CN104498527A - Method for constructing peste des petits ruminant transgenic plant vaccine efficient expression vector - Google Patents
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Abstract
The invention relates to recombinant vector construction in the technical field of genetic engineering, in particular to construction used for recombining an efficient expression vector used for preventing and treating peste des petits ruminants. The expression vector comprises F protein genes of F proteins in PPRV and five kinds of recombinant plasmids connected with the genes, wherein the five recombinant plasmids include the pBI121-F, the 121-C-F-M12-M34, the 121-C-FKDEL-M12-M34, the 121-C-G-F-M12-M34 and the 121-C-G-FKDEL-M12-M34; and the gene sequence can be seen in a sequence table.
Description
Technical Field
The invention relates to construction of a recombinant vector in the technical field of genetic engineering, in particular to construction of a recombinant plant high-efficiency expression vector for preventing Peste des petits ruminants.
Background
Peste des Petits R μminsants (PPR) is an acute and contact epidemic disease caused by Peste des Petits R μminsants vis PPRV, mainly infecting small ruminants such as goats and sheep. The morbidity and mortality of the epidemic can reach 100% and 50%. There is no effective treatment for the epidemic disease, so an effective prevention method is to be found for preventing the epidemic disease. With the development of biotechnology and genetic engineering technology, research on PPRV is more intensive. The PPRV genome is a single-strand negative-strand RNA, and N-P-M-F-H-L6 genes are sequentially arranged from the 3 'end to the 5' end of an RNA strand and respectively code 6 corresponding structural proteins. The F protein is a fiber process forming the surface of the viral envelope, belongs to fusion protein, plays an important role in the aspect of virus-induced cytopathology, and is a key factor for determining the success of viral infection. Devireddy et al found that the F protein of pure PPRV has biological activity and is easy to cause virus-induced hemolysis, cell fusion and initial infection, and has immunogenicity, which is an important basis for the present invention to adopt the F protein gene as a target gene.
The edible vaccine of transgenic plant utilizes molecular biology technology to transfer the antigen coding gene of pathogenic microorganism into plant, and express active protein in plant, so that when human or animal eats the transgenic plant containing the antigen, the intestinal immune system can be excited, thereby generating immunity to pathogenic bacteria such as virus and parasite.
Disclosure of Invention
The invention aims to: the F gene is connected with a plant expression vector pBI121 by a gene recombination method, and a regulatory sequence is added on two sides of the F gene so that the F gene can be efficiently expressed in plants. Finally, the purpose of preventing the Peste des petits ruminants is achieved.
The invention provides a plant expression vector, which is characterized by comprising an F protein gene of an F protein in PPRV and a recombinant plasmid connected with the gene, wherein the total five genes are respectively as follows:
pBI121-F，
121-C-F-M12-M34
121-C-FKDEL-M12-M34
121-C-G-F-M12-M34
121-C-G-FKDEL-M12-M34
the gene sequence is shown in a sequence table.
Wherein the F protein gene is shown in a sequence table 1.
The invention is obtained by modifying the HindIII enzyme cutting site of a pBI121 plant expression vector to the EcoRI enzyme cutting site, the genes or the regulation and control sequences required by the modification comprise GFP, MAR12, MAR34, CsVMV, F and FKDEL, and the corresponding gene sequences are shown in a sequence table 2-7.
The invention adds related genes and sequences to modify 4 vectors, and the sequence of the added genes and the regulatory sequences is as follows:
MAR12-CsVMV-GFP-F-MAR34；
MAR12-CsVMV-GFP-FKDEL-MAR34；
MAR12-CsVMV-F-MAR34；
MAR12-CsVMV-FKDEL-MAR34。
the invention also provides a preparation method of the plant expression vector, which comprises the following steps:
step 1, extracting relevant plasmids by using primers and carrying out PCR amplification,
step 2, the PCR product is ligated to a pUCM-T vector using the T/A cloning method,
and 3, constructing a plant expression vector.
Preferably, the method comprises the following steps:
step 1, amplifying the gene,
MAR sequence: cloning was performed using pMD-MAR plasmid as template and primers MAR12-F, MAR12-R and MAR34-F, MAR34-R, respectively;
GFP sequence: pHL005 is taken as a template, and GFP-F, GFP-R is taken as a template;
PPRV-F, F-KDEL sequence with PPRV-F as template and PPRV-F-F, PPRV-F-R and PPRV-F-F, PPRV-FKDEL-R as template;
CsVMV sequence: taking the synthesized CsVMV as a template, and taking CsVMV-F, CsVMV-R as a template;
step 2, the gene obtained in step 1 was ligated to a pUCM-T vector, and the resulting plasmid was designated as: pGFP, pMAR12, pMAR34, pCsVMV, pF, pFDDLEL.
Step 3, constructing a plant expression vector:
1) construction of expression vector pBI121-CsVMV
2) Construction of expression vector pBI121-CsVMV-GFP
3) Construction of expression vector pBI121-CsVMV-GFP-Mar12
4) Construction of intermediate expression vector pUCM-GUS-GFP
5) Construction of pUCM-GUS-GFP-MAR34 intermediate expression vector
6) Construction of intermediate expression vectors pUCM-F-GFP-MAR34 and pUCM-FKDEL-GFP-MAR34
7) Construction of plant expression vectors pBI121-CsVMV-GFP-F-MAR12-MAR34 and pBI121-CsVMV-GFP-F-KDEL-MAR12-MAR34
8) Construction of plant expression vectors pBI121-CsVMV-F-MAR12-MAR34 and pBI121-CsVMV-F-KDEL-MAR12-MAR34
9) Construction of plant expression vector pBI121-F
After the vector is connected with a module, the verification is carried out by using a PCR verification method, and a sequence on a pBI121 vector of a primer used is verified to be a sequence on the connected module.
More preferably, the method comprises the following steps:
step 1, gene amplification:
MAR sequence: cloning was performed using pMD-MAR plasmid as template and primers MAR12-F, MAR12-R and MAR34-F, MAR34-R, respectively; PCR amplification conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 60s, annealing at 55 ℃ for 60s, extension at 72 ℃ for 5min after 35 cycles, and storing the reaction product at 4 ℃.
GFP sequence: pHL005 is taken as a template, and GFP-F, GFP-R is taken as a template; PCR amplification conditions: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 45s, extension at 72 ℃ for 7min after 30 cycles, and storing the reaction product at 4 ℃.
PPRV-F, F-KDEL sequence: PPRV-F is taken as a template, and PPRV-F-F, PPRV-F-R and PPRV-F-F, PPRV-FKDEL-R are respectively taken as templates; PCR amplification conditions: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 40s, extension at 72 ℃ for 90s, extension at 72 ℃ for 10min after 35 cycles, and storing the reaction product at 4 ℃.
CsVMV sequence: taking the synthesized CsVMV as a template, and taking CsVMV-F, CsVMV-R as a template; PCR amplification conditions: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 40s, extension at 72 ℃ for 10min after 35 cycles, and storing the reaction product at 4 ℃.
Step 2, the sequence obtained in step 1 is ligated to a pUCM-T vector,
the extracted plasmid was named: pGFP, pMAR12, pMAR34, pCsVMV, pF, pFDDLEL.
Step 3, constructing a plant expression vector:
1) construction of expression vector pBI121-CsVMV
The promoter CaMV35S on the expression vector pBI121 is replaced by CsVMV on the cloning vector pCsVMV to improve the expression efficiency of the vector in plants.
2) Construction of expression vector pBI121-CsVMV-GFP
3) Construction of expression vector pBI121-CsVMV-GFP-Mar12
Plasmids pBI121-CsVMV-GFP and pMAR12 were each digested separately with HindIII
4) Construction of intermediate expression vector pUCM-GUS-GFP
5) Construction of pUCM-GUS-GFP-MAR34 intermediate expression vector
6) Construction of intermediate expression vectors pUCM-F-GFP-MAR34 and pUCM-FKDEL-GFP-MAR34
7) Construction of plant expression vectors pBI121-CsVMV-GFP-F-MAR12-MAR34 and pBI121-CsVMV-GFP-F-KDEL-MAR12-MAR34
8) Construction of plant expression vectors pBI121-CsVMV-F-MAR12-MAR34 and pBI121-CsVMV-F-KDEL-MAR12-MAR34
9) Construction of plant expression vector pBI121-F
Most preferably, the plant expression vectors pBI121-CsVMV-GFP-F-MAR12-MAR34 and pBI121-CsVMV-GFP-F-KDEL-MAR12-MAR34 of the present invention are constructed by the following steps:
(1) enzyme digestion:
plasmids pBI121-CsVMV-GFP-MAR12 and pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were double digested with XbaI and EcoRI, respectively
Enzyme digestion system: mu.L of plasmid, 2.5. mu.L of 10 XM buffer, ul of each of the two enzymes, 10.5. mu.L of ddH20, and a total reaction volume of 25. mu.L. The enzyme was cleaved at 37 ℃ for 2.5 h.
(2) Two fragments pBI121-CsVMV-GFP-MAR12, pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were recovered by agarose gel electrophoresis as fragments A, B and C, respectively.
(3) Connecting:
a connection system: mu.L of fragment A, 4. mu.L of fragment B or C, 1. mu.L of 10 XT4DNA ligase buffer, 1. mu. L T4DNA ligase. The total reaction was 10. mu.L, 16 ℃ overnight.
(4) And transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
Most preferably, the plant expression vectors pBI121-CsVMV-F-MAR12-MAR34 and pBI121-CsVMV-F-KDEL-MAR12-MAR34 of the present invention are constructed by the following steps:
(1) enzyme digestion:
the plasmids pBI121-CsVMV-MAR12, pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were double digested with BamHI and EcoRI, respectively
Enzyme digestion system: mu.L of plasmid, 2.5. mu.L.times.K buffer, two enzymes, each ul, 10.5. mu.L of ddH20, and a total reaction volume of 25. mu.L. The enzyme was cleaved at 37 ℃ for 3 h.
(2) Two fragments pBI121-CsVMV-MAR12, pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were recovered by agarose gel electrophoresis as fragments A, B and C, respectively.
(3) Connecting:
a connection system: mu.L of fragment A, 4. mu.L of fragment B or C, L. mu. L L0XT4DNA ligase buffer, L. mu. L T4DNA ligase. The total reaction was 10. mu.L, 16 ℃ overnight.
(4) And transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
Most preferably, the plant expression vector pBI121-F of the present invention is constructed by the following steps:
(1) enzyme digestion:
pBI121 plasmid is digested by SmaI and SacI
The enzyme digestion system is as follows: 10 μ L of plasmid, 2.5 μ L of 10 XT buffer, 2.5 μ L of 1% BSA, two enzymes each μ L, 8 μ L of ddH20, total reaction volume 25. mu.L. The enzyme was cleaved at 30 ℃ for 3 h.
Enzyme PvuII and BstxI step-by-step double enzyme digestion pF
The enzyme digestion system is as follows: mu.L of plasmid, 2.5. mu.L of 10 XM buffer, PvuII L. mu.L, 11.5. mu.L of ddH20, and a total reaction volume of 25. mu.L. The enzyme was cleaved at 37 ℃ for 4 h. After the enzyme digestion is finished, ethanol is carried out to recover precipitated DNA, and the next step of enzyme digestion is carried out.
The enzyme digestion system is as follows: mu.L of ethanol recovered DNA, 2.5. mu.L of 0 XH buffer, BstxI. mu.L, 11.5. mu.L of ddH20, total reaction volume of 25. mu.L. The enzyme was cleaved at 45 ℃ for 4 h.
(2) The cleavage products pBI121 and pF were subjected to agarose gel electrophoresis, and two DNA fragments were recovered, respectively, and designated as fragment A, B.
(3) Connection of
A connection system: 2.5. mu.L of fragment A, 5. mu.L of fragment B, 2.5. mu. L L0XT4DNA ligase buffer, 1. mu.LT 4DNA ligase, 14. mu.L ddH 20. The total reaction was 25. mu.L, 16 ℃ overnight.
(4) Coli competent cells were transformed, and single colony PCR forward and reverse verified with primers MAR12-PCR-IN upstream and F downstream, F upstream and MAR34-PCR-IN downstream, respectively. And (5) extracting plasmid, and carrying out enzyme digestion verification.
To prepare the vector of the present invention, PCR amplification using the following elements is required:
PPRV-F: namely the F protein gene, is provided by the Chinese animal health and epidemiology center;
promoter CsVMV: synthesized by Shanghai Biotechnology engineering services, Inc.;
pHL 005: provided by Qingdao bioenergy and process research institute of Chinese academy of sciences;
the pMD-MAR plasmid was stored in the genetic laboratory of Qingdao university of agriculture.
The primers used in the PCR of the invention are shown in the following table:
TABLE 1 primers used in PCR of the present invention
Note: the first 3 bases of the 5' end are protective bases, the italic and black parts are enzyme cutting sequences, and the name of an enzyme cutting site is shown in brackets.
The invention further carries out enzyme digestion verification on the amplified related plasmids, namely pGFP, pMAR12, pMAR34, pCsVMV, pF and pFDKDEL. The corresponding enzymes are pGFP (BamH I, Xba I) double enzyme digestion; 4, pCsVMV (Hind III, Xba I) double enzyme digestion; 2, pMAR12(Hind III) single enzyme digestion; pMAR34 (MunI, EcoRI) double enzyme digestion; pF (PvuII, Bstx I) step-by-step double enzyme digestion; 6: pFDDLEL (PvuII, Bstx I).
The following is a list of gene sequences in the sequence listing:
sequence 1F Gene sequence
Sequence 2 cloned GFP Forward sequence
Mar12 Forward sequence of sequence 3 clone
Mar34 forward sequence of sequence 4 clone
Sequence 5 cloned CsVMV forward sequence
Sequence 6 cloned F Forward sequence
FKDEL forward sequence of sequence 7 clone
Sequence 8 artificially synthesized primer CsVMV-F
Sequence 9 artificially synthesized primer CsVMV-R
Sequence 10 artificially synthesized primer GFP-F
Sequence 11 Artificial Synthesis primer GFP-R
Sequence 12 Artificial Synthesis of primer MAR12-F
Sequence 13 Artificial Synthesis of primer MAR12-R
Sequence 14 Artificial Synthesis of primer MAR34-F
Sequence 15 Artificial Synthesis of primer MAR34-R
Sequence 16 artificially synthesized primer F-F
Sequence 17 artificially synthesized primer F-R
Sequence 18 artificially synthesized primer FKDEL-F
Sequence 19 artificially synthesized primer FKDEL-R
Sequence 20 primers MAR12-PCR-IN were synthesized
Sequence 21 Artificial Synthesis of primer MAR34-PCR-IN
Sequence 22 sequence of HindIII site-EcoRI site part of vector 121-F
Sequence 23 sequence of portion of MAR12-MAR34 in vector 121-C-G-F-M12-M34
Sequence 24 sequence of the MAR12-MAR34 portion of vector 121-C-G-FKDEL-M12-M34
Sequence 25 sequence of portion of MAR12-MAR34 in vector 121-C-F-M12-M34
Sequence 26 sequence of portion of MAR12-MAR34 in vector 121-C-FKDEL-M12-M34
The process of constructing the plant expression vector of the present invention was constructed according to the procedure shown in FIG. 1.
Wherein,
121-F was obtained by substituting the cloned F gene for the GUS gene on the original pBI121 vector
121-C-F-M12-M34 is the vector pBI121 from HindIII to EcoRI cleavage site to which the MAR12 sequence, CsVMV sequence, F gene, MAR34 sequence are added.
121-C-FKDEL-M12-M34 was prepared by adding the MAR12 sequence, CsVMV sequence, FKDEL gene, and MAR34 sequence from the HindIII cleavage site to the EcoRI cleavage site of vector pBI 121.
121-C-G-F-M12-M34 is the vector pBI121 from HindIII site to EcoRI site to which MAR12, CsVMV, GFP, F, and MAR34 sequences are added.
121-C-G-FKDEL-M12-M34 was prepared by adding a MAR12 sequence, a CsVMV sequence, a GFP gene, an FKDEL gene, and a MAR34 sequence from the HindIII cleavage site to the EcoRI cleavage site of the vector pBI 121.
The plant expression vector can be used for producing transgenic alfalfa for preventing Peste des petits ruminants. And can cause the overexpression of the target gene F.
The F protein encoded by the F gene in PPRV, which is involved in virus-mediated hemolysis, cell fusion and initiation of infection, plays an important role in virus-induced cytopathology, and is a key factor for determining the success of virus infection. Devireddy (1999) et al found that the F protein of pure PPRV has biological activity and is susceptible to virus-induced hemolysis, cell fusion and initial infection, and is immunogenic, so the F gene of the F protein was cloned in this study as a gene of interest.
The promoter on the plant expression vector pBI121 is CaMV35S, and related researches show that the activity of the promoter in alfalfa is not high (Ying Shi Liang, 2009), Deborah AS (2004) and the like compare the activity of five different promoters such AS a cassava vein mosaic virus (CsVMV) promoter, a double 35S promoter, a sugarcane bacilliform virus (ScBV) promoter, a Figwort Mosaic Virus (FMV) promoter, an alfalfa Rubisco small subunit gene promoter and the like in alfalfa through experiments, and the results show that the CsVMV promoter has the highest activity and is worthy of popularization and application in transgenic alfalfa. Therefore, in this experiment, the synthetic CsVMV promoter was used to replace the pBI121 promoter CaMV35S promoter to improve the expression efficiency of the foreign gene.
The method utilizes a Matrix Attachment Region (MAR) sequence to construct a plant high-efficiency expression vector, overcomes transgene inactivation and enhances the expression stability of exogenous genes, and is a new method for overcoming transgene inactivation developed in recent years. As a result of research, the expression level of foreign gene protein with MAR sequence is obviously higher than that without MAR sequence, such as Wudongmei (2007), Lixugang (2000), Sujin (1999), Han (1997), etc. In this experiment, MAR sequences were added at both ends of the exogenous gene expression region to allow efficient expression of exogenous genes in the transformed receptor.
The KDEL sequence is a signal sequence capable of retaining the protein in the endoplasmic reticulum, and the signal has corresponding receptors on the membranes of all parts of the Golgi apparatus. The KDEL sequence is introduced before the stop codon to be fused with the target protein, so that the target protein is retained in the endoplasmic reticulum, the stability of the transgenic plant can be improved, the edible vaccine is not easy to digest and can stay in a digestive system for a long time, and the target protein enters the viscera through intestinal mucosa to generate immune response. M. Bellucci (2000) and the like respectively transform tobacco with gamma-zein gene, gamma-zein gene added with TMV coat protein regulatory sequence and gamma-zein gene added with KDEL sequence, and the result shows that the plant added with KEDL sequence has the highest peripheral protein expression level, and the expressed protein is confirmed to be attached to endoplasmic reticulum. Lorenzo Frigerio (2001) et al also showed that addition of the sequence number KDEL improves the stability of phaseolin. In the experiment, an expression vector added with a KDEL sequence is expected to improve the stability of immune protein and improve the immune effect.
To sum up, the transgenic alfalfa expression vector constructed by the invention can realize the overexpression of the F gene in alfalfa, and finally achieve the purpose of preventing Peste des petits ruminants.
Description of the drawings:
FIG. 1 expression vector construction Process
FIG. 2 shows the amplification results of the target gene. M: a DL2000DNA marker; GFP PCR product; 2 MAR12 PCR product; 3 MAR34PCR product; 4, CsVMV PCR product; f, PCR products; 6: F-KDEL PCR product.
FIG. 3 shows the results of double restriction enzyme digestion of recombinant plasmid. M: DNA Marker DL 15000; pGFP (BamH I, Xba I) is subjected to double enzyme digestion; 4, pCsVMV (Hind III, Xba I) double enzyme digestion; 2, pMAR12(Hind III) single enzyme digestion; pMAR34 (MunI, EcoRI) double enzyme digestion; pF (PvuII, Bstx I) step-by-step double enzyme digestion; 6: pFDDLEL (PvuII, Bstx I) was digested in two steps.
FIG. 4 shows the results of double digestion of pBI121 plasmid and pCsVMV. M1: DNA Marker DL 15000; m2: DNAmarker DL 2000; 1, carrying out double digestion on pBI121 plasmid (Hind III/Xba I); the pCsVMV plasmid (Hind III/Xba I) was double digested.
FIG. 5 restriction enzyme digestion and PCR verification of pBI121-CsVMV plasmid. 1, pBI121-CsVMV plasmid (Hind III/Xba I) is subjected to double enzyme digestion; 2, PCR result of pBI121-CsVMV plasmid; 3: PCR results for control pBI121 plasmid; 4: and (5) negative control.
FIG. 6pBI121-CsVMV plasmid double digestion and pUCM-GFP plasmid double digestion. 1, pBI121-CsVMV plasmid (Xba I/BamH I) is subjected to double enzyme digestion; the pUCM-T-GFP plasmid (Xba I/BamH I) was digested in two steps.
FIG. 7pBI121-CsVMV-GFP plasmid double digestion and PCR validation. M1 DNA Marker DL 15000; m2 DNA Marker DL 2000; 1, pBI121-CsVMV-GFP plasmid (Xba I/BamH I) is subjected to double enzyme digestion; 2, PCR result of pBI121-CsVMV-GFP plasmid; 3: PCR results for control pBI121 plasmid; 4: and (5) negative control.
FIG. 8 restriction enzyme digestion of pBI121-CsVMV-GFP plasmid and pUCM-T-MAR12 plasmid was verified. M1: DNAmarker DL 2000; m2: DNA Marker DL 15000; 1, pBI121-CsVMV-GFP plasmid (Hind III) single enzyme digestion; 2: the pUCM-T-MAR12 plasmid (Hind III) was digested in a single digest.
FIG. 9 forward and reverse PCR validation of pBI121-CsVMV-GFP-MAR 12. M1: DNA Marker DL 2000; m2: DNA Marker DL 15000; 1 to 6 are forward PCR detection; 7 to 12 are inverse PCR assays.
FIG. 10 double cleavage of pBI121-CsVMV-GFP and p UCM-T-GFP. M1: DNA Marker DL 15000; m2: DNA Marker DL 2000; 1, pBI121-CsVMV-GFP plasmid (Xba I/EcoRI) is subjected to double digestion; 2: the plasmid p UCM-T-GFP (Xba I/EcoRI) was digested in two steps.
FIG. 11 PCR assay of pUCM-T-GUS-GFP. M1: DNA Marker DL 2000; m2: DNAmarker DL 15000; 1: amplifying plasmids by using primers at two ends of GFP; 2: a negative control of 1; 3: amplifying plasmids by primers at two ends of GUS; 4: negative control of 3; 5: amplifying plasmids upstream of GFP and downstream of GUS; 6 is a negative control of 5.
FIG. 12pUCM-GUS-GFP plasmid single-cut and pMAR34 plasmid double-cut. M1: DNA MarkerDL 15000; m2: DNA Marker DL 2000; 1, single enzyme digestion of pUCM-GUS-GFP plasmid (EcoRI); 2: the pMAR34 plasmid (MunI/EcoRI) was double digested.
FIG. 13 forward and reverse PCR assay of pUCM-T-GUS-GFP linked MAR 34. M1: DNA Marker DL 2000; m2: DNA Marker DL 15000; 1 to 5 are forward PCR detection; 6 to 10 are inverse PCR assays.
FIG. 14 the double digestion of the p-G-G-34, pF and pFDDLEL plasmids. M1: DNA Marker DL 15000; 1, double digestion of p-G-G-34 plasmid (SmaI and SacI); 2: pFDDLEL plasmid (PvuII and BstxI) is double digested; 3: the pF plasmid (PvuII and BstxI) was double digested.
FIG. 15 PCR validation of p-G-F-34, p-G-FKDEL-34 plasmids. M1 DNA Marker DL 2000; m2 DNA Marker DL 15000; 1, carrying out PCR verification on a p-G-F-34 plasmid; 2, carrying out PCR verification on the p-G-FKDEL-34 plasmid; 3: control water PCR results.
FIG. 16 plasmid double digestion of pBI121-CsVMV-GFP-M12, P-G-F-34, and P-G-FKDEL-34. M1: DNA Marker DL 15000; 1, plasmid pBI121-CsVMV-GFP-M12 (Xba I and EcoRI) is subjected to double digestion; 2: the plasmid P-G-F-34 (Xba I and EcoRI) is subjected to double digestion; 3: the P-G-FKDEL-34 plasmid (Xba I and EcoRI) was digested in two steps.
FIG. 17 PCR validation of pBI121-C-G-F-12-34, pBI 121-C-G-FKDEL-12-34. M1 DNA MarkerDL 15000; m2 DNA Marker DL 2000; 1-4, PCR verification of pBI 121-C-G-F-12-34; 2 PCR validation of pBI 121-C-G-FKDEL-12-34.
FIG. 18 plasmid double digestion of pBI121-CsVMV-M12, P-G-F-34, and P-G-FKDEL-34. M1: DNA Marker DL 15000; 1, carrying out double digestion on pBI121-CsVMV-GFP-M12 plasmids (BamHI and EcoRI); 2: the plasmid P-G-F-34 (BamHI and EcoRI) is subjected to double digestion; 3: the plasmid P-G-FKDEL-34 (BamHI and EcoRI) was digested simultaneously.
FIG. 19 PCR validation of pBI121-C-F-12-34, pBI 121-C-FKDEL-12-34. M1 DNA MarkerDL 2000; m2 DNA Marker DL 15000; 1-5, PCR verification of pBI 121-C-F-12-34; 6-10 PCR validation of pBI 121-C-FKDEL-12-34.
FIG. 20 plasmid pBI121 and pUCM-T-F were double digested. M1: DNA Marker DL 15000; 1, pBI121 plasmid (SmaI/Sac I) is subjected to double digestion; 2 pF plasmid (PvuII/Bstx I) was double digested.
FIG. 21 PCR validation of plasmid pBI 121-F. M1: DNA Marker DL 15000; 1: forward PCR verification 3: reverse PCR verification 2, 4: negative control.
The specific implementation mode is as follows:
the invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.
Example 1
Cloning of the genes and sequences of interest:
PCR amplification System (25. mu.L) 10 XBuffer 2.5. mu.L, dNTP (2.5mM) 2. mu.L, upstream and downstream primers (20. mu.M) 0.5. mu.L each, Taq enzyme (5U/. mu.L) 0.3. mu.L, template DNA 1. mu.L, ddH2O18.2μL。
The PCR amplification procedure was:
cloning of MAR sequences: cloning was performed using pMD-MAR plasmid as template and primers MAR12-F, MAR12-R and MAR34-F, MAR34-R, respectively; PCR amplification conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 60s, annealing at 55 ℃ for 60s, extension at 72 ℃ for 5min after 35 cycles, and storing the reaction product at 4 ℃.
GFP gene amplification, using pHL005 as template and GFP-F, GFP-R as template; PCR amplification conditions: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 45s, extension at 72 ℃ for 7min after 30 cycles, and storing the reaction product at 4 ℃.
Amplifying PPRV-F, F-KDEL gene by using PPRV-F as a template and PPRV-F-F, PPRV-F-R and PPRV-F-F, PPRV-FKDEL-R as templates respectively; PCR amplification conditions: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 40s, extension at 72 ℃ for 90s, extension at 72 ℃ for 10min after 35 cycles, and storing the reaction product at 4 ℃.
Amplification of the promoter CsVMV: taking the synthesized CsVMV as a template, and taking CsVMV-F, CsVMV-R as a template; PCR amplification conditions: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 40s, extension at 72 ℃ for 10min after 35 cycles, and storing the reaction product at 4 ℃.
The PCR results for each fragment are shown in FIG. 2.
Example 2
The PCR product was ligated to pUCM-T vector using the T/A cloning method, and the extracted plasmid was named: pGFP, pMAR12, pMAR34, pCsVMV, pF, pFDDLEL. And enzyme digestion verification is carried out on corresponding enzymes respectively, wherein pGFP (BamH I and Xba I) is subjected to double enzyme digestion; 4, pCsVMV (Hind III, Xba I) double enzyme digestion; 2, pMAR12(Hind III) single enzyme digestion; pMAR34 (MunI, EcoRI) double enzyme digestion; pF (PvuII, Bstx I) step-by-step double enzyme digestion; 6: pFDDLEL (PvuII, Bstx I). The cleavage results are shown in FIG. 3.
Example 3
Construction of plant expression vectors:
1. replacement of promoter, i.e., construction of expression vector pBI121-CsVMV (FIGS. 4 and 5)
The promoter CaMV35S on the expression vector pBI121 is replaced by CsVMV on the cloning vector pCsVMV to improve the expression efficiency of the vector in plants.
(1) Plasmid pBI121 and plasmid XbaI were double digested with restriction enzymes HindIII and XbaI, respectively
pUCM-T-CsVMV
The digestion system was 10. mu.L of plasmid, 2.5. mu.L of 10 XM buffer, 1. mu.L of each of the two endonucleases, 11.5. mu.L of ddH20, and the total reaction volume was 25. mu.L. The fragment was digested at 37 ℃ for 2h, electrophoresed with 1% agarose gel for 35min, and purified with a DNA gel recovery kit to recover a large fragment of pBI121, denoted fragment A, and a small fragment of pUCM-T-CsVMV, denoted fragment B.
(2) Ligation of fragment A and fragment B
The ligation system was as follows, 4. mu.L of fragment A, 4. mu.L of fragment B, 1. mu.L of 0XT4DNA ligase buffer, 1. mu. L T4DNA ligase. The total reaction was 10. mu.L, 16 ℃ overnight.
(3) And transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
2. Construction of plant expression vector pBI121-CsVMV-GFP (FIGS. 6 and 7)
(1) Extraction of plasmids pBI121-CsVMV, pGFP
(2) Double digestion of pBI121-CsVMV, pGFP with XbaI and BamHI
The digestion system was 10. mu.L of plasmid, 2.5. mu.L of 10 XK buffer, two endonucleases, 11.5. mu.L of ddH2O, in a total reaction volume of 25. mu.L. The enzyme was cleaved at 37 ℃ for 3 h. And (3) carrying out electrophoresis on the mixture for 35min by using 1% agarose gel, and purifying and recovering a large fragment of the pBI121-CsVMV, which is marked as a fragment A, and a small fragment of the pGFP, which is marked as a fragment B, by using a DNA gel recovery kit.
(3) The ligation system of fragment A and fragment B was as follows, 4. mu.L fragment A, 4. mu.L fragment B, L10 XT4DNA ligase buffer, 1. mu. L T4DNA ligase. The total reaction was 10. mu.L, 16 ℃ overnight.
(4) And transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
3. Construction of plant expression vector pBI121-CsVMV-GFP-Mar12 (FIGS. 8 and 9)
Plasmids pBI121-CsVMV-GFP and pMAR12 were each digested separately with HindIII
(1) The digestion was carried out in 10. mu.L of plasmid, 2.5. mu.L of 10 XM buffer, 2.5. mu.L of 1% BSA, 1. mu.L of HindIII, 9. mu.L of ddH20, and a total reaction volume of 25. mu.L. The enzyme was cleaved at 37 ℃ for 2.5 h.
(2) Dephosphorylation of enzyme-digested pBI121-CsVMV-GFP
Dephosphorizing system: 25 μ L of pBI121-CsVMV-GFP digested system, 5 μ L of 10 XAlkalinephoshatase Buffer,2 μ L of CIAP,18 μ L of ddH20, and a total reaction volume of 50 μ L.
The method comprises the following steps:
a.37 ℃ water bath for 1 h.
b. Extract 2 times with phenol/chloroform/isoamyl alcohol (25:24: 1).
c. Chloroform/isoamyl alcohol (24: 1).
d. mu.L of 3M NaCl (final concentration 150mM) was added.
e. Adding 125 μ l (2.5 times of) cold ethanol, and standing at-20 deg.C for 30-60 min.
f. The precipitate was dissolved in 20. mu.L of TE Buffer. The dephosphorylated vector is designated fragment A.
After electrophoresis, pMAR12, designated B, was recovered by purification using a DNA gel recovery kit.
(3) And (3) a linking system: mu.L of fragment A, 4. mu.L of fragment B, L. mu.L of 10 XT4DNA ligase buffer, 1. mu. L T4DNA ligase. The total reaction was 10. mu.L, 16 ℃ overnight.
(4) And transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
4. Construction of intermediate plant expression vector pUCM-GUS-GFP (FIGS. 10 and 11)
(1) The plasmids pBI121-CsVMV-GFP and pGFP were digested with Xba I and EcoRI, respectively
(2) Enzyme digestion system: 10 μ L of plasmid, 2.5 μ L of gamma Buffer, 1 μ L of each enzyme, and 10.5 μ L of ddH 20.
(3) After electrophoresis, two enzyme digestion fragments are respectively purified and recovered by a DNA gel recovery kit, and pBI121-CsVMV-GFP and p UCM-T-GFP are respectively marked as fragments A, B.
(4) And (3) a linking system: fragment A4. mu.L, fragment B4. mu. L, L. mu.L 10 XT4DNA ligase buffer, 1. mu. L T4DNA ligase. The total reaction was 10. mu.L, 16 ℃ overnight.
(5) And transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
5. Construction of pUCM-GUS-GFP-MAR34 for the intermediate expression vector in plants (FIGS. 12, 13)
(1) Enzyme digestion: single digestion of pUCM-GUS-GFP with EcoRI
The digestion system was 10. mu.L of plasmid, 2.5. mu.L of 10 XH buffer, 1. mu.L of EcoRI, 11.5. mu.L of ddH20, and the total reaction volume was 25. mu.L. The enzyme was cleaved at 37 ℃ for 2.5 h. Then, dephosphorylation treatment was performed, and the treated fragment was designated as A.
pMAR34 was cleaved stepwise with MunI and EcoRI
Enzyme digestion system: mu.L of plasmid, 2.5. mu.L of 10 XM buffer, 2.5. mu.L of 1% BSA, 1. mu.L of MunI, 9. mu.L of ddH20, and a total reaction volume of 25. mu.L. The enzyme was cleaved at 37 ℃ for 3 h. The pUCM-GUS-GFP fragment was recovered in ethanol and was designated fragment A.
The method comprises the following steps:
a. to the digestion system, 1/10 volumes of sodium acetate (3mol/L, pH 5.2) were added and mixed well in the DNA solution to give a final concentration of 0.3 mol/L.
b. Adding 2 times volume of anhydrous ethanol pre-cooled by ice, mixing, fully and uniformly mixing again, and placing at-20 ℃ for 20-30 minutes.
c.12000rpm centrifugation for 10min, carefully remove the supernatant, suck off all droplets on the tube wall.
d. Add 1/2 centrifuge tube capacity of 70% ethanol, 12000rpm centrifugation for 2min, carefully remove the supernatant, suck off all droplets on the tube wall.
e. The uncapped EP tube was placed on a lab bench at room temperature to evaporate the remaining liquid to dryness.
f. Add 20. mu.L of ddH2O to dissolve the DNA pellet.
The digestion system was composed of 10. mu.L of ethanol-recovered DNA, 2.5. mu.L of 10 XH buffer, L. mu.L of EcoRI, 9. mu.L of ddH20, and a total reaction volume of 25. mu.L. The enzyme was cleaved at 37 ℃ for 2.5 h.
(2) Two fragments, pUCM-GUS-GFP and pUCM-MAR34, were recovered by agarose gel electrophoresis and were designated as fragment A, B.
(3) And (3) a linking system: mu.L of fragment A, 2. mu.L of fragment B, L. mu.L of 10 XT4DNA ligase buffer, L. mu. L T4DNA ligase. The total reaction was 10. mu.L, 16 ℃ overnight.
(4) And transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
6. Construction of intermediate expression vectors pUCM-F-GFP-MAR34 and pUCM-FKDEL-GFP-MAR34 (FIGS. 14, 15)
(1) Enzyme digestion
Double digestion of pUCM-T-GUS-GFP-MAR34 with SmaI and SacI
Enzyme digestion system: mu.L of plasmid, 2.5. mu.L of 10 XT buffer, 2.5. mu.L of 1% BSA, 1. mu.L of each of the two enzymes, 8. mu.L of ddH20, and a total reaction volume of 25. mu.L. The enzyme was cleaved at 30 ℃ for 3 h.
pUCM-T-F and pUCM-T-F-KDEL were cut separately in two steps with PvuII and BstxI.
(2) The products of pUCM-T-GUS-GFP-MAR34, pUCM-T-F and pUCM-F-KDEL cleavage were subjected to agarose gel electrophoresis to recover two DNA fragments, designated as A, B and C, respectively.
(3) Connecting:
a connection system: mu.L of fragment A or B, 5. mu.L of fragment C, 2.5. mu.L of 10 XT4DNA ligase buffer, 1. mu. L T4DNA ligase, 14. mu.L of ddH 20. The total reaction was 25. mu.L, 16 ℃ overnight.
(4) And transforming the strain into escherichia coli competent cells, selecting a single colony for PCR verification, and extracting plasmid for enzyme digestion verification.
7. Plant expression vectors pBI121-CsVMV-GFP-F-MAR12-MAR34 and pBI121-CsVMV-GFP-F-KDEL-MAR12-MAR34 (FIGS. 16, 17)
(1) Enzyme digestion:
plasmids pBI121-CsVMV-GFP-MAR12 and pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were double digested with XbaI and EcoRI, respectively
Enzyme digestion system: mu.L of plasmid, 2.5. mu.L of 10 XM buffer, ul of each of the two enzymes, 10.5. mu.L of ddH20, and a total reaction volume of 25. mu.L. The enzyme was cleaved at 37 ℃ for 2.5 h.
(2) Two fragments pBI121-CsVMV-GFP-MAR12, pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were recovered by agarose gel electrophoresis as fragments A, B and C, respectively.
(3) Connecting:
a connection system: mu.L of fragment A, 4. mu.L of fragment B or C, 1. mu.L of 10 XT4DNA ligase buffer, 1. mu. L T4DNA ligase. The total reaction was 10. mu.L, 16 ℃ overnight.
(4) And transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
8. Plant expression vectors pBI121-CsVMV-F-MAR12-MAR34 and pBI121-CsVMV-F-KDEL-MAR12-MAR34 (FIGS. 18, 19)
(1) Enzyme digestion:
the plasmids pBI121-CsVMV-MAR12, pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were double digested with BamHI and EcoRI, respectively
Enzyme digestion system: mu.L of plasmid, 2.5. mu.L.times.K buffer, two enzymes, each ul, 10.5. mu.L of ddH20, and a total reaction volume of 25. mu.L. The enzyme was cleaved at 37 ℃ for 3 h.
(2) Two fragments pBI121-CsVMV-MAR12, pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were recovered by agarose gel electrophoresis as fragments A, B and C, respectively.
(3) Connecting:
a connection system: mu.L of fragment A, 4. mu.L of fragment B or C, L. mu. L L0XT4DNA ligase buffer, L. mu. L T4DNA ligase. The total reaction was 10. mu.L, 16 ℃ overnight.
(4) And transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
9. Construction of plant expression vector pBI121-F (FIGS. 20 and 21)
(1) Enzyme digestion:
pBI121 plasmid is digested by SmaI and SacI
The enzyme digestion system is as follows: 10 μ L of plasmid, 2.5 μ L of 10T buffer, 2.5. mu.L of 1% BSA, two enzymes, each ul. mu.L of 8. mu.L of ddH20, total reaction volume 25. mu.L. The enzyme was cleaved at 30 ℃ for 3 h.
Enzyme PvuII and BstxI step-by-step double enzyme digestion pF
The enzyme digestion system is as follows: mu.L of plasmid, 2.5. mu.L of 10 XM buffer, PvuII L. mu.L, 11.5. mu.L of ddH20, and a total reaction volume of 25. mu.L. The enzyme was cleaved at 37 ℃ for 4 h. After the enzyme digestion is finished, ethanol is carried out to recover precipitated DNA, and the next step of enzyme digestion is carried out.
The enzyme digestion system is as follows: mu.L of ethanol recovered DNA, 2.5. mu.L of 0 XH buffer, BstxI. mu.L, 11.5. mu.L of ddH20, total reaction volume of 25. mu.L. The enzyme was cleaved at 45 ℃ for 4 h.
(2) The cleavage products pBI121 and pF were subjected to agarose gel electrophoresis, and two DNA fragments were recovered, respectively, and designated as fragment A, B.
(3) Connection of
A connection system: 2.5. mu.L of fragment A, 5. mu.L of fragment B, 2.5. mu.L of L L0XT4DNA ligase buffer, 1. mu.L of LT4DNA ligase, 14. mu.L of ddH 20. The total reaction was 25. mu.L, 16 ℃ overnight.
(4) Coli competent cells were transformed, and single colony PCR forward and reverse verified with primers MAR12-PCR-IN upstream and F downstream, F upstream and MAR34-PCR-IN downstream, respectively. And (5) extracting plasmid, and carrying out enzyme digestion verification.
Claims (10)
1. A plant expression vector is characterized by comprising an F protein gene of an F protein in PPRV and a recombinant plasmid connected with the gene, wherein the five genes in total are respectively as follows:
pBI121-F，
121-C-F-M12-M34
121-C-FKDEL-M12-M34
121-C-G-F-M12-M34
121-C-G-FKDEL-M12-M34
the gene sequence is shown in a sequence table.
2. The plant expression vector of claim 1, wherein the F protein gene is shown in sequence listing 1.
3. The plant expression vector of claim 1, which is obtained by modifying pBI121 plant expression vector from HindIII site to EcoRI site, wherein the genes or regulatory sequences required for modification include GFP, MAR12, MAR34, CsVMV, F, FKDEL, and the corresponding gene sequences are shown in the sequence table.
4. The plant expression vector of claim 3, wherein 4 vectors are transformed after adding related genes and sequences, and the added genes and regulatory sequences are sequentially arranged as follows:
MAR12-CsVMV-GFP-F-MAR34；
MAR12-CsVMV-GFP-FKDEL-MAR34；
MAR12-CsVMV-F-MAR34；
MAR12-CsVMV-FKDEL-MAR34。
5. the method for preparing the plant expression vector of claim 1, comprising the steps of:
step 1, extracting relevant plasmids by using primers and carrying out PCR amplification,
step 2, the PCR product is ligated to a pUCM-T vector using the T/A cloning method,
and 3, constructing a plant expression vector.
6. A method of making the plant expression vector of claim 5, comprising the steps of:
step 1, amplifying the gene,
MAR sequence: cloning was performed using pMD-MAR plasmid as template and primers MAR12-F, MAR12-R and MAR34-F, MAR34-R, respectively;
GFP sequence: pHL005 is taken as a template, and GFP-F, GFP-R is taken as a template;
PPRV-F, F-KDEL sequence with PPRV-F as template and PPRV-F-F, PPRV-F-R and PPRV-F-F, PPRV-FKDEL-R as template;
CsVMV sequence: taking the synthesized CsVMV as a template, and taking CsVMV-F, CsVMV-R as a template;
step 2, ligating the gene obtained in step 1 to a pUCM-T vector,
the resulting plasmid was designated: pGFP, pMAR12, pMAR34, pCsVMV, pF, pFDDLEL,
step 3, constructing a plant expression vector:
1) construction of expression vector pBI121-CsVMV
2) Construction of expression vector pBI121-CsVMV-GFP
3) Construction of expression vector pBI121-CsVMV-GFP-Mar12
4) Construction of intermediate expression vector pUCM-GUS-GFP
5) Construction of pUCM-GUS-GFP-MAR34 intermediate expression vector
6) Construction of intermediate expression vectors pUCM-F-GFP-MAR34 and pUCM-FKDEL-GFP-MAR34
7) Plant expression vectors pBI121-CsVMV-GFP-F-MAR12-MAR34 and
pBI121-CsVMV-GFP-F-KDEL-MAR12-MAR34 construction
8) Plant expression vectors pBI121-CsVMV-F-MAR12-MAR34 and
pBI121-CsVMV-F-KDEL-MAR12-MAR34 construction
9) Construction of plant expression vector pBI121-F
After the vector is connected with a module, the verification is carried out by using a PCR verification method, and a sequence on a pBI121 vector of a primer used is verified to be a sequence on the connected module.
7. A method of making the plant expression vector of claim 6, comprising the steps of:
step 1, gene amplification:
MAR sequence: cloning was performed using pMD-MAR plasmid as template and primers MAR12-F, MAR12-R and MAR34-F, MAR34-R, respectively; PCR amplification conditions: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 60s, annealing at 55 deg.C for 60s, extension at 72 deg.C for 5min after 35 cycles, storing the reaction product at 4 deg.C,
GFP sequence: pHL005 is taken as a template, and GFP-F, GFP-R is taken as a template; PCR amplification conditions: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 45s, extension at 72 deg.C for 45s, 30 cycles, extension at 72 deg.C for 7min, storing the reaction product at 4 deg.C,
PPRV-F, F-KDEL sequence: PPRV-F is taken as a template, and PPRV-F-F, PPRV-F-R and PPRV-F-F, PPRV-FKDEL-R are respectively taken as templates; PCR amplification conditions: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 45s, annealing at 55 deg.C for 40s, extension at 72 deg.C for 90s, extension at 72 deg.C for 10min after 35 cycles, storing the reaction product at 4 deg.C,
CsVMV sequence: taking the synthesized CsVMV as a template, and taking CsVMV-F, CsVMV-R as a template; PCR amplification conditions: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 45s, extension at 72 deg.C for 40s, extension at 72 deg.C for 10min after 35 cycles, storing the reaction product at 4 deg.C,
step 2, the sequence obtained in step 1 is ligated to a pUCM-T vector,
the extracted plasmid was named: pGFP, pMAR12, pMAR34, pCsVMV, pF, pFDDLEL,
step 3, constructing a plant expression vector:
1) construction of expression vector pBI121-CsVMV
The promoter CaMV35S on the expression vector pBI121 is replaced by CsVMV on the cloning vector pCsVMV to improve the expression efficiency of the vector in plants,
2) construction of expression vector pBI121-CsVMV-GFP
3) Construction of expression vector pBI121-CsVMV-GFP-Mar12
Plasmids pBI121-CsVMV-GFP and pMAR12 were each digested separately with HindIII
4) Construction of intermediate expression vector pUCM-GUS-GFP
5) Construction of pUCM-GUS-GFP-MAR34 intermediate expression vector
6) Construction of intermediate expression vectors pUCM-F-GFP-MAR34 and pUCM-FKDEL-GFP-MAR34
7) Plant expression vectors pBI121-CsVMV-GFP-F-MAR12-MAR34 and
pBI121-CsVMV-GFP-F-KDEL-MAR12-MAR34 construction
8) Plant expression vectors pBI121-CsVMV-F-MAR12-MAR34 and
pBI121-CsVMV-F-KDEL-MAR12-MAR34 construction
9) And constructing a plant expression vector pBI 121-F.
8. The method for producing a plant expression vector of claim 7, wherein the plant expression vectors pBI121-CsVMV-GFP-F-MAR12-MAR34 and
pBI121-CsVMV-GFP-F-KDEL-MAR12-MAR34 was constructed as follows:
(1) enzyme digestion:
plasmids pBI121-CsVMV-GFP-MAR12 and EcoRI were double digested with XbaI and EcoRI, respectively
pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34
Enzyme digestion system: mu.L of plasmid, 2.5. mu.L of 10 XM buffer, ul of each of the two enzymes, 10.5. mu.L of ddH20, total reaction volume of 25. mu.L, digestion at 37 ℃ for 2.5h,
(2) the two fragments pBI121-CsVMV-GFP-MAR12, pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were recovered by agarose gel electrophoresis as fragments A, B and C, respectively,
(3) connecting:
a connection system: mu.L of fragment A, 4. mu.L of fragment B or C, 1. mu.L of 10 XT4DNA ligase buffer, 1. mu. L T4DNA ligase, 10. mu.L of total reaction system, and ligation overnight at 16 ℃.
(4) And transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
9. The method for producing the plant expression vector of claim 7, wherein the plant expression vectors pBI121-CsVMV-F-MAR12-MAR34 and
pBI121-CsVMV-F-KDEL-MAR12-MAR34 was constructed as follows:
(1) enzyme digestion:
the plasmids pBI121-CsVMV-MAR12, pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were double digested with BamHI and EcoRI, respectively
Enzyme digestion system: 10. mu.L of plasmid, 2.5. mu.L. times.K buffer, two enzymes, each ul, 10.5. mu.L of ddH20, total reaction volume of 25. mu.L, digestion at 37 ℃ for 3h,
(2) the two fragments pBI121-CsVMV-MAR12, pBI121-GFP-F-MAR34 and pBI121-GFP-F-KDEL-MAR34 were recovered by agarose gel electrophoresis as fragments A, B and C, respectively,
(3) connecting:
a connection system: mu.L of fragment A, 4. mu.L of fragment B or C, L. mu. L L0XT4DNA ligase buffer, L. mu. L T4DNA ligase, 10. mu.L of total reaction system, ligation overnight at 16 ℃,
(4) and transforming escherichia coli competent cells, selecting single colony PCR verification, and extracting plasmid enzyme digestion verification.
10. The method for preparing a plant expression vector of claim 7, wherein the plant expression vector pBI121-F is constructed by the steps of:
(1) enzyme digestion:
pBI121 plasmid is digested by SmaI and SacI
The enzyme digestion system is as follows: 10 μ L of plasmid, 2.5 μ L of 10 XT buffer, 2.5 μ L of 1% BSA, two enzymes each μ L, 8 μ L of ddH20, the total reaction volume is 25 mu L, the enzyme digestion is carried out for 3h at the temperature of 30 ℃,
enzyme PvuII and BstxI step-by-step double enzyme digestion pF
The enzyme digestion system is as follows: 10 mu L of plasmid, 2.5 mu L of 10 XM buffer, PvuII L mu L, 11.5 mu L of ddH20, the total reaction volume is 25 mu L, the enzyme digestion is carried out for 4h at 37 ℃, ethanol is carried out after the enzyme digestion is finished, the precipitated DNA is recovered and is carried out for the next enzyme digestion,
the enzyme digestion system is as follows: 10 u L ethanol recovery DNA, 2.5 u Ll0 xH buffer, BstxI L u L, 11.5 u L ddH20, total reaction volume of 25 u L, 45 degrees C enzyme digestion for 4H,
(2) agarose gel electrophoresis of the restriction products pBI121 and pF, respectively recovering two DNA fragments marked as fragment A, B,
(3) connection of
A connection system: mu.L of fragment A, 5. mu.L of fragment B, 2.5. mu.L of L L0XT4DNA ligase buffer, 1. mu. L T4DNA ligase, 14. mu.L of ddH20, in a total reaction system of 25. mu.L, ligated overnight at 16 ℃,
(4) transforming escherichia coli competent cells, selecting single colony PCR forward and reverse verification, extracting plasmid enzyme digestion verification by primers of the upstream of MAR12-PCR-IN, the downstream of F, the upstream of F and the downstream of MAR34-PCR-IN respectively.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107098979A (en) * | 2017-06-22 | 2017-08-29 | 中国动物疫病预防控制中心 | PPR virus H F fusion proteins and its relevant biological material and application |
| CN107236047A (en) * | 2017-06-22 | 2017-10-10 | 中国动物疫病预防控制中心 | Recombinate the solubility expression method of PPR virus H F fusion proteins |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102178947A (en) * | 2011-03-16 | 2011-09-14 | 中国人民解放军军事医学科学院军事兽医研究所 | Live vector vaccine for expressing peste des petits ruminants virus (PPRV) H gene and preparation method thereof |
-
2014
- 2014-11-20 CN CN201410669120.0A patent/CN104498527A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102178947A (en) * | 2011-03-16 | 2011-09-14 | 中国人民解放军军事医学科学院军事兽医研究所 | Live vector vaccine for expressing peste des petits ruminants virus (PPRV) H gene and preparation method thereof |
Non-Patent Citations (6)
| Title |
|---|
| DEBORAH A. SAMAC等: "A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa)", 《TRANSGENIC RESEARCH》 * |
| 刘霞: "农杆菌介导小反刍兽疫病毒H、F基因转化苜蓿的初步研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 孙娟 等: "小反刍兽疫病毒H、F基因转基因苜蓿表达载体的构建", 《黑龙江畜牧兽医》 * |
| 朱学亮 等: "小反刍兽疫病毒F基因真核载体的构建与表达", 《中国兽医科学》 * |
| 杨国锋 等: "转基因苜蓿植物疫苗的研究", 《中国草学会饲料生产委员会第15次饲草生产学术研讨会论文集》 * |
| 杨辉: "农杆菌介导小反刍兽疫病毒F基因转化苜蓿的研究", 《万方数据 学位论文》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107098979A (en) * | 2017-06-22 | 2017-08-29 | 中国动物疫病预防控制中心 | PPR virus H F fusion proteins and its relevant biological material and application |
| CN107236047A (en) * | 2017-06-22 | 2017-10-10 | 中国动物疫病预防控制中心 | Recombinate the solubility expression method of PPR virus H F fusion proteins |
| CN107236047B (en) * | 2017-06-22 | 2020-05-19 | 中国动物疫病预防控制中心 | Soluble expression method of recombinant peste des petits ruminants virus H-F fusion protein |
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