CN112626037B - Construction and application of green fluorescent protein-labeled recombinant iridovirus - Google Patents
Construction and application of green fluorescent protein-labeled recombinant iridovirus Download PDFInfo
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Abstract
The invention provides a construction method and application of a green fluorescent protein marked recombinant iridovirus. And inserting eGFP and Flag labels into the C end of the main capsid protein coding gene by adopting a homologous recombination method, and further screening out the green fluorescent protein marked recombinant iridovirus by a cell sorter and a limiting dilution method. For the target virus, it was verified by Western Blot whether eGFP and Flag tags were inserted at preset positions, and the exact positions of eGFP and Flag insertion were further determined by viral genome sequencing. Finally, the structural difference between the green fluorescent protein-labeled recombinant iridovirus and the wild type is observed by using a transmission electron microscope and an atomic force microscope, and the result shows that the structural sizes of the two are basically consistent without obvious difference. The recombinant virus is used as a molecular tool for researching the interaction between the virus and a host, has important value in immunological research, and provides a new tool for further researching the antiviral immunity of fish.
Description
Technical Field
The present invention relates to the field of biotechnology.
Background
With the rapid development of economy, the living standard of people is gradually improved, and the demands of high-protein aquatic products are also increasing. The expansion of aquaculture scale is increasingly limited by environmental and cultivation techniques, especially large-scale diseases caused by viruses, resulting in huge economic losses.
Iridovirus (SGIV) is a virus with deadly infectious power to economic fishes, and is one of the most important causes of death of cultured animals in many times in recent years, and the research on immune mechanisms has been carried out more. However, to truly understand the pathogenesis of SGIV and the control method, further research into details of the viral infection process is still needed.
Disclosure of Invention
In the recombinant iridovirus of this item, the green fluorescent protein successfully marks part of the main capsid protein of the virus, and the recombinant virus not only can make infected cells appear green under a fluorescent microscope, but also can normally infect and replicate in the body of garrupa, and the green fluorescent signal is detected by flow sorting analysis of the infected blood cells. The successful construction of the virus enables people to observe the whole process of the iridovirus infecting the host in real time, and is an effective tool for exploring the interaction between the iridovirus and the host.
The technical scheme adopted by the invention is as follows:
according to the invention, wild garrupa iridovirus (Singapore Grouper Iridovirus, SGIV) is taken as a transformation object, the C end of ORF72R gene for expressing main capsid protein (Major Capsid Protein, MCP) is taken as an insertion site, plasmid with eGFP and Flag label and ORF72R fusion expression is constructed, the plasmid is transferred into garrupa embryo stem cells by a liposome transfection method, a flow sorter is utilized to screen out target viruses, namely cells with green fluorescent signals, and the target viruses are purified by a limiting dilution method, so that the obtained viruses are named SGIV-72R-eGFP. And then Western Blot and immunoprecipitation are used for analyzing whether eGFP and Flag labels are correctly inserted into preset positions, and a transmission electron microscope is used for observing that the recombinant green fluorescent iridovirus and the wild garrupa iridovirus have similar characteristics in structure and size, and no obvious difference exists between the two.
Compared with the prior art, the green fluorescent protein label has the following advantages and beneficial effects:
recombinant viruses are of great value in immunological research as a molecular tool for studying the interaction of viruses with hosts. At present, the research on mammal recombinant viruses is more, the recombinant viruses related to aquatic viruses are relatively less, and the green fluorescent protein-marked recombinant iridovirus constructed by the invention provides a new tool for further researching fish antiviral immunity.
Drawings
FIG. 1 is a fluorescence microscope image of green fluorescent protein-labeled recombinant iridovirus infecting garrupa embryonic stem cells;
FIG. 2 is a diagram comparing purified green fluorescent protein-labeled recombinant iridovirus with wild-type virus;
FIG. 3 is a Western Blot diagram of green fluorescent protein labeled recombinant iridovirus versus wild-type virus;
FIG. 4 is a graph of immunoprecipitation analysis of wild-type iridovirus and green fluorescent protein-labeled recombinant iridovirus;
FIG. 5 is a transmission electron microscope image of a green fluorescent protein labeled recombinant iridovirus versus wild-type virus;
FIG. 6 is a flow-based sorting analysis of blood cell green fluorescent protein markers infected with recombinant green fluorescent iridovirus.
Detailed Description
The invention will be described in further detail below with reference to the drawings by means of specific embodiments. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, ind. Molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: construction, screening and purification of green fluorescent protein marked recombinant iridovirus
1) The ORF72R gene expressing MCP in SGIV was selected as a target gene to construct a recombinant plasmid, and the C-terminus of MCP was inserted with eGFP and Flag tags.
2) The recombinant virus is obtained by homologous recombination between wild virus and recombinant plasmid, the recombinant plasmid enters into garrupa embryo stem cells in the growth logarithmic phase by a liposome transfection method, and after the wild virus is inoculated for 48 hours, a fluorescence microscope is used for observing whether the recombinant virus is generated.
3) The infected cells are collected, the cells with green fluorescence are screened by a flow cell sorter, the screening process is repeated for a plurality of times, and the obtained infected cells are prepared into virus suspension by an ultrasonic disruption method.
Example 2: screening and purifying of green fluorescent protein marked recombinant iridovirus
1) The virus suspension obtained in example 1 was subjected to gradient dilution and inoculated in 96-well plates, and after 48h, significant cytopathic effect CPE was present. And screening out holes for detecting single green fluorescent CPE by using an inverted fluorescent microscope, namely, single green fluorescent protein labeled recombinant iridovirus clone. FIG. 1 is a microscopic image of green fluorescent protein-labeled recombinant iridovirus infected grouper embryo cells.
2) Green fluorescent protein markers cell suspensions containing large amounts of virus were obtained by cell culture, using garrupa embryonic stem cells in log phase of growth, and recombinant virus with moi=10 was added. After about 3-4 days, the infected cells were detached, suspended in medium, and the cells were collected and washed several times with 0.01M PBS.
3) The cell suspension was sonicated until the solution was clear, and then sucrose density gradient centrifugation was performed, setting sucrose density gradients of 30%, 40%, 50%, 60%. The cell suspension after ultrasonic disruption is paved on the 30% sucrose solution of the uppermost layer, and is centrifuged for 1h at 20000rpm and 4 ℃, and the green fluorescent protein marked recombinant iridovirus particles are suspended in the 50-60% sucrose layer. Taking out the virus suspension of the layer, diluting with a proper amount of 0.01M PBS, centrifuging at 20000rpm and 4 ℃ for 30min, and obtaining a pale yellow precipitate which is the purified green fluorescent protein-marked recombinant iridovirus, re-suspending with a proper amount of TN buffer, and preserving at-80 ℃. FIG. 2 is a comparison of purified green fluorescent protein-labeled recombinant iridovirus and wild-type iridovirus under UV.
Example 3: identification of green fluorescent protein-labeled recombinant iridovirus
1) Western Blot was performed using purified wild-type iridovirus and green fluorescent protein-labeled recombinant iridovirus, with Anti-MCP, anti-eGFP and Anti-Flag as primary antibodies, respectively. The results showed that in wild-type iridovirus the band position of MCP was around 50kDa, whereas the band of recombinant virus MCP was around 75kDa, i.e. the size of MCP after fusion expression with eGFP and Flag tag, and antibodies to eGFP and Flag also detected this band in recombinant virus, indicating correct insertion of eGFP and Flag tag into MCP. FIG. 3 is a Western Blot plot of wild-type iridovirus and green fluorescent protein-labeled recombinant iridovirus.
2) Immunoprecipitation analysis is carried out on the purified wild iridovirus and the green fluorescent protein-labeled recombinant iridovirus, and the Flag tag antibody is utilized for detecting the interaction protein. The results showed that in recombinant iridovirus, a differential band was detected around 75kDa, i.e.MCP-eGFP-Flag, further demonstrating the correct insertion of eGFP and Flag tags into MCP. FIG. 4 is an immunoprecipitation analysis of wild-type iridovirus and green fluorescent protein-labeled recombinant iridovirus.
3) About 10ul of purified green fluorescent protein marked recombinant iridovirus and wild iridovirus suspension is dripped on a copper mesh of a 200-mesh ordinary carbon film, and is left stand for 10min, after the surplus liquid is absorbed, the solution is subjected to negative dyeing by using 2% PFA, and after the solution is dyed for 5min, the surplus liquid is absorbed. Compared with wild iridovirus, the recombinant green fluorescent iridovirus has basically consistent structure and size, and the green fluorescent protein markers have no obvious difference when observed by a transmission electron microscope. FIG. 5 is a transmission electron microscope image of green fluorescent protein-labeled recombinant iridovirus (a) and wild-type iridovirus (b).
Example 4: preliminary application of green fluorescent protein marked recombinant iridovirus
1) Taking grouper seedlings purchased from a farm and temporarily raised for 30 days, setting a control group and an experimental group, injecting 100 mu L of PBS into the control group, injecting 100 mu L of purified recombinant iridovirus SGIV-72R-eGFP into the experimental group, and attacking the grouper seedlings with a virus concentration of 1 multiplied by 10 6.5 pfu/mL, using a 1mL sterile syringe to inject toxin from the abdominal cavity of the fish;
2) After 14d, the fish blood is drawn by pinching the gill of the fish blood with wet cloth and adopting a tail vein blood drawing method, a proper amount of anticoagulant is added into a 1mL needle tube to prevent coagulation, and then the fish blood cells are collected for flow sorting analysis after centrifugation for 5min at 1000 Xg at 4 ℃.
3) The blood cells of the fish were resuspended in 0.01M PBS and passed through the filters of the flow tube before loading and 10,000 cells were collected for analysis. The blood cells infected by the recombinant iridovirus have obvious green fluorescence signals, which indicates that the recombinant iridovirus marked by the green fluorescence protein can be infected and replicated in the body of the grouper, and the green fluorescence signals can be detected by a flow cytometer. FIG. 6 is a flow-based sorting analysis of blood cells infected with green fluorescent protein-labeled recombinant iridovirus.
Claims (2)
1. The construction method of the green fluorescent protein marked recombinant iridovirus is characterized by comprising the following steps:
1) Constructing a recombinant plasmid, and selecting the C end of the ORF72R gene of the garrupa iridovirus as an insertion site of eGFP and Flag tags, wherein the recombinant plasmid contains the ORF72R gene fused with the eGFP and Flag tags;
2) The recombinant virus is obtained by homologous recombination between wild type virus and recombinant plasmid, the recombinant plasmid enters into garrupa embryo stem cells in the growth log phase by a liposome transfection method, and then the garrupa embryo stem cells are inoculated with wild iridovirus;
3) Collecting infected cells, screening out cells with green fluorescence, and extracting intracellular viruses, namely, green fluorescent protein-labeled recombinant iridovirus;
4) And (3) after the screened and purified green fluorescent protein marked recombinant iridovirus is subjected to amplification culture, separating by a sucrose density gradient centrifugation method, wherein the green fluorescent protein marked recombinant iridovirus is suspended in a 50-60% sucrose solution.
2. The method for constructing a green fluorescent protein-labeled recombinant iridovirus according to claim 1, further comprising the step of, after step 4):
5) And (3) identifying the green fluorescent protein-labeled recombinant iridovirus obtained in the step 4) by a western blotting method.
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| CN102242173A (en) * | 2011-04-29 | 2011-11-16 | 中国科学院南海海洋研究所 | Extraction method for envelope protein of grouper iridovirus |
| WO2012037723A1 (en) * | 2010-09-21 | 2012-03-29 | 中山大学 | Attenuate live vaccine of vsocs gene-deleted megalocytivirus, method to prepare the same and use thereof |
| WO2012122858A1 (en) * | 2011-03-17 | 2012-09-20 | 中国科学院上海巴斯德研究所 | Method for producing virus-like particle by using drosophila cell and applications thereof |
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| US20130177582A1 (en) * | 2010-04-29 | 2013-07-11 | Riemser Arzneimittel Ag | Parapoxvirus expressing the vp60 major capsid protein of the rabbit haemorrhagic disease virus |
| CN102277361A (en) * | 2011-04-29 | 2011-12-14 | 中国科学院南海海洋研究所 | Method for screening protective antigens of Singapore grouper iridovirus |
| CN107794291A (en) * | 2017-04-10 | 2018-03-13 | 中山大学 | A kind of drug screening method of anti-Singapore's grouper irido virus |
| CN108384763B (en) * | 2018-03-14 | 2022-02-11 | 中山大学 | Infectious spleen and kidney necrosis virus ORF074 gene deletion strain and preparation method and application thereof |
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| WO2012037723A1 (en) * | 2010-09-21 | 2012-03-29 | 中山大学 | Attenuate live vaccine of vsocs gene-deleted megalocytivirus, method to prepare the same and use thereof |
| WO2012122858A1 (en) * | 2011-03-17 | 2012-09-20 | 中国科学院上海巴斯德研究所 | Method for producing virus-like particle by using drosophila cell and applications thereof |
| CN102242173A (en) * | 2011-04-29 | 2011-11-16 | 中国科学院南海海洋研究所 | Extraction method for envelope protein of grouper iridovirus |
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