CN111850046A - Preparation method of mandarin fish rhabdovirus glycoprotein expressed by recombinant baculovirus - Google Patents
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract
The invention discloses a preparation method of mandarin fish rhabdovirus glycoprotein expressed by recombinant baculovirus, which comprises the following steps: designing and synthesizing a primer: amplifying and purifying target genes; constructing a recombinant donor plasmid pFastBac-G; constructing a recombinant shuttle plasmid rBacmid-G; preparing recombinant baculovirus; purifying target protein and detecting Western blot. The method can obtain a large amount of soluble recombinant proteins with better antigenicity and immunogenicity and similar functions to natural proteins, and is suitable for efficient expression of mandarin fish rhabdovirus glycoprotein and preparation of DNA vaccines.
Description
Technical Field
The invention relates to a preparation method of mandarin fish rhabdovirus glycoprotein expressed by recombinant baculovirus, belonging to the field of biotechnology.
Background
Baculovirus Expression System (BES) is one of the most widely used protein expression systems at present, and is commonly used in the fields of drug development, vaccines, growth promotion factors, oncogenes, oncogene-suppressor protein products, certain oncogenic virus proteins, immunologically active molecules, gene expression regulation, and the like. The baculovirus is used as carrier to express exogenous gene in high efficiency, and hundreds of genes including animal, plant, virus, bacteria and fungus are expressed in insect cell or larva. The expression system is mainly characterized in that a large amount of soluble recombinant protein with better antigenicity and immunogenicity and similar functions to natural protein can be obtained. After the recombinant baculovirus infects insect cells, the foreign protein can be subjected to post-transcriptional processing of a plurality of eukaryotic cells, including glycosylation, phosphorylation, acylation, correct signal peptide cleavage, proteolysis and proper folding, and the recombinant protein can be aggregated and positioned on the same organelle of the natural protein, and proper oligomerization assembly can be carried out. Therefore, the vector is an ideal vector for expressing a biologically active protein.
Mandarin fish rhabdovirus (SCRV) belongs to the Rhabdoviridae (rhabdiviridae). The morbid mandarin fish is separated from a diseased mandarin fish body in 1999, the SCRV mainly infects the mandarin fish, the mandarin fish with typical symptoms is collected in an epidemic area where the mandarin fish epidemic disease occurs, the symptoms of the diseased mandarin fish are mainly manifested by congestion or bleeding points at the base parts of the lip end, the head skin, the dorsal fin and the tail fin, some fish eyeballs are protruded, the gill and the gill are whitened due to ischemia, the internal organs are locally bled, and the ascites and the internal organs are full of yellow sticky substances and die after a while. There is currently no effective treatment. For this virus, no effective vaccine and therapeutic method has been developed, and glycoprotein as its important antigen has not been studied so far using baculovirus expression system.
Among the various expression systems, prokaryotic expression systems have been the first to be studied, and are currently the most mature expression systems. The main method of this technique is to transform a vector (generally a plasmid) into which a DNA fragment of a target gene has been cloned into a bacterium (usually E.coli is selected), induce it with IPTG and finally purify it to obtain the desired target protein. The advantage is that the gene expression product can be obtained in a shorter time and the required cost is relatively low. However, prokaryotic expression systems also have a number of disadvantages that are difficult to overcome: if the commonly used expression system can not regulate and control the expression time and the expression level, the continuous expression of some genes can generate toxic action on host cells, the over-expression can cause non-physiological reaction, and the target protein is often expressed in the form of inclusion bodies, so that the product is difficult to purify; and the prokaryotic expression system has imperfect post-translational processing and modifying system and lower biological activity of the expression product.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide the preparation method of the mandarin fish rhabdovirus glycoprotein expressed by the recombinant baculovirus, which can obtain a large amount of soluble recombinant protein with better antigenicity and immunogenicity and similar functions to natural protein, and is suitable for the efficient expression of the mandarin fish rhabdovirus glycoprotein and the preparation of DNA vaccine.
In order to solve the technical problems, the invention provides a preparation method of mandarin fish rhabdovirus glycoprotein expressed by recombinant baculovirus, which comprises the following steps:
designing and synthesizing primers GF and GR, wherein the nucleotide sequences of the primers GF and GR are shown in SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;
amplifying glycoprotein genes by using mandarin fish rhabdovirus genomes as templates and GF and GR as primers;
carrying out enzyme digestion on the vector and the glycoprotein gene by adopting a double enzyme digestion method, recovering and purifying the vector and the glycoprotein gene, connecting the vector and the glycoprotein gene by using ligase, transforming the vector and the glycoprotein gene into competent cells, coating and screening positive clones, selecting the positive clones, carrying out amplification culture, extracting plasmids, and identifying the plasmids to obtain recombinant donor plasmids;
transforming the recombinant donor plasmid with correct sequencing into competent cells, coating the competent cells for blue-white screening, and performing PCR (polymerase chain reaction) and sequencing identification after purification to obtain a recombinant shuttle plasmid;
Transfecting the recombinant shuttle plasmid into Sf9 cells, collecting supernatant to obtain recombinant baculovirus and performing amplification culture;
infecting Sf9 cells with the recombinant baculovirus, centrifuging, harvesting infected cells, washing, repeatedly freezing and thawing, and harvesting supernatant to obtain the mandarin fish rhabdovirus glycoprotein.
Preferably, the amplification procedure of the glycoprotein gene is: 5min at 95 ℃, 30s at 55 ℃, 1min at 72 ℃ and 30 cycles; extension at 72 ℃ for 5 min.
Preferably, the amplification system of the glycoprotein gene is: cDNA template × 1 μ L, primer GF × 1 μ L, primer GR × 1 μ L, Premix Taq × 10 μ L, ddH2O.times.7. mu.L, total 20. mu.L.
Preferably, the enzyme is cut by using BamH I and Hind III restriction enzymes, and the cut vector is pFastBac1 vector.
Preferably, the method for screening blue and white spots is as follows: coating a three-antibody, X-gal and IPTG plate for blue-white screening, and selecting white colonies for 3 times of streak purification.
Preferably, the recombinant baculovirus is used for infecting Sf9 cells, after 72 hours, the infected cells are obtained by centrifugation at 3000 r.min < -1 > for 10min, PBS is used for centrifugal washing, PBS is added according to 10% of the volume of the culture solution to suspend the cells, the cells are repeatedly frozen and thawed for 3 times, after centrifugation, the supernatant is obtained, the cell lysis antigen solution is prepared to be subjected to SDS-PAGE electrophoresis, and is electrically transferred to a PVDF membrane, and the confining liquid is used for 2 hours at room temperature.
Preferably, the method for detecting the mandarin fish rhabdovirus glycoprotein comprises the following steps: adding a His tag antibody as a primary antibody into the mandarin fish rhabdovirus glycoprotein, and culturing at 4 ℃ overnight; and taking IgG as a secondary antibody, incubating at room temperature for 1h, washing with PBS for three times, and detecting by a chemiluminescence method.
The invention achieves the following beneficial effects:
(1) the invention selects a baculovirus expression system, introduces a prokaryotic gene regulation system into the field of eukaryotic gene regulation, and designs the prokaryotic gene regulation system according to the high specificity of the action between prokaryotic proteins and target DNA, and the target DNA has no homology with the eukaryotic gene regulation sequence basically, so that the non-specific activation or inhibition of genes does not exist; can induce the high-efficiency expression of genes; can strictly regulate gene expression, i.e. not only can control the 'switch' of gene expression, but also can artificially regulate gene expression quantity.
(2) In contrast to prokaryotic expression systems, this system enables the correct folding and post-translational modifications of proteins, including disulfide bond formation and glycosylation, phosphorylation, etc., to produce recombinant proteins with a structure similar to the native protein. The invention constructs a recombinant vector rBacmid-G by cloning the G protein gene of the SCRV and successfully expresses the G protein of the SCRV in Sf9 insect cells by utilizing a Bac-to-Bac baculovirus expression system, thereby establishing a foundation for the future research and development of mandarin rhabdovirus vaccines.
Drawings
FIG. 1 shows the results of PCR detection: m: marker DL 2000; 1. 2, 3, 4: PCR products;
FIG. 2 is a double restriction enzyme identification of pFastbac-G plasmid: m: marker (200,500,800,1200,2000,3000,4500 bp); 1: plasmid before enzyme digestion; 2: carrying out enzyme digestion on the plasmid;
FIG. 3 is PCR identification analysis of recombinant genes: m: DNA molecular mass standard; 1: negative control; 2: rBacmid-G-1; 3: rBacmid-G-2; 4: rBacmid-G-3; 5: rBacmid-G-4;
FIG. 4 shows diseased cells (A) and normal cells (B);
FIG. 5 is Western Blot identification and analysis of recombinant protein expression: m: protein molecular mass standard; 1: secretion medium (negative); 2: cell lysis supernatant (negative); 3: secretion medium (SCRV-G); 4: cell lysis supernatant (SCRV-G).
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Experimental materials and reagents: the pFastBac1 plasmid, the TOP10 strain and the pFastBac1 vector are available from Hongxin Biotechnology Ltd, Suzhou. Restriction enzymes BamH I and Hind III (Baori physician's technology (Beijing) Co., Ltd.); pfu DNA polymerase (Nanjing Dingding Biotechnology Co., Ltd.); bacterial LB medium (Qingdao Haibo Biotechnology, ltd); agarose (shanghai gene corporation); DNA gel purification kit, plasmid miniprep kit (thinking about biotechnology limited); DNA gel recovery kit, Ampicillin (Amp) (sanshu biotechnology limited, tokyo); kanamycin (Kanamycin, Kan) (Beijing Soilebao Tech., Ltd.), Gentamicin (Gentamicin, Gen) (Suzhou Hongxin Biotech Co., Ltd.), Tetracycline (Tetracycline, Tet), X-gal (Guangzhou Xiangbo Biotech Co., Ltd.), isopropyl-beta-D-thiogalactoside (Isop) Copy 1 beta-D-thiogalactoside, IPTG) (Beijing gold Markson Biotech Co., Ltd.), baculovirus expression system (
Baculovir Expression System (Invitrogen Life technologies, Inc., USA), Insect
Reagent (seimer feishell science). Other reagents are all domestic analytical purifiers.
Example 1 preparation of recombinant baculovirus expressed Mandarin Fish rhabdovirus glycoprotein
1. Primer design and Synthesis
Primers were designed based on the published sequence of the SCRV-G gene. Wherein the primer is designed after the signal peptide 5 'and the hydrophobic region 3' of the G gene are removed.
An upstream primer GF: GGATCCATGTACCCACTGTTTGTTCCGAT (SEQ ID NO: 1),
downstream primer GR: GAATTCCTAGTGATGGTGGTGATGATGAGTTCCCACCCACTCA (SEQ ID NO: 2).
The primers were synthesized by Nanjing Optimalaceae Biotechnology, Inc.
2. Amplification and purification of target gene
Amplifying glycoprotein gene (G gene) by taking the SCRV genome as a template and GF and GR as primers, wherein an amplification reaction system refers to a kit specification and comprises the following steps: cDNA template × 1 μ L, primer GF × 1 μ L, primer GR × 1 μ L, PremixTaq × 10 μ L, ddH2O.times.7. mu.L, total 20. mu.L. Amplification of reaction program: 5min at 95 ℃, 30s at 55 ℃, 1min at 72 ℃ and 30 cycles; extension at 72 ℃ for 5 min. The PCR product was detected by electrophoresis on 1% agarose, recovered and stored at 4 ℃.
The experimental results are as follows: after the gene of the partial region of the SCRV glycoprotein is amplified, the amplified product is detected by 1% agarose electrophoresis to obtain an amplified fragment with the visible size of about 1500bp (figure 1), which is consistent with the expected result. G is inserted into a pFastBac1 vector through conventional DNA enzyme digestion and connection, and sequencing shows that the pFastBac-G recombinant plasmid is successfully constructed.
3. Construction of recombinant donor plasmid pFastBac-G
Carrying out double enzyme digestion on a pFastBac1 vector and a G gene by using BamH I and Hind III restriction endonucleases respectively, recovering and purifying, connecting by using DNA ligase, transforming into an E.coli DH10 alpha competent cell, coating a plate containing AMP (100mg/mL), screening positive clones, selecting the positive clones, carrying out amplification culture, extracting a plasmid by using a plasmid extraction kit, carrying out enzyme digestion identification and sequencing, and naming the correctly identified recombinant plasmid as pFastBac-G.
The experimental results are as follows: the recombinant plasmid pFastbac-G was identified by double digestion with BamH I and Hind III, which resulted in target bands of about 1500bp and 4800bp (FIG. 2), consistent with the predicted results. The sequencing result shows that the recombinant plasmid contains a target gene fragment, the reading frame is correct, and the recombinant plasmid pFastbac-G is successfully constructed.
4. Construction of recombinant shuttle plasmid rBacmid-G
Reference to
The Baculovir Expression System operation manual is characterized in that a recombinant plasmid pFastBac-G with correct sequencing is transformed into E.coli DH10 alpha competent cells, a three-antibody, X-gal and IPTG plate is coated to carry out blue-white screening, white colonies are selected for 3 times of streaking and purification, and after the primary identification of a specific primer PCR, positive colonies are sent to Nanjing engine department biotechnology limited company for further identification by sequencing. The positive recombinant transposon whose result was confirmed to be correct was designated as rBacmid-G.
The experimental results are as follows: transforming DH10 alpha Bac colon bacillus by recombinant plasmid pFastbac-G, coating a three-antibody, X-gal and IPTG plate, culturing for 48h at 37 ℃ in the dark, selecting a white single colony for streak culture, then selecting the white single colony, inoculating the white single colony in a liquid culture medium containing LB of three antibiotics for amplification, and directly taking a bacterial liquid as a template to carry out PCR identification by using a universal primer, wherein the result is shown in figure 3, a band with the size of about 4000bp can be seen, and the result is consistent with the expected result.
5. Preparation of recombinant baculovirus
According to instect
The method of Reagent instructions, rBacmid-G is transfected into Sf9 cells, the cells are cultured for 72h at 28 ℃, after the cells are diseased, cell supernatant is collected to obtain recombinant baculovirus, and the recombinant baculovirus is marked as P1 generation recombinant baculovirus. Continuously infecting Sf9 cells with the P1 generation recombinant virus, and carrying out 3 generation amplification culture; meanwhile, a genome DNA extraction kit is used for extracting virus genome DNA, universal primers and specific primers are used for verifying whether the SCRV-G gene obtains stable recombination in purified virus or not respectively by PCR, and the recombinant virus is named as rpFB-G and is stored at 4 ℃ for later use.
The experimental results are as follows: under serum-free conditions, Sf9 cell monolayers in the logarithmic growth phase are transfected by liposome-mediated rBacmid-G DNA, and the observation day by day shows that the cells obviously have swelling and enlargement phenomena after being transfected for about 72 hours, and part of the cells are exfoliated (FIG. 4-A), while the control normal cells (FIG. 4-B) grow well, which indicates that the recombinant baculovirus rpFB-G is obtained. Collecting the culture supernatant of the pathological cells, and centrifuging at low speed to remove cells and debris to obtain the recombinant virus.
6. Purification of target protein and Western blot detection
According to an operation instruction, infecting normal Sf9 cells with the rpFB-G virus, centrifuging at 3000 r.min < -1 > for 10min after 72h to obtain infected cells, centrifuging and washing with PBS, adding PBS to suspend the cells according to 10% of the volume of a culture solution, repeatedly freezing and thawing for 3 times, centrifuging to obtain a supernatant, preparing a cell lysis antigen solution, performing SDS-PAGE electrophoresis, and performing electric transfer to a PVDF membrane, wherein the confining solution is at room temperature for 2 h; adding His tag antibody as primary antibody into SCRV-G protein, and culturing at 4 deg.C overnight; IgG (1: 200-fold dilution) was used as a secondary antibody, incubated at room temperature for 1h, washed three times with PBS, and detected by ECL (chemiluminescence).
The experimental results are as follows: carrying out Western-blot identification on rpFB-G72 h infected Sf9 cells after ultrasonic disruption, and taking normal cells as a control. The Western-blot result (figure 5) shows that a specific band exists at the position of 60kDa, but the control group does not, which indicates that the recombinant protein is successfully expressed, and the expression product can specifically react with the SCRV serum, so that the recombinant protein has good immunological activity.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Yangzhou university
<120> preparation method of mandarin fish rhabdovirus glycoprotein expressed by recombinant baculovirus
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Claims (7)
1. The preparation method of the mandarin fish rhabdovirus glycoprotein expressed by the recombinant baculovirus is characterized by comprising the following steps:
designing and synthesizing primers GF and GR, wherein the nucleotide sequences of the primers GF and GR are shown in SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;
amplifying glycoprotein genes by using mandarin fish rhabdovirus genomes as templates and GF and GR as primers;
carrying out enzyme digestion on the vector and the glycoprotein gene by adopting a double enzyme digestion method, recovering and purifying the vector and the glycoprotein gene, connecting the vector and the glycoprotein gene by using ligase, transforming the vector and the glycoprotein gene into competent cells, coating and screening positive clones, selecting the positive clones, carrying out amplification culture, extracting plasmids, and identifying the plasmids to obtain recombinant donor plasmids;
transforming the recombinant donor plasmid with correct sequencing into competent cells, coating the competent cells for blue-white screening, and performing PCR (polymerase chain reaction) and sequencing identification after purification to obtain a recombinant shuttle plasmid;
Transfecting the recombinant shuttle plasmid into Sf9 cells, collecting supernatant to obtain recombinant baculovirus and performing amplification culture;
infecting Sf9 cells with the recombinant baculovirus, centrifuging, harvesting infected cells, washing, repeatedly freezing and thawing, and harvesting supernatant to obtain the mandarin fish rhabdovirus glycoprotein.
2. The method of claim 1, wherein the glycoprotein gene is amplified by the method comprising: 5min at 95 ℃, 30s at 55 ℃, 1min at 72 ℃ and 30 cycles; extension at 72 ℃ for 5 min.
3. The method of claim 1, wherein the glycoprotein gene is amplified by a system comprising: cDNA template × 1 μ L, primer GF × 1 μ L, primer GR × 1 μ L, PremixTaq × 10 μ L, ddH2O.times.7. mu.L, total 20. mu.L.
4. The method of claim 1, wherein the digestion is performed with BamH I and Hind III restriction enzymes, and the vector for digestion is pFastBac1 vector.
5. The method of producing the recombinant baculovirus-expressed mandarin fish rhabdovirus glycoprotein according to claim 1, wherein the blue-white spot screening method comprises: coating a three-antibody, X-gal and IPTG plate for blue-white screening, and selecting white colonies for 3 times of streak purification.
6. The method of claim 1, wherein the recombinant baculovirus-expressed mandarin fish rhabdovirus glycoprotein is prepared by infecting recombinant baculovirus into Sf9 cells, centrifuging at 3000r min-1 for 10min after 72 hours to obtain infected cells, centrifuging and washing with PBS, adding PBS to the culture medium in an amount of 10% by volume to suspend the cells, repeatedly freezing and thawing for 3 times, centrifuging to obtain supernatant, preparing a cell lysis antigen solution, performing SDS-PAGE electrophoresis, electrically transferring to a PVDF membrane, and sealing the solution at room temperature for 2 hours.
7. The method of claim 1, wherein the method of detecting the rhabdovirus glycoprotein of mandarin fish comprises: adding a His tag antibody as a primary antibody into the mandarin fish rhabdovirus glycoprotein, and culturing at 4 ℃ overnight; and taking IgG as a secondary antibody, incubating at room temperature for 1h, washing with PBS for three times, and detecting by a chemiluminescence method.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114621970A (en) * | 2021-12-09 | 2022-06-14 | 中国水产科学研究院珠江水产研究所 | Fusion gene, protein coded by fusion gene and application of fusion gene in fish rhabdovirus oral vaccine |
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| CN114432435A (en) * | 2022-01-25 | 2022-05-06 | 苏州大学 | SARS-Cov-2 vaccine based on polyhedron nano structure and its preparing method and use |
| CN114432435B (en) * | 2022-01-25 | 2024-05-17 | 苏州大学 | SARS-Cov-2 vaccine based on polyhedra nano structure and its preparation method and application |
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