CN102933713A - Viral-based transient-expression vector system that allows multiple applications - Google Patents
Viral-based transient-expression vector system that allows multiple applications Download PDFInfo
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Abstract
Disclosed herein are viral vectors suitable for transfection into woody trees for purposes of delivering and expressing beneficial genes. Specifically exemplified herein are vectors for transfecting citrus trees. The vectors allow for the expression of useful proteins, such as those that can protect the tree from disease. Specifically exemplified herein are methods of transfecting woody trees that allow multiple applications of vectors while avoiding superinfection exclusion.
Description
The cross reference of related application
The application requires the right of priority of No. the 61/440th, 445, the U.S. Patent application of No. the 61/321st, 970, U.S. Patent application submitting on August 8th, 2010 and submission on February 8th, 2011 according to United States Code the 35th piece the 119th (35USC 119).
Invention field
Initial embodiment and the transient expression based on virus of invention are carrier related, and this carrier can be expressed alien gene for a long time in subsystem, thereby allow at one time or after a while together individual subsystem to be used a similar carrier.Other embodiment are relevant with virus vector structure and method, and these methods can avoid getting rid of double infection, thereby make the application variation of carrier.
Background technology
The used common tool of molecular biology of plants experiment in the world based on the transient expression carrier of virus, it can express fast or reticent plant in gene.They also are the important tool in the Research of Plant Genomics, can screen the position sequence of certain function.But, can only from the similar virus of herbal limited quantity, develop available carrier.More famous example has based on the carrier of tobacco mosaic virus (TMV) (TWV) (Dawson et al., 1989; Donson et al., 1991; Shivprasad et al., 1999; Rabindran and Dawson, 2001).The challenge of xylophyta is more special.Even there is the carrier can infect trees, system infects and the required time of expressing gene in the trees analyzed has generally all exceeded the stability of known carrier based on virus.But the challenge that reproduction restriction and improvement required time decades of trees bring has increased the demand of useful carrier based on virus.
Oranges and tangerines Te Lisidecha virus (CTV) is a member in the complicated long line virus family, the virus of this family has the genome of single minute, two minutes and three minutes, by the Some Insects carrier transfer, comprises aphid, Bemisia tabaci and mealybug (Bar-Joseph et al., 1979; Dolja et al., 1994; Agranovsky, 1996; Karasev, 2000).The crooked virion of the length of CTV (2000nm X 10-12nm) is coated by two coat protein: main coat protein (CP) covers about 97% virion, and non-staple coat protein (CPm) is responsible for another end is wrapped in the shell.The single stranded RNA gene of CTV is approximately 19.3kb, is divided into 12 open reading frame (ORFs) (Pappu et al., 1994; Karasev et al., 1995) (Fig. 1).The polyprotein matter of a 349kDa size of ORF 1a coding, this albumen comprise proteolytic enzyme zone and class methylferase and the class helicase zone of two class papoids.The translation of polyprotein is considered to by proceeding in the a+1 frameshit of the synthase zone of birdsing of the same feather flock together (ORF 1b).ORFs 1a and 1b add that the non-coding end all is that protoplastis copies necessary (Satyanarayana et al., 1999).Ten 3 ' ORFs express (Hilf et al., 1995 by 3 ' altogether terminal subgene (sg) mRNAs; Karasev et al., 1997).Except two coat proteins, p65 (HSP70 homology) and p61 are that effective virosome assembling is required, and it is required to be that virus goes down to posterity between protoplastis, thereby enrich budder (Satyanarayana et al., 2000) that lemon infects.P6 protein is that plant infection is required, and p20 and p23 protein and CP are inhibition (Lu et al., 2004) of RNA silence simultaneously.
CTV can infect and be mobile in the citrus mutation of some deleted part virogenes.CTV comprises five genes in the unwanted genomic 3 ' part in virosome copies or forms, i.e. p6, p33, p18, p13 and p20.P33, p18 and p13 are conservative in other members of this type of virus groups, thereby and are considered to evolve and guarantee special interaction with the Citrus host.We find that the deletion among p33, p18 or the p13ORF all can cause respectively virus infection, amplification and the transmission capacity between citrus that not serious losing (Tatineni et al., 2008) occurs.And the deletion of arbitrary combination can make virus infect on the whole citrus in p33, p18 or the p13 gene, comprises the deletion of all three genes.The virus type that deletion among the CTV sudden change of Green Fluorescent Protein and p33ORF or p33, p18 and the p13ORF proves the Dispersal behavior and distribution of these deletion variations and wild-type seemingly.
In double infection eliminating or homology interference, the infection of preexist can stop the superinfection with individual or close virus, but can not affect irrelevant Viral infection.This phenomenon is at first by McKinney (McKinney, 1926; 1929) between two genotypic tobacco mosaic virus (TMV)s (TMV), find, subsequently also in phage (Dulbecco, 1952; Visconti, 1953) the middle discovery.Since then, often also in animal (Adams and Brown, 1985; Bratt and Rubin, 1968; Delwart and Panganiban, 1989; Geib et al., 2003; Johnston et al., 1974; Karpf et al., 1997; Lee et al., 2005; Singh et al., 1997; Steck and Rubin, 1966; Strauss and Strauss, 1994; Whitaker-Dowling et al., 1983; Wildum et al., 2006) and plant (Bennett, 1951; Fulton, 1978; Gal-On and Shiboleth, 2005; Hull and Plaskitt, 1970; Hull, 2002; Lecoq et al., 1991; Salaman, 1933; Walkey et al., 1992) observe this phenomenon in the virus.In plant virology, homology is disturbed the detection that is used as at first viral dependency, is used for determining two virus isolated strains with a virus " strain " or is representing different virus (McKinney, 1929; Salaman, 1933).Subsequently; this technology is developed into a management tool; by with the moderate virus strain isolated on purpose infection plant reduce the infection of more serious virus isolated strain and the loss that causes; thereby reduced the loss of farm crop; this is called as " cross protection " and (sees Gal-On and Shiboleth; 2005 and Hull, 2002 summary).
Description of drawings
(A) is the sketch of wild-type CTV T36 (T36CTV9R) gene organization among Fig. 1.(B) and (C) the hybrid structure diagram that is respectively Δ p33CTV structure and is replaced by front proteolytic enzyme zone.Blank box indicating ORF and translation product thereof.PRO, class papoid zone; MT, methylferase; HEL, helicase; RdRp, the RNA polymerase that RNA relies on; HSP70h, the HSP70 homologue; CPm, non-staple coat protein; CP, major cat protein matter.Black surround represents respectively T68-1 sequence alternative in the T36 genome.Arrow is pointed out the position of p33 ORF deletion.
Fig. 2 has shown the diagram that is entered into the alternative hybrid virus of T36 genome from T68-1 strain isolated (black surround) proteolytic enzyme zone.Below: infect the T36 strain isolated or infect separately the propagation of hybridization L1L2h (the 2nd, 3 road) detection virus in plant; What the 4th road represented is the propagation of L1L2h in the plant of the pre-T36 of infection.React to analyze virus amplification by reverse transcription PCR, 1 cover primer is wherein arranged in each reaction mixture: a cover is T36 proteolytic enzyme specificity, and another set of is regional for the proteolytic enzyme of T68, thereby distinguishes T36 and L1L2h.
Detailed Description Of The Invention
Virus is tended to prevent double infection by relevant virus.Adding generally can stop this carrier of use or related vector in same trees based on the Instantaneous Carrier of virus.The inventor recognizes also now that target organism is infected to be had after the similar carrier, sometimes adds carrier and still have meaning in target organism (for example trees or plant).For example, lost the alien gene of expressing if the inventor finds a plant, can add so carrier will be very necessary; If finding a better gene expresses in trees; If and/or need to express a more than gene.Equally, the inventor recognizes that also the carrier that is necessary a viral strain is applied in the plant that has infected the wild-type that this strain is arranged.The inventor finds that the corresponding section of virus vector is made with clearly defined objective modification can avoid double infection to get rid of phenomenon.
Corresponding embodiment of the present invention described herein is that they can carry out double infection in by the plant of the virus infection of a similar strain based on the virus structure of oranges and tangerines Te Lisidecha virus (CTV) and the long line style virus of a positive chain RNA structure.The inventor finds can solve the problem that double infection is got rid of by the virus structure that modification wild-type CTV virus is constructed, such as, allow their front proteolytic enzyme be substituted by the front proteolytic enzyme sequence of a different virus kind.
According to other specific embodiment, the present invention also has been related to the virus structure based on other oranges and tangerines Te Lisidecha virus (CTV) and the long line style virus of a positive chain RNA structure, the double infection based on the CTV virus structure of introducing after these viruses can realize.The inventor finds can't provide protection to the infection of wild-type virus by the virus structure that modification wild-type CTV virus is constructed, and lacks the gene of encoding function p33 protein such as these viruses.When continuous plant was buddered the virus structure that use to lack p33 protein and wild-type CTV, the plant primary infection had the virus structure of deletion variation, and this subinfection is on the not significant impact of the foundation of the superinfection of wild-type virus.Thereby deletion p33ORF can produce a CTV " non-cross protection " variation.
Before, the inventor has checked the relation of oranges and tangerines Te Lisidecha virus (CTV) different genotype between other virus isolated strain double infection abilities of prevention.Only have the double infection eliminating just can occur between the strain isolated with a CTV strain.When carrying out in-order plant with the strain isolated with strain when buddering, first infection can provide comprehensive protection of dangerous strain isolated.An example noticing the complete intersection protection between the CTV virus be in primary infection belong in the plant with the wild-type CTV of T36 strain, the CTV that expresses GFP infects and is suppressed fully.
According to an embodiment, the inventor has checked based on the double infection of the virus structure of the infection cDNA clone structure of the T36 strain of CTV and has got rid of.The result shows, can not provide double infection to get rid of for wild-type virus infects by the virus structure of revising wild-type CTV virus structure, for example these viruses lack the gene of encoding function p33 protein or a front proteolytic enzyme zone surrogate that comes from other CTV strains (T68) are arranged.When a kind of virus structure wherein is used to buddering for the first time of citrus, use afterwards wild-type CTV to budder, the variant viral of plant primary infection is on the not significant impact of the superinfection of wild-type virus.Thereby, alternative " non-cross protection " variation that can produce CTV in p33ORF deletion and front proteolytic enzyme zone.These virus structures can be used as plant vector, thereby realize the variation application.
In another embodiment, invention is related to a virus vector structure, and this virus structure front proteolytic enzyme of being designed to be used in the strain isolated that comes from a viral kind different lines commonly used substitutes the endogenous front proteolytic enzyme of virus vector herein.The strain of CTV is defined as according to the different lines (Hilf et al., 2005) of 1a ORF nucleotide sequence analysis virus on the system.Use this definition, T36 and T68 are strains.Each Virus Sample is called as one strain isolated in these strains.Each strain is named as " type strain isolated ", and by forming with the less strain isolated of type member sequence difference.At one more specifically among the embodiment, virus vector is the CTV strain isolated that front proteolytic enzyme sequence is replaced by the strain isolated of another CTV strain.One than previous more specific embodiment in, the CTV carrier is according to T36 strain strain isolated structure, its front proteolytic enzyme sequence is substituted by the front proteolytic enzyme sequence of a strain isolated of T68CTV strain.At one more specifically among the embodiment, replaced proteolytic enzyme sequence is class papoid zone.
According to another embodiment, invention with one to alleviate the budder method of the CTV virus vector double infection eliminating that brings of continuous virus relevant.The method comprise allow target plant budder first p33ORF loses or destroyed CTV virus vector, thereby make first CTV viral vector infection target plant and produce a plant that has infected, then allow this plant that has infected second CTV virus vector of buddering, this carrier or be with or without the p33 gene of pss gene or destroyed.Second CTV virus vector can infect infected plant.In specific embodiment, target plant is citrus.In another specific embodiment, first and/or secondary CTV carrier be configured to comprise the expressible nucleotide sequence of a coding heterologous protein.
According to another embodiment, invention with one to alleviate the budder method of the CTV virus vector double infection eliminating that brings of continuous virus relevant.The method comprises allows target plant in succession budder first and second virus vector.First virus vector be configured to a front proteolytic enzyme sequence by a homology that comes from another CTV strain strain isolated before the proteolytic enzyme sequence substitute.In specific embodiment, first and second virus vector come from a viral kind commonly used.At one more specifically among the embodiment, first includes the front proteolytic enzyme zone of the strain isolated that comes from different CTV strains with second virus vector.In specific embodiment, target organism is plant, and at one more specifically among the embodiment, this plant is citrus.In another specific embodiment, first and/or second CTV carrier are configured to comprise the expressible nucleotide sequence of a coding heterologous protein.
The inventor recognize sometimes target organism infected a similar carrier is arranged after, at target organism
Add carrier in (for example trees or plant) and still have meaning.For example, lost the alien gene of expressing if the inventor finds the carrier in the plant, can add so carrier will be very necessary; If finding a better gene expresses in trees; If and/or need to express a more than gene.Equally, the inventor recognizes that also the carrier that is necessary a viral strain is applied in the plant that has infected the wild-type that this strain is arranged.The inventor finds that the corresponding section of virus vector is made with clearly defined objective modification can avoid double infection to get rid of phenomenon.
The inventor finds can solve the problem that double infection is got rid of by the virus structure that modification wild-type CTV virus is constructed, such as, allow their front proteolytic enzyme be substituted by the front proteolytic enzyme sequence of a different virus kind.
Checked the relation of different genotype oranges and tangerines Te Lisidecha virus (CTV) between the ability that stops other virus isolated strain double infections.The result shows that the double infection eliminating only occurs between the strain isolated with a CTV strain.When carrying out in-order plant with the strain isolated with strain when buddering, infecting for the first time for dangerous strain isolated provides comprehensive eliminating.An example noticing the complete intersection protection between the CTV virus be in primary infection belong in the plant with the wild-type CTV of T36 strain, the CTV that expresses GFP infects and is suppressed (Folimonova et al., 2010) fully.
According to an embodiment, the inventor has checked based on the double infection of the virus structure of the infection cDNA clone structure of the T36 strain of CTV and has got rid of.The result shows, by revising the virus structure of wild-type CTV virus structure, the virus vector of structure for example L1L2 proteolytic enzyme zone is replaced into one from the homologous sequence of different virus strain, even so that also can avoid double infection to get rid of in by the plant that infected with a viral strain.Correspondingly, can be with virus vector embodiment of the present invention as the carrier that had infected before with the trees of strain virus, for example afield in the trees of growth by the natural propagation of virus infected or since one of early application based on a trees infection that the CTV carrier of virus product series structure causes, thereby avoid the eliminating of secondary viral vector infection.
In another embodiment, invention is related to a virus vector structure, and this virus structure front proteolytic enzyme of being designed to be used in the strain isolated that comes from a viral kind different lines commonly used substitutes the endogenous front proteolytic enzyme of virus vector herein.The strain of CTV is defined as according to the different lines (Hilf et al., 2005) of 1a ORF nucleotide sequence analysis virus on the system.Use this definition, T36 and T68 are strains.Each Virus Sample is called as one strain isolated in these strains.Each strain is named as " type strain isolated ", and by forming with the less strain isolated of type member sequence difference.At one more specifically among the embodiment, virus vector is the CTV strain isolated that front proteolytic enzyme sequence is replaced by the strain isolated of another CTV strain.One than previous more specific embodiment in, the CTV carrier is according to T36 strain strain isolated structure, its front proteolytic enzyme sequence is substituted by the front proteolytic enzyme sequence of a strain isolated of T68CTV strain.At one more specifically among the embodiment, replaced proteolytic enzyme sequence is the L1L2 zone.
According to another embodiment, invention with one to alleviate the budder method of the CTV virus vector double infection eliminating that brings of continuous virus relevant.In a specific embodiment, the method may comprise allows target plant first CTV virus vector based on first product series structure of CTV of buddering, thereby make first CTV viral vector infection target plant and produce a plant that has infected, then allow this plant that has infected budder second based on a CTV strain but be modified to the CTV virus vector of the front proteolytic enzyme sequence that comprises a different virus strain (second).Second CTV virus vector also can comprise expresses the gene that can obtain useful result's protein.In a specific embodiment, first strain is T36, and second strain is T68.Second CTV virus vector is allowed to infect the plant that had infected.In a specific embodiment, target plant is citrus.In another specific embodiment, first and/or second CTV carrier are configured to comprise the expressible nucleotide sequence of a coding heterologous protein.
According to another embodiment, invention is relevant with a method that alleviates the eliminating of virus vector double infection.The method comprises that allowing target plant budder one had infected target Plant virus carrier strain.Second virus vector be configured to a front proteolytic enzyme sequence by a homology that comes from another CTV strain strain isolated before the proteolytic enzyme sequence substitute.In specific embodiment, first and second virus vector come from a viral kind commonly used.At one more specifically among the embodiment, first includes the front proteolytic enzyme zone of the strain isolated that comes from different CTV strains with second virus vector.At one more specifically among the embodiment, front proteolytic enzyme sequence comprises a complete front protein sequence segment, respectively by 800 base pairs (bp) or still less, 700bp or still less, 600bp or still less, 500bp or still less, 400bp or still less, 300bp or still less, 200bp or still less or 100bp or still less form.Perhaps, carrier is comprised of a complete front proteolytic enzyme sequence that is at least 100bp, 200bp, 300bp, 400bp or 500bp fragment.The reference chapters and sections that unrestrictedly are described in hereinafter of known front proteolytic enzyme sequence example are discussed.In a specific embodiment, target organism is plant, and target organism is tree more specifically among the embodiment at one, and another is more in the specific embodiment, and target organism is citrus.In another embodiment, first/second CTV carrier be configured out the expressible nucleotide sequence that comprises a coding heterologous protein.
Viral kind used herein is the virus that a group has similar features, and can infect host's kind of identical (perhaps approximate).Be divided into the virus of a class with reference to " viral strain " expression, for example CTV or other viral kinds, but their gene order or some other features are to be divided into of a sort other viruses different.
Embodiment:
Embodiment 1:CTV Δ P33 structure
The inventor has checked several virus structures, and all virus all comprises based on the infection cDNA of T36CTV clone and the p33ORF deletion (Tatineni et al., 2008) of structure, thereby can prevent from expressing the double infection of the CTV of GFP.The result shows that these deletions make a variation and can increase, and also can infect most Citrus mutation tree (Tatineni et al., 2008) on the whole in trees.For the ability of host plant GFP labeling CT V of double infection in a host of CTV p33 deletion variation of having assessed a primary infection, little Da Ye comes at first the budder virus of variation of lemon tree.As the control group of this experiment, with the other one group plant wild-type CTV that budders, referring to Fig. 1, represented the diagram of wild-type CTV (A) and p33 deletion variation structure (B).For these two groups, set up primary infection on the trunk by being organized into of grafting virus infection.The leaf of pruning the top impels new leaf to generate.In six weeks after buddering, confirm that by ELISA the system of new leaf infects.Then in plant, add second bark tissue that contains CTV-BC5/GFP.After graft union, again prune the leaf of top and induce new growth.After for the second time new leaf grows up to (starting from for 6 weeks), determine that by the GFP fluorescence in the bark tissue of observing new first portion virus is to the double infection of trees.Wild-type CTV has fully stoped GFP to express the double infection of virus as a result: infected first in the plant of CTV9R not detecting GFP fluorescence.On the contrary, the plant that has infected first the variant viral that lacks functional p33 protein has all shown GFP fluorescence, with do not have primary infection and also the trees of testing viral CTVBC5/GFP of only having buddered the same.This shows that the deletion variation is on the not impact of CTV infection of GFP mark.
Embodiment 2: the substituted CTV structure in front proteolytic enzyme zone
In one is similarly tested, the inventor has checked that front proteolytic enzyme zone (nucleotide position 108-3040 in the CTV genome) is replaced to the hybrid virus structure of T68-1 genome respective regions, and the rest part of hybrid structure comprises the sequence of T36 (diagram of this structure is seen Fig. 1 (C)).T68-1 represents a strain isolated in the CTV T68 strain.Similar with above-mentioned experiment, this structure is used to budder for the first time citrus plant, (confirms to set up after the infection by ELISA) afterwards to re-use CTV-BC5/GFP.Wild-type CTV be used to budder for the first time the contrast trees (such as above-mentioned experiment).In 6 weeks after buddering for the second time, determine that by the GFP fluorescence in the bark tissue of observing new first portion virus is to the double infection of trees.The same with expection, wild-type CTV has fully stoped GFP to express viral double infection: infected first in the plant of CTV9R not detecting GFP fluorescence.By contrast, the plant that has infected first the virus that contains alternative front proteolytic enzyme zone has all shown GFP fluorescence, with do not have primary infection and also the trees of testing viral CTV BC5/GFP of only having buddered the same.This shows that hybrid virus infects not impact to the CTV of GFP mark.
Embodiment 3: the replaced virus in proteolytic enzyme zone can be removed eliminating.Recently, experimental study the modification in front proteolytic enzyme zone whether can provide the ability of getting rid of of removing for virus.We have constructed a hybrid virus structure, in this virus, a zone of containing among the T36cDNA clone of two front proteolytic enzyme L1 and L2 (the nts position position 108-3039 in the CTV genome) is replaced by the respective regions of the T68-1 strain isolated of T68 strain, and the rest part of hybrid structure then comprises the sequence (Fig. 2) of T36.Again budder with the virus structure of describing and to have infected the plant that T36CTV virus is arranged.As proof before us, after the primary infection CTV strain isolated, can fully get rid of reinfect with another strain isolated of a strain.For example, infect a strain isolated of T36 strain and can get rid of other strain isolateds of reinfect T36 strain, and can get rid of the virus that infects based on the GFP mark of T36.And, infect the T36 strain isolated and can get rid of superinfection fully based on the hybrid virus of T36 strain isolated structure, the sequence of 8 genes of 3 ' genome part is substituted the corresponding sequence that (sequences of individual gene sequence or several assortments of genes) become T30 or T68 strain strain isolated in this virus.Significantly, the proteolytic enzyme zone is replaced to the genomic hybrid virus of T36 and is proved to be a special behavior before the T68: the virus of variation can infect the plant that has been crossed by maternal T36 virus infection on the whole, and the same (Fig. 1 of viral accumulating level of the level of virus accumulation and the health plant that infects identical virus; Compare other roads that the 4th road and contrast are buddered).
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Not with the conflicting prerequisite of instruction herein under, the announcement of any reference of quoting is in this article quoted in conjunction with in the present invention by complete.Need to know example described herein and embodiment just being illustrated as better purpose, and suggestion those skilled in the art can make various modifications or change thus, and this kind revised or change should be within the spirit and scope of the present invention.
Claims (29)
1. one kind alleviates the method for getting rid of to the relevant CTV virus vector double infection of target plant with continuous transfection virus, and described method comprises:
Make target plant first CTV virus vector of buddering, this carrier is lost or a destroyed p33 gene;
Make first CTV viral vector infection target plant, thereby produce the plant that infects first, and
Make the plant of primary infection second the CTV virus vector of buddering, the p33 gene that this carrier contains or do not contain the p33 gene or destroy, and second CTV virus vector further infects the plant of primary infection to produce the plant of a superinfection.
2. according to claim 1 method, wherein said target plant is citrus.
3. according to claim 1 method, wherein first and/or second CTV carrier are configured to comprise the expressible nucleotide sequence of a coding heterologous protein.
4. one kind alleviates and the method for target organism being carried out virus vector double infection that first and second virus vector budder relevant continuously and getting rid of, it is replaced with another CTV strain that first virus vector is configured to front proteolytic enzyme sequence, and described method comprises:
Make target organism first CTV virus vector of buddering;
Make first CTV viral vector infection target organism, thereby produce the biology that infects first, and
Make the biology of primary infection second virus vector of buddering, and second CTV virus vector further infects the biology of primary infection to produce the biology of a superinfection.
5. according to claim 4 method, wherein said target organism is plant.
6. according to claim 4 method, wherein said target organism is tree.
7. according to claim 6 method, wherein said target organism is citrus.
8. according to claim 4 method, wherein said first and second virus vector are configured to contain the front proteolytic enzyme zone from the different lines of viral kind commonly used.
9. according to claim 8 method, wherein said viral kind commonly used is CTV.
10. according to claim 9 method, wherein said first virus vector are based on the T36 product series structure of CTV.
11. method according to claim 9, wherein said second virus vector are based on the T36 product series structure of CTV, wherein before the proteolytic enzyme zone be replaced to homology zone from CTV T68 strain.
12. method according to claim 4, wherein said first and/or second virus vector are configured to comprise the expressible nucleotide sequence of a coding heterologous protein.
13. method according to claim 9, class papoid of wherein said front proteolytic enzyme sequence encoding.
14. a virus vector is configured to endogenous front proteolytic enzyme sequence and is substituted by the front proteolytic enzyme from the different lines of commonly using viral kind, and comprises the expressible nucleotide sequence of a coding heterologous protein.
15. virus vector according to claim 14, wherein said virus vector are the CTV strains, and are replaced by a front proteolytic enzyme sequence that comes from different CTV strains.
16. virus vector according to claim 15, wherein said CTV strain thing T36, its front proteolytic enzyme sequence is replaced by the front proteolytic enzyme sequence of T68CTV strain.
17. virus vector according to claim 15, wherein replaced proteolytic enzyme sequence is a class papoid zone.
18. one kind alleviates the method that the virus vector double infection relevant with the target plant that has infected before first viral strain being carried out continuous transfection got rid of, described method comprises:
Budder and make it the front plant that has infected the virus vector of above-mentioned first viral strain, described virus vector structure comprises the proteolytic enzyme sequence of second viral strain, and comprise the purpose heterologous protein, and virus vector further infects the plant of primary infection to produce the plant of a superinfection.
19. method according to claim 18, wherein said purpose heterologous protein is expressed in the plant of described superinfection.
20. method according to claim 18, wherein said virus vector is CTV.
21. method according to claim 18, wherein said target plant is citrus.
22. method according to claim 18, wherein said first viral strain is T36.
23. method according to claim 18, wherein said second viral strain is T68.
24. method according to claim 18, wherein said proteolytic enzyme sequence are the front proteolytic enzyme sequences in second viral strain homology zone.
25. method according to claim 18, wherein said proteolytic enzyme sequence are the sequences in L1L2 zone.
26. a virus vector is configured to endogenous front proteolytic enzyme sequence and is replaced to a front proteolytic enzyme that comes from viral kind different lines commonly used, and comprises the expressible nucleotide sequence of the heterologous protein of can encoding.
27. virus vector according to claim 26, wherein said virus vector are strain isolateds of CTV strain, its front proteolytic enzyme sequence by one from different CTV strains replace.
28. virus vector according to claim 27, wherein the CTV strain is T36, and its front proteolytic enzyme sequence is substituted by the front proteolytic enzyme sequence of a T68CTV strain.
29. virus vector according to claim 27, the proteolytic enzyme sequence that wherein substitutes are the L1L2 zones.
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| MARKUS U EHRENGRUBER ET AL.: "Semliki Forest virus vectors with mutations in the nonstructural protein 2 gene permit extended superinfection of neuronal and non-neuronal cells", 《JOURNAL OF NEUROVIROLOGY》 * |
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