CN116064649B - Method for expressing classical swine fever virus E2 protein in Nicotiana benthamiana - Google Patents
Method for expressing classical swine fever virus E2 protein in Nicotiana benthamiana Download PDFInfo
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Abstract
The invention provides a method for efficiently expressing a classical swine fever virus E2 protein in Nicotiana benthamiana. The E2 protein can be visually observed to be expressed in leaves under the ultraviolet irradiation condition by fusion expression of the classical swine fever virus C strain protein E2, 6xHis tag, GS connecting sequence and GFPuv fluorescent protein in Nicotiana benthamiana. After extracting tobacco leaf protein, the total protein is treated by tobacco etching virus protease, and purified by Ni-NTA nickel column, thus obtaining high purity E2-6XHis protein.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for efficiently expressing classical swine fever virus E2 protein in Nicotiana benthamiana and application thereof.
Background
Swine fever (classical swine fever, CSF) is a highly contagious, lethal infectious disease in pigs caused by swine fever virus (classical swine fever virus, CSFV). The classical swine fever virus E2 protein is a main immunogenic protein and is a first choice antigen for establishing CSFV serological detection. The E2 protein is present on the surface of a viral particle or host cell and plays an important role in viral adsorption, invasion into host cells, antibody production, induction of protective immune responses, and virulence. The E2 protein of classical swine fever virus contains an O-glycosylation site (OLG) at position 75 and a total of 6N-glycosylation sites at positions 116, 121, 185, 229, 260 and 297, mutations in these glycosylation sites resulting in reduced viral virulence. Studies have shown that the glycosylation core pathway is quite conserved among animals and plants, so that the expression of classical swine fever envelope glycoprotein E2 in tobacco is feasible.
With rapid development of pharmaceutical biotechnology and plant genetic engineering, the use of transgenic plants as bioreactors for expressing important recombinant protein antigens has become a big hotspot, not only greatly reducing the production costs of these expensive drugs, but also simplifying the way in which they are stored, transported and used. For example, the first worldwide plant-derived new crown vaccine COVIFENZ developed by the biopharmaceutical company, medicinal go and Ghatti (GSK) in tandem was produced by the model plant "ben's smoke.
The Nicotiana benthamiana (Nicotiana benthamiana) belongs to the genus Nicotiana of the family Solanaceae, is a heterotetraploid, and its genome consists of 19 chromosomes. The Nicotiana benthamiana plant is small but has large and luxuriant leaves, short culture period, easy planting under laboratory conditions, and is beneficial to collecting a large amount of experimental materials; the Nicotiana benthamiana as a model plant can finish gene expression regulation and control and protein post-translational modification in a plant body, and is more suitable for functional verification of heterologous genes than E.coil and yeast; along with the development of visual reporter genes (fluorescent protein genes and the like) and the great development of agrobacterium-mediated gene transient transformation systems, the functional research of expressing a large amount of exogenous proteins in the Nicotiana benthamiana is more convenient, so that the Nicotiana benthamiana is also widely applied to the aspects of protein subcellular localization, protein interaction, biological medicine production and the like at present.
The green fluorescent protein gene (Green fluorescent protein, abbreviated as GFP) is a reporter gene commonly used in molecular biology research, is a protein consisting of about 238 amino acids, can be excited by blue light to ultraviolet light to emit green fluorescence, and in plant research, various microscopes are usually required to determine whether the gene is expressed or not, and false positive misjudgment is caused occasionally due to plant autofluorescence. GFPuv is a variant of GFP protein which not only has strong fluorescence, but also excitation light in the invisible ultraviolet spectrum region, which can be observed with the naked eye without any filter, which greatly facilitates our observation of the expression of the protein of interest in the host.
Disclosure of Invention
The present invention provides a recombinant vector for producing an immunogenic substance from Nicotiana benthamiana, comprising: a) a classical swine fever virus E2 protein sequence, b) a tag sequence, c) a Tev recognition sequence, d) a junction sequence, E) a reporter gene sequence, f) a plant expression vector.
The present invention also provides a method for expressing a classical swine fever virus E2 protein using Nicotiana benthamiana, comprising the steps of constructing a recombinant vector for producing an immunogenic substance from Nicotiana benthamiana, and transforming, infecting or inducing a host cell with the recombinant vector, wherein the recombinant vector comprises a) a classical swine fever virus E2 protein sequence, b) a tag sequence, c) a Tev recognition sequence, d) a linker sequence, E) a reporter gene sequence, and f) a plant expression vector.
In some embodiments, the recombinant vector has a) a swine fever virus E2 protein sequence shown in SEQ ID NO. 1.
In some embodiments, the b) tag sequence is a 6XHis tag sequence, d) linker sequence is a GS sequence, and the 6XHis-Tev site-GS sequence is set forth in SEQ ID NO. 2.
The present invention provides a recombinant vector for producing an immunogenic substance from Nicotiana benthamiana, comprising: a) a classical swine fever virus E2 protein sequence SEQ ID NO:1, b) a 6xHis tag sequence, c) a Tev recognition sequence, d) a junction sequence GS, E) a reporter gene sequence, f) a plant expression vector, wherein b) the 6xHis tag sequence, c) the Tev recognition sequence, d) the sequence 6xHis-Tev site-GS of the junction sequence GS is as shown in SEQ ID NO:2.
The invention also provides a method for expressing a classical swine fever virus E2 protein by using Nicotiana benthamiana, comprising the steps of constructing a recombinant vector for producing an immunogenic substance from Nicotiana benthamiana, and transforming, infecting or inducing a host cell, wherein the recombinant vector comprises a) a classical swine fever virus E2 protein sequence SEQ ID NO:1, b) a 6xHis tag sequence, c) a Tev recognition sequence, d) a junction sequence GS, E) a reporter gene sequence, f) a plant expression vector, the b) a 6xHis tag sequence, c) a Tev recognition sequence, d) a junction sequence GS, and the sequence 6xHis-Tev site-GS of which is shown as SEQ ID NO:2.
In some embodiments, in the recombinant vector, the e) reporter gene sequence is a green fluorescent protein gene sequence. In some embodiments, the green fluorescent protein gene is a mutated green fluorescent protein gene having the sequence set forth in SEQ ID NO. 4.
The present invention provides a recombinant vector for producing an immunogenic substance from Nicotiana benthamiana, comprising: a) a classical swine fever virus E2 protein sequence SEQ ID NO:1, b) a 6XHis-Tev-GS sequence SEQ ID NO:2, c) a uvGFP sequence SEQ ID NO:4, d) a plant expression vector.
The present invention also provides a method for expressing a swine fever virus E2 protein using Nicotiana benthamiana, comprising the steps of constructing a recombinant vector for producing an immunogenic material from Nicotiana benthamiana, and transforming, infecting or inducing a host cell with the recombinant vector, wherein the recombinant vector comprises: a) a classical swine fever virus E2 protein sequence SEQ ID NO:1, b) a 6XHis-Tev-GS sequence SEQ ID NO:2, c) a uvGFP sequence SEQ ID NO:4, d) a plant expression vector.
In some embodiments, the plant expression vector in the recombinant vector is pCAMBIA1300. In some embodiments, the pCAMBIA1300 is a pCAMBIA1300-UQ recombinant vector engineered to include a UQ10 promoter.
The invention also provides host cells transformed, infected or induced with the recombinant vectors of the invention. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is agrobacterium tumefaciens.
The invention also provides plant cells infiltrated, transformed, infected or induced with the host cells of the invention. In some embodiments, the plant cell is nicotiana benthamiana.
The invention also provides a swine fever virus E2 protein produced by the plant cell.
In some embodiments, the host cell transformed, infected or induced with the recombinant vector in the method of expressing a classical swine fever virus E2 protein using nicotiana benthamiana is a bacterial cell. In some embodiments, the host cell is agrobacterium tumefaciens.
The method for efficiently expressing the classical swine fever virus E2 protein by utilizing the Nicotiana benthamiana provided by the invention further comprises the steps of host cell infiltration, transformation, infection or induction of plant cells. In some embodiments, the plant cell is nicotiana benthamiana.
The method for efficiently expressing the classical swine fever virus E2 protein by utilizing the Nicotiana benthamiana further comprises the steps of extracting tobacco leaf protein, treating total protein by using tobacco etching virus protease, and purifying by using a Ni-NTA nickel column to obtain high-purity E2-6xHis protein.
In some embodiments, the method for efficiently expressing the swine fever virus E2 protein BY using the Nicotiana benthamiana provided BY the invention further comprises constructing a transgenic tobacco strain or a tobacco suspension cell BY2 cell line for stably expressing the swine fever E2 protein.
The E2 protein obtained by the invention can be used for preparing vaccines, and can induce animal organisms to generate antibodies against swine fever viruses so as to prevent the viruses from infecting the animal organisms. The E2 protein obtained by the invention can also be used for preparing a reagent for diagnosing or detecting the infection of the swine fever virus.
The invention provides a recombinant vector for producing an immunogenic substance, namely, the classical swine fever virus E2 protein, by optimizing a nucleotide sequence encoding the classical swine fever virus E2 protein (the codon is optimized to be a Benshi tobacco preference type codon) so as to adapt the nucleotide sequence to Benshi tobacco. The substance can induce immune response against swine fever virus in mammals. The recombinant vector at least comprises a nucleotide sequence for encoding a classical swine fever virus E2 protein. The recombinant vector is introduced into plant cells by agrobacterium, so that the plant cells express the E2 protein, and the protein can be used as an immunogenic substance. The expression system provided by the invention can be used for efficiently expressing the swine fever virus E2 protein in Nicotiana benthamiana, and the E2 protein provided by the invention has obvious antigen reactivity. When the E2 protein disclosed by the invention is used in a detection reagent, the analysis sensitivity and the clinical compliance rate are higher.
Terminology
As used herein, the terms "transformation," "infection," "induction," and "infiltration" refer to any process of introducing or delivering exogenous DNA into a host cell using methods well known in the art.
Description of the drawings:
FIG. 1: corresponding base sequence alignment of E2 protein extracellular region before and after codon optimization;
fig. 2: agarose gel electrophoresis of GFPuv and E2 fragments obtained by PCR amplification;
fig. 3: schematic diagram of recombinant binary expression vector E2-1300 UQ;
fig. 4: expression of E2-GFPuv fusion protein in tobacco under ultraviolet lamp;
fig. 5: the efficiency of E2 protein expression in tobacco was compared before and after codon optimization.
Detailed Description
The present invention will be described in detail below with reference to examples, which are intended to illustrate the present invention only, and are not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions are within the scope of the present invention.
Example 1: construction of recombinant vector
The operation steps are as follows:
1. the construction method of the plant binary expression vector pCAMBIA1300-UQ comprises the following steps: the plant binary expression vector pCAMBIA1300 (GenBank: AF 234296.1) is a common plasmid in laboratory, and we firstly design a primer pair UQ-F/UQ-R, and uses the UQ10 sequence (GenBank: KY 080693.1) as a templateMax (Vazyme #P505) high-fidelity polymerase amplification gave a UQ10 promoter fragment, and then UQ10 was assembled onto pCAMBIA1300 using ClonExpress MultiS One Step Cloning Kit (Vazyme #C113); furthermore, we designed primer pair tNOS-F/R, amplified with the above high fidelity enzyme to obtain tNOS termination sequence fragment using NOS termination sequence as template (GenBank: MK 896896.1), and assembled into recombinant vector obtained in the previous step to obtain the whole protein tableThe pCAMBIA1300-UQ recombinant vector reaching the framework.
UQ-F：5'-CTATGACATGATTACGAATTCGACGAGTCAGTAATAAACGGCGT-3'
UQ-R：5'-CTAGAGGATCCCCCGGGTACCCGCACTCGAGCTGTTAATCAGA-3'
tNOS-F：5'-GACCTGCAGGCATGCAAGCTTAGCTCGAATTTCCCCGATCG-3'
tNOS-R：5'-ACGACGGCCAGTGCCAAGCTTCCGATCTAGTAACATAGATGACACCG-3'
2. The amino acid sequence of E2 protein of the classical swine fever virus strain C is obtained by searching in NCBI (https:// www.ncbi.nlm.nih.gov /) of the national center for biotechnology information (AF 531433.1);
3. e2 is subjected to transmembrane domain prediction by using a deep TMHMM (https:// dtu. Biological. Com/deep TMHMM) tool, and the result shows that amino acids 1-341 of E2 protein are extracellular regions of the protein;
4. using a gold GenSmart TM Codon Optimization the codon optimization tool optimizes the codon corresponding to the amino acid sequence of the extracellular region of the E2 protein into the Nicotiana benthamiana preference type codon (the optimized codon sequence is shown as SEQ ID NO: 1). The corresponding base sequences of the extracellular region of the E2 protein before and after codon optimization are compared by using ESPrip 3.0 on-line software, and the sequence consistency is 75%. As shown in the attached figure 1, 1 is a base sequence before optimization, the GC content is 48%,2 is a base sequence after optimization, and the GC content is reduced to 43%;
SEQ ID NO:1
ATGAGGTTGGCCTGCAAGGAAGATTATCGTTATGCTATTAGTTCAACTGATGAAATTGGGTTACTTGGAGCTGGAGGCCTCACAACGACTTGGAAAGAGTACAATCATGATTTGCAGCTAAATGATGGAACTGTGAAAGCTAGTTGTGTTGCAGGATCCTTCAAGGTTACTGCGTTGAACGTGGTTAGCCGACGGTACTTGGCAAGTCTGCACAAGAAGGCACTGCCAACCAGCGTCACCTTTGAGCTCCTTTTTGATGGAACAAATCCATCTACGGAGGAGATGGGGGATGATTTCCGGAGCGGCTTGTGCCCATTTGACACATCACCCGTCGTGAAAGGAAAATATAACACAACATTGCTGAATGGATCTGCATTTTATCTTGTTTGTCCTATTGGATGGACAGGTGTTATTGAATGCACAGCAGTATCTCCTACTACTCTACGTACTGAGGTTGTTAAAACATTTAGGCGAGACAAGCCTTTTCCTCACAGAATGGATTGTGTAACTACAATTGTTGAAAATGAGGATCTTTTCTATTGTAAATTAGGGGGAAATTGGACTTGTGTGAAGGGTGAACCTGTAGTGTACACCGGTGGGGTCGTCAAGCAATGTAGATGGTGTGGTTTCGATTTTGATGGTCCCGACGGTTTACCACATTATCCAATCGGTAAATGTATCCTCGCCAATGAAACTGGTTACAGAATAGTTGACTCCACCGATTGCAACAGAGATGGCGTAGTTATATCTACAGAAGGCTCTCATGAATGTTTGATTGGCAATACCACAGTAAAAGTTCATGCTTCAGATGAGAGGTTAGGGCCTATGCCTTGCAGACCAAAAGAAATAGTGTCATCTGCTGGACCGGTGATGAAGACTTCGTGCACTTTCAACTATACCAAAACTTTGAAGAATCGCTATTATGAACCAAGAGACAGTTATTTTCAACAATACATGCTTAAGGGCGAATATCAGTATTGGTTTGACCTTGATGCAACAGATAGGCATTCAGATTACTTCGCTGAG
5. optimizing codons corresponding to a 6XHis tag, a Tev recognition site sequence ENLYFQG and a GS linker sequence (HHHHHHENLYFQGGS) into a Nicotiana benthamiana preference type codon (the optimized codon sequence is shown as SEQ ID NO: 2);
SEQ ID NO:2
CATCATCATCACCACCATGAAAATCTTTATTTTCAAGGAGGTTCT
6. the synthetic codon is optimized into the base sequence of tobacco preference type coding E2-6xHis-Tev site-GS linker (the sequence is shown as SEQ ID NO: 3), the primer pair F1/R1 and F2/R2 is designed, then the nucleotide coding sequence of SEQ ID NO:3 (the nucleotide coding sequence of E2-6xHis-Tev site-GS) and the nucleotide coding sequence of SEQ ID NO:4 (the nucleotide coding sequence of uvGFP, which is derived from GenBank: U62637.1) are used as templatesMax (Vazyme#P505) high fidelity polymerase amplifies E2 and uvGFP;
F1:5’-AACAGCTCGAGTGCGGGTACCATGAGGTTGGCCTGCAAGG-3’
R1:5’-TGCTAGCCATAGAACCTCCTTGAAAATAAAGATTTTC-3’
F2:5’-AGGAGGTTCTATGGCTAGCAAAGGAGAAGAACTT-3’
R2:5’-CAGGTCGACTCTAGAGGATCCTTATTTGTAGAGCTCATCCATGCC-3’
SEQ ID NO:3
ATGAGGTTGGCCTGCAAGGAAGATTATCGTTATGCTATTAGTTCAACTGATGAAATTGGGTTACTTGGAGCTGGAGGCCTCACAACGACTTGGAAAGAGTACAATCATGATTTGCAGCTAAATGATGGAACTGTGAAAGCTAGTTGTGTTGCAGGATCCTTCAAGGTTACTGCGTTGAACGTGGTTAGCCGACGGTACTTGGCAAGTCTGCACAAGAAGGCACTGCCAACCAGCGTCACCTTTGAGCTCCTTTTTGATGGAACAAATCCATCTACGGAGGAGATGGGGGATGATTTCCGGAGCGGCTTGTGCCCATTTGACACATCACCCGTCGTGAAAGGAAAATATAACACAACATTGCTGAATGGATCTGCATTTTATCTTGTTTGTCCTATTGGATGGACAGGTGTTATTGAATGCACAGCAGTATCTCCTACTACTCTACGTACTGAGGTTGTTAAAACATTTAGGCGAGACAAGCCTTTTCCTCACAGAATGGATTGTGTAACTACAATTGTTGAAAATGAGGATCTTTTCTATTGTAAATTAGGGGGAAATTGGACTTGTGTGAAGGGTGAACCTGTAGTGTACACCGGTGGGGTCGTCAAGCAATGTAGATGGTGTGGTTTCGATTTTGATGGTCCCGACGGTTTACCACATTATCCAATCGGTAAATGTATCCTCGCCAATGAAACTGGTTACAGAATAGTTGACTCCACCGATTGCAACAGAGATGGCGTAGTTATATCTACAGAAGGCTCTCATGAATGTTTGATTGGCAATACCACAGTAAAAGTTCATGCTTCAGATGAGAGGTTAGGGCCTATGCCTTGCAGACCAAAAGAAATAGTGTCATCTGCTGGACCGGTGATGAAGACTTCGTGCACTTTCAACTATACCAAAACTTTGAAGAATCGCTATTATGAACCAAGAGACAGTTATTTTCAACAATACATGCTTAAGGGCGAATATCAGTATTGGTTTGACCTTGATGCAACAGATAGGCATTCAGATTACTTCGCTGAGCATCATCATCACCACCATGAAAATCTTTATTTTCAAGGAGGTTCT
SEQ ID NO:4
ATGGCTAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCTACATACGGAAAGCTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA
7. the size of the E2-6XHis-Tev site-GS linker and uvGFP sequences obtained was confirmed by agarose gel electrophoresis. And 5 mu L of PCR amplification products of GFPuv and E2 are respectively absorbed, 1% agarose gel is prepared, agarose gel electrophoresis is carried out under the constant voltage of 140V by using 1 xTAE as an electrophoresis buffer, gel blocks are taken out for imaging after 20 minutes, and the result shows that the band size of GFPuv is about 600bp, and the band size of E2 is about 1000bp, so that the method meets the expectations. The results are shown in FIG. 2.
8. E2-6xHis-Tev site-GS linker and uvGFP were assembled into a modified plant binary expression vector pCAMBIA1300-UQ10 by ClonExpress MultiS One Step Cloning Kit (Vazyme#C113) to obtain a recombinant plasmid UQ10pro: E2-6xHis-Tev site-GS linker-uvGFP-tNOS/pCAMBIA1300 (abbreviated as E2-1300 UQ), and the sequence was confirmed to be accurate by sequencing. A schematic representation of recombinant plasmid E2-1300UQ is shown in FIG. 3.
9. E2-1300UQ was transformed into Agrobacterium tumefaciens GV3101 by freeze thawing. The specific operation method is as follows: (1) Thawing GV3101 Agrobacterium competent cells stored at-80deg.C on ice or at room temperature; (2) Under aseptic condition, adding 1 μg of E2-1300UQ plasmid DNA to be transformed into the freshly frozen competent cell suspension, gently mixing, and standing in ice bath for 10min; (3) placing the centrifuge tube in liquid nitrogen and freezing for 5min; (4) rapidly placing the centrifuge tube in a water bath at 37 ℃ and keeping for 5min; (5) placing the centrifuge tube back into the ice bath, and keeping for 5min; (6) Adding 800 mul of LB liquid culture medium without antibiotics under aseptic condition, and shake culturing for 2-3 hours at 28 ℃; (7) The cells were harvested by centrifugation at 5000rpm for 1min, about 100. Mu.l of supernatant was left to resuspend, and the cells were spread evenly on LB solid medium plates containing Rifampicin (Rifampicin), gentamicin (Gentamicin) and Kanamycin (Kanamycin). After the liquid in the waiting plate is completely absorbed, the waiting plate is inverted and is cultivated for 2 to 3 days at the temperature of 28 ℃; (8) 3 single clones were randomly picked up for 24h of liquid culture, then 1. Mu.l of bacterial liquid was taken, amplified with F1/R2 primer pair and 2 XTaq Master Mix (Vazyme#P111), run on agarose gel electrophoresis, and confirmed that the recombinant plasmid had been successfully transformed into GV3101.
F1:5’-AACAGCTCGAGTGCGGGTACCATGAGGTTGGCCTGCAAGG-3’
R2:5’-CAGGTCGACTCTAGAGGATCCTTATTTGTAGAGCTCATCCATGCC-3’
10. The helper plasmid pSoup-p19 purchased from Shanghai Utility company was transferred into GV3101 in the same manner as described above to obtain GV3101 containing pSoup-p 19.
Example 2: transient transformation of tobacco
The E2 protein was transiently expressed by mixing the E2-1300UQ and pSoup-p 19-containing Agrobacterium GV3101 in equal proportions and injecting tobacco.
The specific operation method is as follows:
(1) 1ml of overnight culture of the above two agrobacteria containing E2-1300UQ and pSoup-p19 were transferred to 25ml of LB liquid medium containing the corresponding antibiotics and acetosyringone, and cultured overnight at 28 ℃;
(2) Detecting the OD600 value of the bacterial liquid cultured overnight;
(3) Centrifuge 5000g for 15min, removing supernatant, and adding heavy suspension (10 mM MgCl) 2 The method comprises the steps of carrying out a first treatment on the surface of the 10mM Mes-KOH, pH5.6;100 μm acetosyringone (As)) was resuspended to a final OD600 of 0.4;
(4) Mixing E2-1300UQ/GV3101 and pSoup-p19/GV3101 in equal proportion, and standing for 2-3h;
(5) The injector reverse plate is pressed by a thumb to inject liquid into tobacco leaves from the lower epidermis of the leaves, and the tobacco leaves are wet after successful injection;
(6) After 2-5 days, the uvGFP fluorescent signal was detected under a portable long wavelength uv lamp.
The results showed that the E2-1300UQ/GV3101 Agrobacterium-injected tobacco leaves were able to visually observe a green fluorescent signal under UV light, indicating that the classical swine fever virus E2 protein was successfully expressed in tobacco, see FIG. 4.
Example 3: genetic transformation of tobacco
The tobacco transgenic plant stably expressing E2 is obtained, and the specific operation method is as follows:
(1) Preparation: aseptically culturing (30 days) a Nicotiana benthamiana plant; forceps, scissors, scalpel, petri dish (diameter 12 cm) with 5-6 layers of filter paper;
(2) Preparing a co-culture medium: MS medium+NAA (0.1 mg/L) +6-BA (1.5 mg/L);
(3) The blade was cut into about 0.5-1cm pieces in a glass dish with a scalpel 2 The blade is rapidly scratched through the blade, and the blade block is cut off, so that the incision is ensured to be tidy as much as possible. Clamping the cut leaves into E2-1300UQ/GV3101 agrobacterium liquid with OD value of 0.4-0.6 by forceps, and soaking for 8-10min;
(4) Pouring out the bacterial liquid, transferring the leaves to filter paper in a glass dish by using tweezers to suck the bacterial liquid on the leaves, and placing the leaves on an ultra-clean workbench for a period of time to suck the bacterial liquid on the surfaces of the leaves;
(5) Placing the leaves of the fungus liquid to the co-culture medium, and culturing in dark at 25 ℃ for three days;
(6) Transferring tobacco leaves after three days of co-culture to a culture medium (MS+1.5 mg/L6-BA+0.1 mg/LNAA+30mg/L hygromycin+100 mg/L carbenicillin), enabling the leaves to contact and adhere to the surface of the culture medium as much as possible, leaving a certain gap between the leaves, and culturing under the illumination condition of 25 ℃;
(7) Culturing in an illumination culture room for two to three weeks to obtain tobacco buds, cutting the buds out, and transferring the tobacco buds to a strong seedling culture medium (MS+0.1 mg/L6-BA+0.01mg/LNAA+30mg/L hygromycin+100 mg/L carbenicillin);
(8) About one week later, picking out tobacco seedlings growing out roots, and transferring the tobacco seedlings to a rooting medium (MS+30 mg/L hygromycin+100 mg/L carbenicillin);
(9) The E2-uvGFP was observed with an ultraviolet lamp to determine whether it was expressed in tobacco seedlings, and DNA of tobacco seedlings was extracted for PCR identification.
(10) Collecting leaves, extracting total protein, treating with TEV protease, cutting fusion protein E2-6xHis-Tev-GFPuv, and purifying with Ni-NTA nickel column to obtain high purity E2-6xHis protein. Westernblot immunoblotting experiments are carried out to detect the size of the target protein E2-6XHis, and comparison shows that the size of the E2-6XHis protein is about 45 kDa. For the complete E2 protein, the relative molecular weight of deglycosylated protein is 42kDa, and the molecular weight of deglycosylated protein is about 48kDa after glycosylation modification. The molecular mass of the E2-6xHis protein expressed by the method is predicted to be 39kDa by analysis software, but the actual running and cementing fruits show that the size of the E2 protein is about 45kDa, which indicates that the E2 protein can be subjected to complete glycosylation modification after being expressed in Nicotiana benthamiana.
Example 4: transformation of tobacco suspension cells BY2
(1) 4ml of fresh BY2 suspension cells were pipetted into a 250ml sterile Erlenmeyer flask containing 40ml of liquid medium and cultured with shaking at 28℃at 130rpm (first day);
(2) 1L liquid medium contains 4.03g MS basal salt, 0.2g KH 2 PO 4 30g of sucrose; furthermore, at 1:1000, adding vitamin mother liquor according to the formula: 50ml of mother liquor containing 0.02g of 2,4-D (1 ml of absolute ethyl alcohol is added for dissolution assisting), 0.05g of Thiamine and 5g of inositol (myo-inositol), filtering and sterilizing the mother liquor, and then sub-packaging in 1.5ml of EP tube, and preserving at-20 ℃;
(3) E2-1300UQ/GV3101 was selected and cultured overnight (third day) with 5ml LB medium containing rifampicin, gentamicin and kanamycin;
(4) 100 μl of E2-1300UQ/GV3101 bacteria solution was aspirated, transferred to 10ml of LB medium containing antibiotics, and cultured overnight;
(5) Sucking 4ml BY2 suspension cells by using a 10ml pipettor, transferring to a sterile culture dish, adding 500 mu l of bacterial liquid to mix with the suspension cells, sealing by using a sterile adhesive tape, standing at 25-28 ℃ and incubating for 2 days (fourth day);
(6) Spreading the suspension cell and E2-1300UQ/GV3101 mixed solution on a culture medium containing hygromycin and carbenicillin, and culturing for 10-14 days;
(7) After small opaque callus pieces were observed on the petri dish, the callus pieces were mashed and transferred to new selection medium for further screening;
(8) Observing the expression condition of E2-uvGFP in the calli under an ultraviolet lamp, and selecting the calli with better expression for suspension culture;
(9) Taking a suspension cell line successfully expressing E2-uvGFP, and performing subculture every 5-7 days;
(10) Taking E2-uvGFP/BY2 suspension cells in an exponential growth phase, centrifuging at 4000rpm for 5 minutes, discarding the supernatant, repeatedly freezing and thawing cell sediment for 5 times BY liquid nitrogen, adding an extracting solution, extracting total protein, treating BY TEV protease, cutting fusion protein E2-6xHis-Tev-GFPuv, and purifying BY a Ni-NTA nickel column to obtain high-purity E2-6xHis protein.
Comparative example 1: prokaryotic expression of CSFV E2 protein (E.coli)
(1) Synthesizing the extracellular region of the original nucleotide sequence of E2, designing a primer, adding an initiation codon ATG at the 5' -end, and then usingAmplifying E2 by Max high-fidelity polymerase, and recombining to a protein expression vector pET-28a to obtain a recombinant plasmid E2-6xHis-pET-28a;
(2) Mu.l of the recombinant plasmid was added to 100. Mu.l of BL21 (DE 3) competent bacteria and placed on ice for 20min;
(3) Heat-shock at 42 ℃ for 90sec, rapidly placing in ice for 5min, and adding 600 μl of LB culture solution;
(4) Shaking at 37 ℃ for 1h at 220rpm/min, centrifuging, coating on LB plates containing 50 mug/mL Kan, and culturing at 37 ℃ in an inverted manner overnight;
(5) The monoclonal on the transformation plate is picked and inoculated in a test tube containing 50 mug/mL Kan of 3mL LB culture solution, and is shaken at 37 ℃ for 220rpm/min for overnight;
(6) Inoculating the strain into 30mL LB culture solution of 50 mug/mL Kan at the ratio of 1:100 in the next day, and shaking at 37 ℃ for 220r/min until the OD600 of the strain is 0.6-0.8;
(7) Taking out 1mL of culture, centrifuging at 10000rpm/min at room temperature for 2min, discarding the supernatant, and re-suspending the bacterial precipitate with 100 μl of 1 Xloading buffer;
(8) IPTG was added to the remaining culture to a final concentration of 0.5mM, and the mixture was shaken at 37℃for 4 hours at 220rpm/min to induce expression of the fusion protein;
(9) 1mL of the culture was removed, centrifuged at 10000r/min at room temperature for 2min, and the supernatant was discarded, and the bacterial pellet was resuspended in 100. Mu.L of 1 Xloading buffer. Centrifuging the rest culture at 4000r/min for 10min, discarding supernatant, and re-suspending thallus sediment with PBS;
(10) After the heavy suspension is crushed by ultrasonic waves, respectively taking supernatant and precipitation liquid, and adding loading buffer solution for heavy suspension;
(11) 12% SDS-PAGE detection analysis is carried out, and coomassie brilliant blue staining is carried out to develop a band;
(12) Performing a Westernblot experiment, namely using an anti-His mouse antibody as a primary antibody and a horseradish peroxidase-marked sheep anti-mouse antibody as a secondary antibody, and detecting a target protein;
(13) Purifying E2-6XHis protein by Ni column, and directly coating the purified E2 protein.
Comparative example 2: HEK293T cell expressing CSFV E2 protein
(1) Designing primer, adding initiation codon ATG at 5' end, usingAmplifying E2-6xHis by Max high-fidelity polymerase, recombining to an expression vector pcDNA3.0, and obtaining a recombinant vector E2-6xHis-pcDNA3.0;
(2) HEK293T cells were routinely cultured in DMEM medium containing 10% FBS, trypsinized 1 day prior to transfection, passaged cells, plated in 6-well plates;
(3) Transfecting pcDNA3.0-EGFP and pcDNA3.0-E2-6xHis recombinant vector plasmid by PEI method when the cell confluency is about 80%;
(4) After 24h of transfection, the culture medium is changed into a serum-free DMEM culture medium, after 48h of transfection, cell fluorescence is observed, the transfection efficiency is judged, and a Westernblot experiment is carried out to detect the expression of target proteins;
(5) And (3) performing affinity purification on the culture solution supernatant by using a Ni column to obtain the E2-6xHis protein.
Comparative example 3: pichia pastoris system expression CSFV E2 protein
(1) Designing primer, adding initiation codon ATG at 5' end, usingAmplifying E2-6xHis by Max high-fidelity polymerase, and inserting the E2-6xHis into pMCO-AOXalpha to obtain a recombinant plasmid pMCO-AOXalpha-E2-6 xHis;
(2) Transforming the recombinant plasmid pMCO-AOX alpha-E2-6 xHis into Pichia pastoris, inoculating the Pichia pastoris into YPD culture medium, and culturing at 28 ℃ at 200rpm/min for overnight;
(3) Resuspension with an equal volume of BMMY culture medium, at 28 ℃, continuing to induce and culture for 96 hours at 200rpm/min, and supplementing methanol to a final concentration of 0.5% every 24 hours during the culture period;
(4) And (3) performing affinity purification on the culture solution supernatant by using a Ni column to obtain the E2-6xHis protein.
Example 5: comparison of E2 protein expression efficiency in Nicotiana benthamiana before and after codon optimization
(1) The original E2 and the E2 sequence with optimized codons into tobacco preference type are respectively imported into 1300UQ, and then the respective E2-1300UQ is transformed into agrobacterium GV 3101;
(2) Selecting monoclonal, shaking, and taking GV3101 injection tobacco with the same volume and concentration;
(3) Respectively cutting tobacco leaves with the same mass, extracting total protein, treating with TEV protease, cutting fusion protein E2-6xHis-Tev-GFPuv, and purifying with Ni-NTA nickel column to obtain E2-6xHis protein;
(4) The concentration of the E2-6XHis protein obtained was determined using the Rapid Gold BCA Rapid protein quantification kit (cat. No. A53225) from Thermo Fisher;
(5) 4 times of repeated experiments, statistical data show that under the same condition, the original E2 sequence is used for expressing in tobacco, the concentration of the obtained E2 protein is 0.26mg/ml, and after the optimized E2 sequence is expressed in the tobacco, the concentration of the obtained E2 protein is 1.45mg/ml, which shows that the expression efficiency of the E2 in the tobacco can be greatly improved by optimizing the codon of the E2 (figure 5).
Example 6: reactivity of E2 antigen obtained by different expression systems
The reactivity of E2 antigens expressed by tobacco, E.coli BL21, HEK293T and Pichia pastoris was evaluated using the monoclonal antibodies in the classical swine fever virus antibody detection kit of IDEXX and Beijing. Our results found that tobacco expressed E2 was cross tested with mab enzyme in both kits, and the blocking rate of strong positive and negative samples was tested at different concentrations, and that strong positive samples were effectively blocked with tobacco expressed E2 as antigen (tables 1 and 2). The cross test of the prokaryotic expression E2 protein of BL21 and two competing products shows that the strong positive sample can not be well blocked (table 1 and table 2). After crossing the E2 antigen expressed by HEK293T cells with the monoclonal antibody of IDEXX, neither the positive nor the negative samples reacted (Table 1), and the reactivity after crossing with the monoclonal antibody of Jinno was consistent with the control, which suggests that there may be partial deletion of the epitope of the E2 antigen expressed by HEK293T cells (Table 2). The E2 protein expressed by pichia pastoris and the IDEXX monoclonal antibody enzyme are subjected to cross test, the blocking rate of a positive sample is slightly lower (table 1), and the blocking rate of the positive sample is higher (table 2) after cross test with Jin Nuomei. In conclusion, the results show that the tobacco expression E2 protein is used as a coating antigen, and compared with antigens obtained by other expression systems, the tobacco expression E2 protein has relatively complete structure and better reactivity with both the IDEXX monoclonal antibody enzyme and the kinoform monoclonal antibody enzyme.
TABLE 1 evaluation of antigen reactivity of E2 protein using classical swine fever virus antibody detection kit of IDEXX in the United states
TABLE 2 evaluation of antigen reactivity of E2 protein Using hog cholera Virus antibody detection kit of Beijing kinoform
Example 7: sensitivity of analysis of E2 antigen produced by different expression systems
And (3) using E2 proteins expressed by tobacco, HEK293T and pichia pastoris as coating antigens, matching with monoclonal antibody enzyme in a detection kit of the antibody of the swine fever, and analyzing the analysis sensitivity of the E2 antigens produced by different expression systems. The E2 antigen expressed by tobacco is detected by carrying out gradient dilution on the strong positive specimen monitored by the medium, and has higher analysis sensitivity (table 3) compared with E2 antigens of other expression systems.
TABLE 3 analytical sensitivity of E2 antigens produced by different expression systems
Example 8: clinical compliance of E2 antigen produced by different expression systems
The clinical coincidence rate of E2 antigen expressed by tobacco, HEK293T and pichia pastoris is further analyzed, ELISA blocking method is used for testing, and monoclonal antibody enzyme in the kit is selected as monoclonal antibody enzyme. The clinical samples are 200 negative samples and 300 positive samples of known detection information. The overall compliance of the ELISA reaction system consisting of tobacco-expressed E2 antigen was 96.2%, while the clinical compliance of the ELISA reaction system consisting of HEK293T and pichia pastoris-expressed E2 antigen was 87.2% and 92.80%, respectively, which was lower compared to the reaction system consisting of tobacco-expressed E2 antigen (table 4).
TABLE 4 clinical compliance of E2 antigens produced by different expression systems
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Claims (8)
1. A method of expressing a classical swine fever virus E2 protein using nicotiana benthamiana, comprising the steps of constructing a recombinant vector for producing an immunogenic agent from nicotiana benthamiana and transforming the recombinant vector into a host cell, and transforming nicotiana benthamiana with the host cell, wherein the recombinant vector comprises, a) a classical swine fever virus E2 protein nucleotide sequence, b) a tag sequence, c) a Tev recognition sequence, d) a linker sequence, E) a reporter gene sequence, f) a plant expression vector; the carrier structure is shown in figure 3;
wherein, the nucleotide sequence of the E2 protein of the hog cholera virus is shown as SEQ ID NO. 1, b) the tag sequence is a 6xHis tag sequence, c) the Tev recognition sequence is a Tev site, d) the connecting sequence is a GS sequence, and the 6xHis-Tev site-GS sequence is shown as SEQ ID NO. 2;
the host cell is Agrobacterium tumefaciens.
2. A recombinant vector for producing an immunogenic material from nicotiana benthamiana, comprising: a) a nucleotide sequence of a classical swine fever virus E2 protein, b) a tag sequence, c) a Tev recognition sequence, d) a junction sequence, E) a reporter gene sequence, f) a plant expression vector; the carrier structure is shown in figure 3;
wherein, the nucleotide sequence of the E2 protein of the classical swine fever virus is shown as SEQ ID NO. 1, b) the tag sequence is a 6xHis tag sequence, c) the Tev recognition sequence is Tev site, d) the connecting sequence is a GS sequence, and the 6xHis-Tev site-GS sequence is shown as SEQ ID NO. 2.
3. The method of claim 1, wherein e) the reporter gene sequence is uvGFP, which is shown in SEQ ID No. 4.
4. The recombinant vector according to claim 2, wherein the e) reporter gene sequence is uvGFP, the sequence of which is shown in SEQ ID NO. 4.
5. A host cell transformed with the recombinant vector of claim 2, said host cell being agrobacterium tumefaciens.
6. A plant cell transformed with the host cell of claim 5, said plant cell being nicotiana benthamiana.
7. The method of claim 1, further comprising extracting tobacco leaf protein, and treating the total protein with tobacco etch virus protease and purifying with a Ni-NTA nickel column to obtain high purity E2-6xHis protein.
8. A gene for encoding the E2 protein of hog cholera virus has the nucleotide sequence shown in SEQ ID No. 1.
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| CN103626852A (en) * | 2012-08-23 | 2014-03-12 | 中国科学院微生物研究所 | AraC mutant protein and application thereof |
| CN107533066A (en) * | 2015-03-05 | 2018-01-02 | 勃林格殷格翰动物保健公司 | Mk system, particularly for baculovirus expression subunit antigen Mk system |
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| CN109563518A (en) * | 2016-05-12 | 2019-04-02 | 巴伊沃爱普有限公司 | The vaccine composition and its manufacturing method for classical swine fever from plant |
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