CN108251356B - Fish egg cleaning solution for resisting fish nervous necrosis virus - Google Patents
Fish egg cleaning solution for resisting fish nervous necrosis virus Download PDFInfo
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Abstract
The invention provides a fish egg cleaning solution for resisting fish nervous necrosis viruses, which comprises a de-mucus and a virus neutralizing solution, wherein the de-mucus comprises a detergent, and the virus neutralizing solution comprises a fusion protein of a single-chain antibody and a polypeptide for resisting the fish nervous necrosis viruses. The fish egg cleaning solution for resisting the fish nervous necrosis virus can effectively remove the fish nervous necrosis virus possibly existing in supporting mucus.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a fish egg cleaning solution for resisting fish nervous necrosis virus and a preparation method of the fish egg cleaning solution.
Background
Before the fish eggs are fertilized artificially, the fish eggs are washed many times and quickly to remove pathogens including Nervous Necrosis Virus (NNV) as a main pathogen and prevent the pathogens from being brought into fertilized eggs during fertilization. NNV infection of parent fish (6-10kg) will not cause damage to the parent fish, but low levels of virus proliferation will support virus-carrying mucus in the blood and roe of the parent fish. Normally, NNV can not directly enter fish eggs, but if virus is contained in fish egg supporting mucus, the virus can be brought into fertilized eggs by sperms in the fertilization process, and viral neuronecrosis can be developed in the larval stage.
In order to remove NNV that may be present in the supporting mucus prior to fertilization and to provide eggs for artificial fertilization free of NNV contamination, the eggs need to be washed with an egg wash against NNV to effectively remove the virus.
Disclosure of Invention
The invention aims to solve the technical problems and provide a fish egg cleaning solution which can effectively remove fish nervous necrosis virus possibly existing in supporting mucus.
In order to achieve the above object, the present invention provides the following technical solutions:
a fish egg cleaning solution for resisting fish nervous necrosis viruses comprises a de-mucus and a virus neutralizing solution, wherein the de-mucus comprises a detergent, and the virus neutralizing solution comprises a fusion protein of a single-chain antibody and a polypeptide for resisting the fish nervous necrosis viruses.
According to the fish egg cleaning solution, the detergent is 0.2% of Triton X-100. It is diluted 100 times when used.
When cleaning viscous roe (roe with much mucus, especially roe of freshwater fish), raw powder can be added to enhance the effect of debonding. As referred to herein, "raw meal" is ready-to-use raw meal, including but not limited to potato starch or corn starch. In a preferred embodiment, the raw meal is used in the following amounts: for every 100g of roe, 0.2g of raw meal was added to 300ml of diluted (100-fold dilution of stock solution) 0.2% Triton X-100, and the mixture was stirred well and used.
According to the fish egg cleaning solution, the virus neutralizing solution further comprises Phosphate Buffered Saline (PBS). Preferably, the virus neutralizing solution consists of a fusion protein of a single-chain antibody and a polypeptide of the anti-fish nervous necrosis virus and a phosphate buffer solution, and more preferably, the concentration of the fusion protein is 20 mug/mL.
The fusion protein comprises three polypeptide 12c proteins (used for combining NNV) in series, maltose binding protein (MBP, which is a plasmid self-contained protein) and two single-chain antibody ScFv (single-chain antibody of anti-CP) proteins (used for combining NNV) in series.
The sequence of the encoded fusion protein is shown in SEQ ID NO. 1.
The invention also provides application of the roe cleaning solution in removing the fish nervous necrosis virus in roe supporting mucus.
In the fish egg cleaning solution for resisting the fish nervous necrosis virus, the de-adhesive solution is used for removing the fish egg viscous supporting solution, and the fusion protein of the virus neutralizing solution can be effectively combined with the virus and is not easy to separate, so that the virus can be removed and neutralized. Experimental results show that the fish egg cleaning solution for resisting the fish nervous necrosis virus can effectively remove the fish nervous necrosis virus possibly existing in supporting mucus.
Drawings
FIG. 1 is a comparison of the ability of different fusion proteins to bind NNV-VLPs.
FIG. 2 is a comparison of the binding capacity of 12C-MBP-scFv to NNV of different serotypes.
FIG. 3 is a comparison of the ability of different fusion proteins to prevent the invasion of SB cells by NNV-VLPs.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative of the present invention only, and are not intended to limit the scope of the present invention. The experimental procedures involved in the following examples are well known to those skilled in the art, unless otherwise specified. For some steps where specific reagents or operating parameters are not specified, those skilled in the art can also determine the procedure according to routine practice.
EXAMPLE 1 preparation of Fish egg cleaning solution
The fish egg cleaning solution for resisting the fish nervous necrosis virus comprises mucus removal liquid (liquid A) and virus neutralization liquid (liquid B).
Preparation of liquid A (mucus removal)
0.2% Triton X-100 (Triton) was formulated as a delipidated mucus. It is diluted 100 times when used.
The composition can also be added with starch (potato starch or corn starch). The raw powder is used in the following amount: adding 0.2g of raw powder into 300ml of diluted (100 times diluted stock solution) of the de-binding solution per 100g of roe, and stirring uniformly for use. When cleaning viscous roe (roe with much mucus, especially roe of freshwater fish), raw powder is added to assist debonding to enhance debonding effect.
Preparation of solution B (virus-neutralizing solution)
The virus neutralizing solution consists of fusion protein of a single-chain antibody and polypeptide of the anti-fish nervous necrosis virus and PBS (phosphate buffer solution), wherein the concentration of the fusion protein is 20 mu g/mL.
The fusion protein of the single-chain antibody and the polypeptide for resisting the fish nervous necrosis virus comprises three polypeptide 12c proteins (used for combining NNV) in series, maltose binding protein (MBP, which is a plasmid self-contained protein) and two single-chain antibody ScFv (single-chain antibody for resisting CP) proteins (used for combining NNV) in series.
Specifically, the virus neutralization solution contains a 12C-MBP-scFv fusion protein. The protein is obtained by prokaryotic expression and purification of pMal-C2-t12C-MBP-dscFv plasmid.
The sequence of the fusion protein of the 12C-MBP-scFv is shown in SEQ ID NO. 1.
In the DNA sequence shown in SEQ ID NO.1, a sequence comprising 12C (i.e., a 12 peptide consisting of 12 amino acids, the amino acid sequence of LHWDFQSWVPLL, shown in SEQ ID NO. 2), three sequences in which 12C are connected in series (SEQ ID NO.3) was proposed to be synthesized by the same company, the MBP sequence was carried by the pMAL-C2 plasmid, and the scFv sequence was shown in SEQ ID NO. 4. The three sequences are constructed together by overlap PCR and homologous recombination, and the flexible peptide sequences at intervals are introduced by overlap PCR.
The specific preparation steps of the 12C-MBP-scFv fusion protein are as follows:
RBS-T12c construction. And amplifying by using the constructed T12c (namely three sequences in series of 12c) as a template to obtain RBS-T12c, wherein the 5 'end of the RBS is provided with a homologous recombination sequence, an EcoRI enzyme cutting site and a sequence of RBS, and the 3' end of the RBS is provided with a sequence matched with the previous segment of MBP. The primers are as follows:
EcoRI-RBS-T12c-F：
AAAACGACGGCCAGTGAATTCatgagaggatcgCTACATTGGGATTTTCAGTCTTGG
(15 bp sequence designated for homologous recombination underlined) (SEQ ID NO.5)
T12c-R：
ccagatccgcctccgcctgaaccgccacctccTAGTAGAGGCACCCAAGACT(SEQ ID NO.6)
MBP construction. The amplification was carried out using pMAL-C2 as a template, and flexible peptide sequences were attached to both the front and back MBPs.
MBP-F:
gttcaggcggaggcggatctggcggtggcggatcgATGAAAATCGAAGAAGGTAAACTGGTAAT(SEQ ID NO.7)
MBP-R:
TGCAGCTGGACCTGGGCCATcgatccgccaccgccagatccgcctccgcctgaaccgccacctccCCTTCCCTCGATCCCGAGGT (scFv sequence of 20bp underlined for homologous recombination) (SEQ ID NO.8)
RBS-T12c was constructed with MBP. RBS-T12c and MBP are used as templates, EcoRI-RBS-T12c-F and MBP-R are used as primers to perform overlap PCR, and 1,451bp fragments are selected for recovery and cloning.
dscFv construction. The gene sequence of the ScFv is 765bp, and protein analysis shows that the ScFv contains two antigen complementarity determining regions and three framework structures, and conforms to the basic structural characteristics of immunoglobulin. Two monomers were connected in series by a flexible peptide to form a bivalent dscFv. And performing amplification by using the scFv as a template and the scFv1-F and the scFv1-G4S-R as primers to obtain the scFv 1-G4S. And performing amplification by using scFv as a template and scFv2-G4S-F and scFv2-R as primers to obtain G4S-scFv 2. And then, using scFv1-G4S and G4S-scFv2 as templates, and using scFv1-F and scFv2-R as primers to amplify to obtain dscFv.
scFv1-F：ATGGCCCAGGTCCAGCTGCAGGAGTCAGGAC(SEQ ID NO.9)
scFv1-G4S-R：
cgccaccgccagatccgcctccgcctgaaccgccacctccTGGGAAGATGGATCCAGTTGGTGCAGCATC (lower case incomplete G4S sequence) (SEQ ID NO.10)
scFv2-G4S-F：
gcggttcaggcggaggcggatctggcggtggcggatcgATGGCCCAGGTCCAGCTGCAGGAGTCAGGAC (lower case incomplete G4S sequence) (SEQ ID NO.11)
scFv2-R：
CACCAACTGGATCCATCTTCCCAtgaAAGCTTGGCGTAATCATGGTC
(15 bp sequence designated for homologous recombination underlined) (SEQ ID NO.12)
5. RBS-T12c-MBP-dscFv was constructed. With nunoprazan
Homologous recombination was performed with the MultiS One Step Cloning Kit to obtain recombinant plasmids containing the complete RBS-T12c-MBP-dscFv sequence (EcoRI-RBS-12c- (GGS) 3-12c- (GGGGS) 3-MBP- (GGGGS) 3-scFv- (GGGGGGS) 3-scFv-HindIII, as shown in SEQ ID No. 1). The recombinant plasmid was digested with EcoRI and HindIIIEnzyme cutting, recovering 3009bp fragment, cloning the same enzyme cutting site with pQE30, obtaining the plasmid which can be used for expression: pQE-RBS-T12 c-MBP-dscFv.
6. Expression was induced in e.coli (e.coli).
Example 2 comparison of the ability to bind NNV-VLPs (virus like particles)
The scFv protein resisting NNV has strong binding and NNV neutralizing capacity, and the 12C polypeptide resisting NNV can bind to NNV to prevent the NNV from invading cells. Therefore, two anti-NNV molecules are combined and connected through a larger protein, so that the NNV can be combined or neutralized, a net-shaped complex structure can be formed with the NNV, and the infectivity of the NNV is further reduced. In addition, the protein MBP can provide a reducing environment for the periphery of the protein MBP, and is more favorable for the soluble expression of the fusion protein.
Details of ELISA binding experiments: NNV-VLPs were first coated at a concentration of 50 ng/. mu.l and then bound at different concentrations (20 ng/. mu.l, 40 ng/. mu.l, 100 ng/. mu.l, 200 ng/. mu.l) of the fusion protein shown in FIG. 1, including MBP (negative control), MBP-12C, MBP-scFv and 12C-MBP-scFv. And adding an anti-MBP antibody after standard rinsing, and performing enzyme-labeling instrument reading after color development to obtain binding data. The higher the reading at the same concentration the more surface fusion proteins bound to the NNV-VLPs, i.e. the binding capacity was stronger. This result demonstrates the binding strength of the fusion protein to NNV.
As shown in FIG. 1, it was demonstrated by ELISA binding experiments that the 12C-MBP-scFv protein has a stronger binding ability to NNV-VLPs than the 12C-MBP protein and the MBP-scFv protein, which is 2.7 times that of the 12C-MBP protein and 1.4 times that of the MBP-scFv protein.
NNVs have a total of four serotypes, namely RGNNV, SJNNV, BFNNV and TPNNV. The original MBP-scFv protein binds only to the three serotypes RGNNV, SJNNV and TPNNV, and not to the serotype BFNNV, whereas the 12C-MBP protein binds only to RGNNV, SJNNV and BFNNV, and not to TPNNV.
As shown in FIG. 2, the 12C-MBP-scFv protein fused with the advantages of scFv and 12C can bind to four serotypes, shows the broad spectrum of NNV binding, and has good binding capacity at the concentration of 40 ng/. mu.l.
Example 3 evaluation of actual antiviral Effect of fusion protein
The actual antiviral effect of the fusion protein was evaluated by neutralizing the Virus Like Particles (VLP) and preventing the invasion of Sea Bass (SB) cells with the fusion protein.
Specifically, 50 ng/. mu.l of NNV-VLP (equivalent to TCID) was conjugated with three fusion proteins MBP-scFv, MBP-12C, 12C-MBP-scFv at a concentration of 40 ng/. mu.l50＝1.1×105Viral load of (c) was subjected to in vitro neutralization assay (incubation at 28 ℃ for 1h after mixing), invasion assay was performed, supernatant was centrifuged and untreated SB cells were added, incubated for 1h, and then fresh culture medium was replaced to continue culture. After 4 hours, the culture medium was removed, the cells were collected and lysed, and the lysate was subjected to SDS-PAGE and Western blot (using an antibody against RGNNV) to detect the amount of viral protein present in the cells.
As shown in FIG. 3, the VLP sample treated with the 12C-MBP-scFv protein did not substantially enter SB cells (12C-scFv in FIG. 3), and the invasion-preventing ability was the strongest in all samples, showing the effect of binding to virions in practical use. Compared to the negative control (NC, i.e., the sample incubated with VLP in water), the MBP-scFv protein (scFv in FIG. 3) and the 12C-MBP (12C in FIG. 3) protein also prevented invasion of VLP to some extent, and the MBP-scFv protein was more potent in preventing invasion than the 12C-MBP protein. It can be seen that the 12C-MBP-scFv protein contained much smaller (barely detectable) amounts of viral structural proteins in infected cells than the 12C-MBP protein and MBP-scFv protein treated cells, i.e.had a greater capacity to prevent the invasion of SB cells by NNV-VLPs.
Through practical application experiments, the roe cleaning solution disclosed by the invention is used for cleaning roes, can effectively remove the fish nervous necrosis viruses in roe supporting mucus, and does not damage the roes.
Sequence listing
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Claims (7)
1.A fish egg cleaning solution for resisting fish nervous necrosis viruses is characterized by comprising a de-mucus and a virus neutralizing solution, wherein the de-mucus comprises a detergent, the virus neutralizing solution comprises a fusion protein of a single-chain antibody and a polypeptide for resisting the fish nervous necrosis viruses, and a sequence for coding the fusion protein is shown as SEQ ID No. 1.
2. The fish egg wash of claim 1, wherein the detergent is 0.2% Triton X-100.
3. The fish egg cleaning solution as claimed in claim 1, wherein the mucus removing solution further comprises raw meal.
4. The fish egg wash according to claim 1, wherein the virus neutralizing solution further comprises a phosphate buffer.
5. The fish egg cleaning solution according to claim 1, wherein the virus neutralizing solution comprises a fusion protein of a single-chain antibody against a fish nervous necrosis virus and a polypeptide, and a phosphate buffer.
6. The roe wash according to claim 5, wherein the concentration of the fusion protein is 20. mu.g/mL.
7. Use of the fish egg cleaning solution of any one of claims 1 to 6 for preparing an agent for removing nervous necrosis virus of fish.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711479754.XA CN108251356B (en) | 2017-12-29 | 2017-12-29 | Fish egg cleaning solution for resisting fish nervous necrosis virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711479754.XA CN108251356B (en) | 2017-12-29 | 2017-12-29 | Fish egg cleaning solution for resisting fish nervous necrosis virus |
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