years confirms, botanical system has the ability of expression activity mammalian proteins, and shows clear superiority at quality product, cost and secure context.In plant, expression alien gene mainly contains two kinds of approach: one with transgenic method, exogenous origin gene integrator is entered Plant Genome to carry out stably express, another kind is that foreign gene is inserted viral genome, utilizes plant viral vector system to carry out the expression of foreign protein.

Compared with transgenic method, utilize plant virus-based expression vector to express foreign protein and there is simple and quick, with low cost, foreign protein obtains high level expression with virus replication feature, therefore, since phase early 1980s proposes to utilize plant viral vector system to make plant bioreactor, develop multiple plant viral vector system at present, comprise DNA virus expression vector system and plant positive chain RNA virus expression vector system (Peng Yan, 2002).Due to RNA comparatively DNA difficulty operation, initial be mostly DNA virus for plant viral vector system, as cauliflower mosaic virus (cauliflower moscia virus, CaMV), but find that the complicacy that DNA virus copies makes it be not suitable for high level expression foreign protein (Porta etc. subsequently, Biotechnol Genet Eng Rev, 2002,19:245-291).Along with discovery and the widespread use of reversed transcriptive enzyme, the operation of RNA is made to become easy, positive chain RNA virus is also used to the research of virus expression carrier, as tobacco mosaic virus (TMV) (Tobacco mosaic virus, TMV), cowpea mosaic virus (Cowpea mosaic virus, CPMV), potato virus X (Potato virus X, (the Gleba etc. such as PVX), Cur Opin Plant Biol, 2004,7:182-188.).

Although utilize plant positive chain RNA virus virus vector to express foreign protein have very high utility value, but its application still has certain limitation, be mainly manifested in the following aspects: the positive chain RNA virus vector stabilisation one, carrying exogenous sequences is poor, gene recombination is fast virus expression carrier instability and loses the major cause of foreign gene.Two, external source Insert Fragment limited size, the maximum plant viral vector of current foreign gene-carrying is PVX carrier, and expressed foreign gene is b galactosidase gene (GUS), and size is 1800 base pairs.Three, safety issue, virus is one of important pathogen of plant, although virus vector is modified, can not cause serious symptoms or by Vector transmission, but still there is certain environmental safety hidden danger in releasing virus carrier, therefore, the application of plant virus-based expression vector is greatly limited (Gleba etc., Curr Opin Biotechnol, 2007,18:134-141).

Find in the research of minus-stranded rna virus, this viroid may be outstanding gene delivery system, compared with positive chain RNA virus, its advantage is mainly manifested in three aspects: one, the genome of this viroid is always wrapped up by capsid protein, never exist with in cell with exposed form, thus recombination frequency is extremely low, has good genetic stability good; Reading frame on the genome of two, non-minus-stranded rna virus is stage by stage modularization distribution, is easy to insert external source reading frame, and virus particle be linearly, can hold larger exogenous genetic fragment; Three, such virus vector has higher biological safety; existing result of study shows, the mutant virus that deleted virus coating forms albumen can not form high propagated virus (Bukreyev etc., J Virol in the process of cells infected; 2006,80:10293-12306; Pfaller etc., Virology, 2015,479:331-344).Therefore, if transform plant minus-stranded rna virus as virus expression carrier, set up a kind of novel plant virus-based expression vector system, be expected to overcome the defect that plant positive chain RNA virus carrier exists at present, have great importance.

But, there is no the report of plant minus-stranded rna virus reverse genetics in the world, genetic manipulation cannot be carried out to this viroid.

Sonchus yellow net virus (sonchus yellow net virus, SYNV) belong to Rhabdoviridae ( rhabdoviridae) nucleus Rhabdovirus ( nucleorhabdovirus), be the negative adopted RNA viruses (Jackson etc., Annu Rev Phytopathol, 2005,43:623-660) of non-segmented negative (nonsegmented).SYNV virus particle is shaft-like or bullet shaped, and typical virus particle is about 248 nm, diameter 70 nm, and virus particle has obvious regular cross band and axle core, helical symmetry, virus particle tool coating, and envelope membrane surface has the long spike projection of about 6 nm.SYNV full-length genome about 13.7 kb, each gene order of encoding is followed successively by 3'-N-P-sc4-M-G-L-5', comprise 5 kinds of structural protein, nucleocapsid protein (Nucleocapsid respectively, N), phosphorylated protein (Phosphoprotein, P), stromatin (Matrix protein, M), glycoprotein (Glycoprotein, and the large subunit of RNA polymerase (Large subunit of polymerase G), L), wherein, N protein, P albumen and L albumen and geneome RNA are combined closely, composition ribonucleoprotein cone mixture (ribonucleoprotein core complex, RNP), RNP core is by M protein encapsulation, and M protein outer layer is by membrane bound, adventitia is primarily of deriving from the adipose membrane of host cell and the G-protein composition be dispersed in adipose membrane that interts.In addition, this virus is also encoded 1 Nonstructural Protein sc4, infers that its function is motor protein.The genomic two ends of SYNV comprise 3'-leader and 5'-trailer sequence respectively.Leader and trailer sequence is non-protein encoding sequence, but transcribes for the genome duplication of SYNV and mRNA and play important regulating and controlling effect.In transcription, with virus genome RNA (vRNA) for template, the mRNA of leader mRNA and 6 encodes viral protein is transcribed out successively from 3' end, leader mRNA not proteins encoded, article 6, the mRNA that encodes transcribes from genomic 3 ' end successively to 5 ' end, and the amount of transcribing is successively decreased gradually from N to L.First copying of geneome RNA be the positive-sense strand RNA(cRNA of templated synthesis and its complete complementary with vRNA), cRNA and N, P and L tri-core proteins combine the RNP being formed and comprise cRNA, cRNA-RNP as template again for the synthesis of vRNA.Form vRNA-RNP after vRNA capsidation, vRNA-RNP, by interacting with M albumen and G-protein, is assembled into complete virion (Jackson etc., Annu Rev Phytopathol, 2005,43:623-660) further.

Summary of the invention

In order to overcome the deficiencies in the prior art, the invention provides a kind of recombinant plant Rhabdovirus vector and construction process thereof.

A kind of recombinant plant Rhabdovirus vector, comprising: an a) plant rhabdovirus genome modified; B) transcriptional units newly introduced, it is inserted into the nucleotide sequence of expressing heterologous in plant rhabdovirus genome; C) heterologous nucleic acid sequence, it is displaced to the glycoprotein transcriptional units of described recombinant plant rhabdovirus, wherein said recombinant plant rhabdovirus has copies and infection ability, and described heterologous nucleic acid sequence is encoded an antigenic polypeptide or other albumen.

The plant rhabdovirus genome of described modification is a sonchus yellow net virus carrier modified.

Described heterologous nucleic acid sequence encoding green fluorescent protein, or encoding green fluorescent protein and b galactoside enzyme fusion proteins.

Wherein said heterologous nucleic acid sequence is the antigen protein deriving from animal virus.

Described heterologous nucleic acid sequence inserts the intergenic region of sonchus yellow net virus with independently transcriptional units.

Described sonchus yellow net virus is the deletant of glycoprotein gene, and wherein said exogenous nucleic acid sequences is inserted into raw sugar protein gene coding frame.

The anti-genomic isolated nucleic acid molecule of described sonchus yellow net virus of encoding is the mosaic in one or more genomes or anti-genome source.

Described recombinant plant Rhabdovirus vector, can infect plant host, comprise sonchus oleraceus ( sonchus oleraceus), lettuce ( lactuca sativa), the cross-fertilize seed of Ben Shi cigarette (Nicotiana benthamiana), Nicotiana glutinosa (Nicotiana glutinosa), kirschner cigarette (Nicotiana clevelandii), Nicotiana glutinosa and kirschner cigarette, youth-and-old-age ( zinnia elegans), Herba Bidentis Bipinnatae ( bidens pilosa) and lamb's-quarters wheat ( chenopodium quinoa).

Described recombinant plant Rhabdovirus vector can be expressed and the polypeptide of production external source insertion nucleic acid sequence encoding in vegetable cell.

A kind of method building recombinant plant Rhabdovirus vector described in any one.

The invention has the beneficial effects as follows: the sonchus yellow net virus that restructuring 1) can be produced after described recombinant virus inoculation at least one host plant in a large number; 2) described recombinant virus comprises the expression cassette of foreign gene, and can transcribe with recombinant virus and copy, and express foreign protein efficiently and stably, the foreign protein of expression can reach 94 kD; 3) utilize recombinant virus expression vector provided by the invention can express foreign protein in plant more than at least one, without the need to the instrument, simple to operate, reproducible of costliness, have broad application prospects.4) recombinant virus expression vector that the present invention relates to can express at least two foreign genes simultaneously, and by increasing transcriptional units, can express multiple foreign gene.

Accompanying drawing explanation

Fig. 1. SYNV transcription vector pSYNV, SYNV recombinant virus expression vector pSYNV-GFP, pSYNV-GUS-GFP, pSYNV-GFP-G/DsRed build schematic diagram; (A) pSYNV is SYNV cDNA transcription vector, under being controlled by 35S promoter, has HDV_Rz ribozyme in the downstream of cDNA, can shear removing at the non-viral sequence held by transcribe rna product 3 '; Restructuring GFP virus vector pSYNV-GFP, GFP expression cassette is inserted between N and P gene as independent transcription unit; (B) GUS-GFP virus vector pSYNV-GUS-GFP, fusion gene GUS-GFP is recombinated as independent transcription unit between N and P; (C). express the recombinant viral vector pSYNV-GFP-G/DsRed of GFP and DsRed, GFP is between N and P, and DsRed substituted for the G gene of virus itself simultaneously.

Fig. 2. RNP core protein and silencing suppressors expression vector schematic diagram; (A) RNP core protein expression vector pGD-NPL, N, P and L are controlled by different 35S promoters, and the expression cassette of three albumen inserts same plasmid; (B) RNA silencing suppressors expression vector pGD-HC-Pro, pGD-P19 and pGD-γ b, HC-Pro, P19 and γ b is inserted into different expression vector plasmids.

Fig. 3. the symptom that recombinant viral vector pSYNV-GFP inoculation Ben Shi cigarette produces and GFP expression.A left side is part A, and the right side is part B, the symptom after (A) recombinant virus clone pSYNV-GFP and infectious clone pSYNV inoculation Ben Shi cigarette under visible ray and ultraviolet lamp; (B) RT-PCR detection and western-blot analyze the stability analysis of recombinant virus SYNV-GFP offspring GFP.

Fig. 4. recombinant viral vector pSYNV-GUS-GFP inoculates Ben Shi symptom that cigarette produces and GFP detection of expression.Be divided into ABC tri-part, the symptom after (A) recombinant virus clone pSYNV-GUS-GFP and pSYNV-GFP inoculation Ben Shi cigarette under visible ray and ultraviolet lamp; (B) western-blot detects GFP expression.(C) SYNV virus vector of recombinating expresses fluoroscopic examination and the GUS Activity determination of GUS-GFP fusion rotein.

Fig. 5. the virus symptoms that recombinant viral vector pSYNV-GFP-G/DsRed inoculation Ben Shi cigarette produces and GFP, DsRed detection of expression.Be divided into AB two portions, the symptom after (A) recombinant virus clone pSYNV-GFP-G/DsRed and pSYNV-GFP inoculation Ben Shi cigarette under visible ray and ultraviolet lamp; (B) western-blot detects GFP and RFP expression.

Embodiment

SYNV recombinant viral vector provided by the invention imports corresponding agrobacterium strains respectively to helper plasmid carrier, this agrobacterium strains mix in specific proportions rear inoculation at least more than one host plant can produce restructuring sonchus yellow net virus in a large number, recombinant virus comprises more than one exogenous gene expression frame, these foreign genes can be transcribed with recombinant virus and be copied, and express foreign protein efficiently and stably, simple to operate, reproducible.

It is carry out on the basis of SYNV infectious clone that SYNV recombinant virus expression vector builds, SYNV infectious clone comprises three class plasmid vectors, one is that cDNA containing full-length gene group coding information transcribes plasmid vector, two is protein expression plasmid carriers of the accessory protein of composition RNP core, and three is the expression plasmid carriers of RNA silencing suppressors albumen from other viruses.

The structure of SYNV recombinant viral vector in fact only needs to transcribe on the basis of plasmid vector at SYNV infectious clone cDNA, transform, Reconstruc-tion policy comprises: 1) insert single or multiple independently exogenous gene expression frame, 2) more than one gene is passed to independently expression cassette and insert SYNV genome, 3) one or more genes of SYNV self are replaced with foreign gene, 4) foreign gene is inserted upstream or the downstream of certain gene of SYNV self by the mode of gene fusion, such as, merge in the upstream of N gene or downstream.5) arbitrary combination of four kinds of vector modification strategy two or more strategies any more than.

1) and 2) described on position can be any one or more positions in SYNV genome in multiple position.These on position are before or after any one expression cassette, such as, can be inserted in before or after N gene expression frame, and the expression cassette referred to here is that the positions transcription initiation of this gene is to all sequences between Transcription Termination position.

In SYNV genome, all protein coding genes include transcriptional initiation sequence and transcription termination sequence, intervening sequence between any two genes is the transcription termination sequence of a gene above and the transcription termination sequence of a rear gene, in addition in the middle of these two kinds of sequences, two conservative cytosine(Cyt)s (CC) are the absolute separation lines of any two genes, these two bases there will not be in the transcriptional initiation sequence or transcription termination sequence of any mRNA, and these three kinds of sequences intergenic are collectively referred to as intergenic region arbitrarily.Therefore, foreign gene is with above-mentioned 1) or 2) independent expression cassette form is when inserting SYNV genome, the corresponding sequence of foreign gene and extra transcriptional initiation sequence must be inserted simultaneously, CC, and transcription termination sequence, i.e. an intergenic region, here intergenic region (the NP gene junction between N and P is selected, NPJ) be example, but selected intergenic region including but not limited to NPJ, can be the multiple intergenic regions on SYNV genome any one.

In addition, in transcriptional initiation sequence and transcription termination sequence, the several bases separately near CC are comparatively conservative, can not lack, and other non-conserved sequences can convert or lack.Such as, intergenic region between N and P just comprises the transcription termination sequence of N gene, the transcriptional initiation sequence of CC and P gene (namely from after the terminator codon of N to one section of sequence before P initiator codon) comes to 119 nt, but only has the conserved sequence of 16 bases (tataagaaaaaCCaac) to be all that absolute conservation is consistent between any two genes.Therefore selecting, in the intergenic region of inserting, at least must comprise the sequence that these are conservative, and other sequences can be suddenlyd change, brachymemma or lengthening.

In a word, the exogenous gene expression frame of insertion be made to observe the genes encoding strategy of SYNV genome itself.

(insert a NPJ while inserting GFP gene) for independent insertion between foreign gene (such as GFP gene) expression cassette to N and P gene, the building process of SYNV recombinant virus expression vector is as follows:

1) the SYNV Full_length cDNA of amplification is cloned into plant expression vector, obtains SYNV cDNA transcription vector plasmid pSYNV;

2) foreign gene (as GFP gene, comprising a NPJ) is inserted in pSYNV carrier, obtain the recombinant vectors (as pSYNV-GFP) comprising exogenous gene expression frame;

3) plant expression vector of SYNV ribonucleoprotein RNP core protein N, P and L is built;

4) plant expression vector from the RNA silencing suppressors albumen of other viruses is built;

5) by electric shock method by above-mentioned 2) ~ 4) described in vector plasmid import agrobacterium strains GV3101 respectively;

6) the bacterium liquid process before inoculation: the Agrobacterium inoculation 20 mL YEP liquid nutrient medium (containing 25 μ g/mL Rifampins, 50 μ g/mL kantlex, 25 μ g/mL gentamicins) comprising above-mentioned vector plasmid, 28 DEG C of 220rpm shake bacterium and are cultured to OD ₆₀₀for 0.8-1.2, through 12,000 rpm 1min is centrifugal, abandons supernatant, with containing 10 mM MgCl2,10 mM MES, the infiltration damping fluid Eddy diffusion thalline of 200 mM Syringylethanones, and adjustment OD ₆₀₀be about 1.0, leave standstill 2-3 hour;

7) Agrobacterium combination: specific Agrobacterium is mixed in desired concn ratio according to required;

8) infiltrate inoculation: the plant selecting the 4-6 leaf fully extended seedling age phase, utilize the 1 ml syringe not with syringe needle to infiltrate at the plant leaf back side in 3-4 week, a strain plant generally inoculates 3-4 sheet leaf;

9) inoculate plant and be placed in 25 DEG C of Isolation warm houses cultivations, within 20 days, observe the symptom of inoculation plant afterwards, the system lobus cardiacus gathering the plant producing virus symptoms and do not produce virus symptoms carries out RT-PCR detection, identifies whether have virus infection.

10) extract disease plant total protein, utilize the modes such as western blot to detect the protein expression level of foreign gene.

Below in conjunction with embodiment and accompanying drawing, the invention will be further described.

Embodiment 1. SYNV cDNA transcription vector builds

SYNV genome is mononegavirale RNA, is about 13.7 kb.Extract total serum IgE from the Ben Shi Tobacco Leaves having infected SYNV, and with carry out reverse transcription PCR for template, obtain Full_length cDNA.By full length cDNA clone to plant expression vector pCB301, namely obtain SYNV cDNA transcription vector, called after pSYNV, pSYNV import Agrobacterium GV3101 by electric shock.

The building process infecting pSYNV transcription vector comprises the steps:

1) from the Ben Shi Tobacco Leaves infecting SYNV, total serum IgE is extracted:

Get 0.1 g blade, add liquid nitrogen grind into powder; By powder fast transfer to 1.5 mL centrifuge tubes of precooling, add rapidly 1 mL TRIzol extract of precooling; Mix up and down, abundant cracking, room temperature places 5 min; Add 0.2 mL chloroform, cover tightly centrifuge tube lid, concuss 15 sec, room temperature places 2 min; 4 DEG C, centrifugal 15 min of 12,000 rpm, get supernatant and add the Virahol of equivalent volumes, put upside down mixing, room temperature places 10 min; 4 DEG C, centrifugal 10 min of 12,000 rpm; Abandon supernatant; Add 1 mL 75% ethanol, vortex washing and precipitating; 4 DEG C, centrifugal 5 min of 7,500 rpm; Abandon supernatant, residue white precipitate is the blade total serum IgE just carried, and makes RNA throw out at room temperature dry; Add 30 μ L without the water of RNA enzyme, RNA to be precipitated all to dissolve.

2) full-length cDNA amplification

Design primer SYNV/1F and SYNV/1R(primer sequence are in table 1), and with the blade total serum IgE extracted for template amplification SYNV Full_length cDNA.Article two, 5 ' end of primer is each with 15 bases (lowercase), these bases respectively can with the 35S promoter of carrier and HDV_Rz ribozyme complementary pairing, the cDNA product two ends of corresponding amplification are respectively with these bases, cDNA with these Extra bases sequences can be inserted into carrier pCB301 by In-Fusion cloning reaction, insert position between 35S promoter and HDV_Rz ribozyme, the transcription vector plasmid called after pSYNV (Fig. 1 .A) obtained.

3) screening positive clone

Utilize restriction enzyme to carry out enzyme and cut qualification, the errorless clone of qualification is cut to enzyme and checks order, determine that SYNV full-length genome is 13726 nt;

4) by the method for electric shock, pSYNV is imported agrobacterium strains GV3101.

Embodiment 2. RNP core protein expression vector establishment

The ribonucleoprotein RNP core of SYNV comprises N, P and L tri-kinds of albumen, these three kinds of albumen are inserted in same expression vector simultaneously, obtain pGD-NPL, in this expression vector, three albumen are controlled by different 35S promoters and NOS transcription terminator, that is same expression vector contains three expression cassettes, express N, P and L(Fig. 2 .A respectively).

Above-mentioned four expression vectors are shocked by electricity respectively and are imported Agrobacterium GV3101.

Embodiment 3. viral RNA silencing suppressors protein expression vector builds

Three from the RNA silencing suppressors coded by other viruses: marmor erodens (Tobacco etch virus, TEV) Hc-Pro gene, tomato bushy stunt virus (Tomato bushy stunt virus, TBSV) P19 gene and barly strip mosaic virus (Barley stripe mosaic virus, BSMV) γ b gene.These three silencing suppressors are inserted into pGD carrier, obtain three expression vectors: pGD-Hc-Pro, pGD-P19 and pGD-γ b(Fig. 2 .B).

Above-mentioned three silencing suppressors expression vectors are shocked by electricity respectively and are imported Agrobacterium GV3101.

Embodiment 4. SYNV-GFP recombinant viral vector builds

Inserted in SYNV genome with the form of independent expression cassette by GFP, on position can be any one on SYNV genome in multiple position, but on position must be in the upstream of any one encoding gene of SYNV or downstream.

To insert between N and P gene by GFP expression cassette in detail, the building process of pSYNV-GFP is described in detail.Being NP intergenic region (NP gene junction, NPJ) between N and P, in order to follow above-mentioned SYNV genes encoding strategy, while inserting GFP, additionally inserting a NPJ.Here select NPJ to be example, but selected intergenic region includes but not limited to NPJ, can be the multiple intergenic regions on SYNV genome any one, and these intergenic regions can suddenly change or brachymemma or lengthening.

With SYNV-GFP-DsRed minireplicon(Genbank Accession:JN038403) for template, design primer GFP-NPJ/NcoI/F and GFP-NPJ/NcoI/R(primer sequence are in table 1) sequence between amplification GFP to NP gene junction, article two, primer respectively carries NcoI, and amplified production inserts and cuts in the pSYNV carrier of process through same enzyme after NcoI enzyme is cut.NcoI in pSYNV is positioned at the initiator codon place of P gene, the front and back of GFP are made respectively to have a NPJ herein afterwards so GFP-NPJ fragment is inserted, namely respectively there is a NPJ between N and GFP and between GFP and P, make the GFP expression cassette of insertion follow the genes encoding strategy (Fig. 1 .A) of SYNV self like this.

Screening positive clone also carries out enzyme and cuts qualification, cuts the errorless clone of qualification check order to enzyme, determines that GFP expression cassette inserts between N and P gene with correct direction;

By the method for electric shock, pSYNV-GFP is imported agrobacterium strains GV3101;

The structure of embodiment 5. pSYNV-GUS-GFP recombinant viral vector

GUS and GFP is inserted SYNV genome with the form of fusion gene (GUS-GFP), and on position can be any one on SYNV genome in multiple position, but on position must be in the upstream of any one encoding gene of SYNV or downstream.

Insert between N and P gene for GUS-GFP, the building process of pSYNV-GUS-GFP is described in detail in detail.Equally, insert GUS-GFP fusion gene also to need to insert an intergenic region simultaneously, here NPJ is selected to be example, but selected intergenic region includes but not limited to NPJ, can be any one of the multiple intergenic regions on SYNV genome, and these intergenic regions can suddenly change or brachymemma or lengthening.

Design primer GUS/NcoI/F and GUS/R(primer sequence are in table 1, and wherein 5 ' the end of GUS/R carries out adding phosphorus reaction, and GUS/NcoI/F is with NcoI restriction enzyme site), amplification GUS.Design primer GFP/F and GFP-NPJ/NcoI/R(primer sequence are in table 1, wherein 5 ' the end of GFP/F carries out adding phosphorus reaction, GFP-NPJ/NcoI/R is with NcoI restriction enzyme site), with SYNV-GFP-DsRed minireplicon(Genbank Accession:JN038403) for template, the sequence between amplification GFP to NPJ.Two sections of PCR primer directly carry out Ligation in vitro reaction, to connect product for template, and carry out pcr amplification with GUS/NcoI/F and GFP-NPJ/NcoI/R, product (i.e. GUS-GFP-NPJ) is cut with NcoI enzyme, insert in the pSYNV carrier cut through through NcoI enzyme, obtain pSYNV-GUS-GFP.NcoI in pSYNV is positioned at the initiator codon place of P gene, the front and back of GUS-GFP are made respectively to have a NPJ herein afterwards so GUS-GFP-NPJ fragment is inserted, namely respectively there is a NPJ between N and GUS-GFP and between GUS-GFP and P, make the GUS-GFP expression cassette of insertion follow the genes encoding strategy (Fig. 1 .B) of SYNV self like this.

Screening positive clone also carries out enzyme and cuts qualification, cuts the errorless clone of qualification check order to enzyme, determines that GUS-GFP expression cassette inserts between N and P gene with correct direction;

By the method for electric shock, pSYNV-GUS-GFP is imported agrobacterium strains GV3101;

Embodiment 6. pSYNV-GFP-G/DsRed

The G gene of recombinant virus is replaced to DsRed gene by the basis of pSYNV-GFP described in embodiment 4.G-protein is a kind of glycoprotein, to tomato spotted wilf virus (tomato spotted wilt virus, TSWV) find in research, in G-protein, nonsynonymous mutation (C1375A) does not affect the systemic infection of TSWV, but can not be propagated by amboceptor thrips, illustrate that plant bears the G-protein possibility involved in insect amboceptor of adopted RNA viruses to the propagation of virus.

In the present embodiment, the recombinant virus formed after G-protein being replaced to DsRed still systematically can infect the host plant of more than at least one, but recombinant virus probably can not effectively by insect Vector transmission, limit recombinant virus in natural diffusibility, effectively improve the biological safety of recombinant viral vector.

Concrete building process is as follows:

Design primer G/DsRed/F1 and G/DsRed/R1(primer sequence are in table 1), take pSYNV as template amplification PCR fragment S1, design primer G/DsRed/F2 and G/DsRed/R2 be template amplification PCR fragment S2 with pSYNV, design primer DsRed/F and DsRed/R amplification DsRed gene, carry out In-Fusion after three PCR primer reclaim to merge single stage method be connected with the pSYNV-GFP cutting process through PmlI and BstBI enzyme, namely obtain final carrier pSYNV-GFP-G/DsRed(Fig. 1 .C).

Screening positive clone also carries out enzyme and cuts qualification, cuts the errorless clone of qualification and checks order, determine that G gene is correctly replaced by DsRed to enzyme;

By the method for electric shock, pSYNV-GFP-G/DsRed is imported agrobacterium strains GV3101;

Embodiment 7. Agrobacterium is cultivated and pre-treatment before inoculation

The Agrobacterium comprising above-mentioned cDNA transcription vector plasmid or protein expression vector inoculates 3 mL YEP liquid nutrient mediums (containing 25 μ g/mL Rifampin+50 μ g/mL kantlex+25 μ g/mL gentamicins) respectively, and it is 0.8-1.2 that 28 DEG C of 220rpm shake bacterium overnight incubation to OD600; Centrifugal through 12000 rpm 1 minute, abandon supernatant, with containing 10 mM MgCl2,10 mM MES, the infiltration damping fluid Eddy diffusion thalline of 200 mM Syringylethanones, adjustment OD600 is about 1.0, leaves standstill 2-3 hour;

Embodiment 8. agroinfiltration inoculation plant

After the Agrobacterium combination in table 2 and the mixing of required Agrobacterium concentration, infiltrate inoculation Ben Shi cigarette respectively, inoculation plant selects the 4-6 leaf phase to be advisable.Infiltrating to inoculate uses the 1 ml disposable syringe not with syringe needle to carry out blade back infiltration at 4-6 leaf phase plant leaf, and every strain plant infiltrates 3-4 sheet blade, and after inoculation, plant is placed in 25 DEG C of Isolation warm houses cultivations.

Embodiment 9. sonchus yellow net virus of recombinating expresses GFP gene

The Ben Shi cigarette that pSYNV infectious clone (Agrobacterium inoculation combination one) is inoculated showed virus symptoms at about 20 days, and symptom comprises the serious last volume of lobus cardiacus, gathered the lug (Fig. 3 A) of epirelief in blade face.Gather recombinant viral vector pSYNV-GFP inoculate and show the system leaf of virus symptoms, extract leaves total protein, Western Blot analysis is carried out respectively by SYNV polyclonal antibody and GFP monoclonal antibody, result shows that the accumulation volume of SYNV-GFP recombinant virus in system leaf and wild-type virus SYNV do not have notable difference, and special GFP band (Fig. 3 B) also can be detected in the Ben Shi Tobacco Leaves of inoculation recombinant viral vector pSYNV-GFP.In order to verify the stability of external source gene insert after viral persistence infects and goes down to posterity in SYNV recombinant virus expression vector, gather recombinant viral vector pSYNV-GFP to inoculate and the system leaf showing virus symptoms carries out friction goes down to posterity, found that, after 5 continuous passages, GFP still stable existence, in SYNV genome, and obtains stably express (Fig. 3 B).

Embodiment 10. sonchus yellow net virus of recombinating expresses GFP-GUS fusion gene

Recombinant viral vector pSYNV-GFP (Agrobacterium inoculation combination two) and pSYNV-GUS-GFP(Agrobacterium inoculation combination three) Ben Shi cigarette, the virus symptoms formed and pSYNV infectious clone inoculate the symptom produced does not have significant difference (Fig. 4 A).Gather recombinant viral vector pSYNV-GUS-GFP inoculate and show the system leaf of virus symptoms, extract leaves total protein, Western Blot analysis is carried out by GFP monoclonal antibody, result shows the GUS-GFP fusion rotein band that 94 kD sizes can be detected in the Ben Shi Tobacco Leaves of inoculation recombinant viral vector pSYNV-GUS-GFP, and contrasts the GFP band (Fig. 4 B) that 27 kD sizes can be detected in the Ben Shi Tobacco Leaves of pSYNV-GFP inoculation.In order to detect the expression of GUS, choosing frank system leaf and carrying out staining analysis, all can occur GUS staining reaction in the master pulse of discovery system leaf and secondary arteries and veins peripheral cell, and be consistent (Fig. 4 C) with the expression pattern of GFP.

Embodiment 10. sonchus yellow net virus of recombinating expresses GFP and DsRed gene simultaneously

The recombinant virus SYNV-GFP-G/DsRed simultaneously expressing GFP and DsRe clones (inoculation combination four) inoculation Ben Shi cigarette plant, and the virus symptoms formed and pSYNV-GFP infectious clone inoculate the symptom produced does not have significant difference (Fig. 5 A)

Gather recombinant viral vector pSYNV-GFP-G/RFP inoculate and show the system leaf of virus symptoms, extract leaves total protein, Western Blot analysis is carried out respectively by GFP monoclonal antibody and RFP monoclonal antibody, result show inoculate recombinant virus clone pSYNV-GFP-G/RFP Ben Shi Tobacco Leaves in GFP and RFP band can be detected simultaneously, and contrast pSYNV-GFP inoculation Ben Shi Tobacco Leaves in GFP band (Fig. 5 B) only can be detected.

SEQUENCE LISTING

 

<110> Zhejiang University

 

<120> recombinant plant Rhabdovirus vector and construction process thereof

 

 

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<170> PatentIn version 3.5

 

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<220>

<223> GFP-NPJ/NcoI/F

 

<400> 3

catgccatgg tgagcaaggg cgag 24

 

 

<210> 4

<211> 26

<212> DNA

<213> Artificial Sequence

 

<220>

<223> GFP-NPJ/NcoI/R

 

<400> 4

catgccatgg cctagacaaa taatac 26

 

 

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence

 

<220>

<223> GUS/NcoI/F

 

<400> 5

catgccatgg tgttacgtcc tgtagaaacc 30

 

 

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence

 

<220>

<223> GUS/R

 

<400> 6

ttgtttgcct ccctgctg 18

 

 

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

 

<220>

<223> GFP/F

 

<400> 7

atggtgagca agggcgagga 20

 

 

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence

 

<220>

<223> G/DsRed/F1

 

<400> 8

aacgacgtag gtcacgtgtc agtc 24

 

 

<210> 9

<211> 47

<212> DNA

<213> Artificial Sequence

 

<220>

<223> G/DsRed/R1

 

<400> 9

ctcggaggag gccattacga aaagttctta ataataaact atcaaag 47

 

 

<210> 10

<211> 36

<212> DNA

<213> Artificial Sequence

 

<220>

<223> G/DsRed/F2

 

<400> 10

cacctgttcc tgtaaatcca ccccataaac acgacc 36

 

 

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence

 

<220>

<223> G/DsRed/R2

 

<400> 11

gatgtgacac caacgttcga at 22

 

 

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

 

<220>

<223> DsRed/F

 

<400> 12

atggcctcct ccgagaacgt 20

 

 

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence

 

<220>

<223> DsRed/R

 

<400> 13

ttacaggaac aggtggtgg 19

 

 

Claims (10)

1. a recombinant plant Rhabdovirus vector, is characterized in that, comprising: an a) plant rhabdovirus genome modified; B) transcriptional units newly introduced, it is inserted into the nucleotide sequence of expressing heterologous in plant rhabdovirus genome; C) heterologous nucleic acid sequence, it is displaced to the glycoprotein transcriptional units of described recombinant plant rhabdovirus, wherein said recombinant plant rhabdovirus has copies and infection ability, and described heterologous nucleic acid sequence is encoded an antigenic polypeptide or other albumen.

2. recombinant plant Rhabdovirus vector as claimed in claim 1, is characterized in that, the plant rhabdovirus genome of described modification is a sonchus yellow net virus carrier modified.

3. recombinant plant Rhabdovirus vector as claimed in claim 1, is characterized in that, described heterologous nucleic acid sequence encoding green fluorescent protein, or encoding green fluorescent protein and b galactoside enzyme fusion proteins.

4. recombinant plant Rhabdovirus vector as claimed in claim 1, it is characterized in that, wherein said heterologous nucleic acid sequence is the antigen protein deriving from animal virus.

5. recombinant plant Rhabdovirus vector as claimed in claim 2, is characterized in that, described heterologous nucleic acid sequence inserts the intergenic region of sonchus yellow net virus with independently transcriptional units.

6. recombinant plant Rhabdovirus vector as claimed in claim 2, it is characterized in that, described sonchus yellow net virus is the deletant of glycoprotein gene, and wherein said exogenous nucleic acid sequences is inserted into raw sugar protein gene coding frame.

7. recombinant plant Rhabdovirus vector as claimed in claim 2, it is characterized in that, the anti-genomic isolated nucleic acid molecule of described sonchus yellow net virus of encoding is the mosaic in one or more genomes or anti-genome source.

8. recombinant plant Rhabdovirus vector as claimed in claim 1, is characterized in that, can infect plant host, comprise sonchus oleraceus ( sonchus oleraceus), lettuce ( lactuca sativa), the cross-fertilize seed of Ben Shi cigarette (Nicotiana benthamiana), Nicotiana glutinosa (Nicotiana glutinosa), kirschner cigarette (Nicotiana clevelandii), Nicotiana glutinosa and kirschner cigarette, youth-and-old-age ( zinnia elegans), Herba Bidentis Bipinnatae ( bidens pilosa) and lamb's-quarters wheat ( chenopodium quinoa).

9. recombinant plant Rhabdovirus vector as claimed in claim 1, is characterized in that, described recombinant plant Rhabdovirus vector can be expressed and the polypeptide of production external source insertion nucleic acid sequence encoding in vegetable cell.

10. one kind builds the method for the recombinant plant Rhabdovirus vector as described in any one of claim 1-9.