Cage structure protein-based standard substance loaded with oyster herpesvirus nucleic acid as well as preparation method and application of cage structure protein-based standard substance loaded with oyster herpesvirus nucleic acid

Abstract

The invention belongs to the field of marine product epidemic disease detection, and particularly relates to a standard substance loaded with oyster herpesvirus nucleic acid based on cage-type structural protein as well as a preparation method and application of the standard substance. The standard substance is characterized in that the charge property of the surface of a hollow inner cavity of a cage-shaped structure of scapharca broughtonii ferritin is changed from negative to positive; the change from negative to positive is that amino acid residues with negative charges on the surface of the hollow inner cavity of the scapharca broughtonii ferritin are mutated into amino acid with positive charges. The creation of the OsHV-1 nucleic acid standard substance provided by the invention can improve the accuracy and comparability of the detection result of the pathogenic acid. The standard substance provided by the invention can be widely applied to the fields of shellfish disease monitoring and early warning, risk assessment and the like, lays a foundation for building a shellfish disease monitoring network, and promotes green and healthy development of shellfish breeding industry.

Description

Technical Field

[0001] The present invention belongs to the field of seafood disease detection, and particularly relates to a reference substance based on a cage-like structural protein loaded with oyster herpesvirus nucleic acid, a preparation method thereof, and an application thereof. Background Art

[0002] The aquaculture of shellfish is one of the important pillars of the marine aquaculture industry and occupies an important economic and industrial position. Since the first discovery of herpesvirus infection in American oysters ( Eastern oyster ) in the 1990s, the prevalence of such viruses has continuously caused large-scale deaths of cultured shellfish. In 2014, the International Committee on Taxonomy of Viruses (ICTV) officially named this type of herpesvirus as oyster herpesvirus (OsHV-1), which belongs to the order Herpesvirales ( Herpesvirales ), the family Malacoherpesviridae ( Malacoherpesviridae ). OsHV-1 has latent infectivity, and the virus in latent infection can be reactivated and replicated under the stimulation of water temperature or other environmental factors. At present, OsHV-1 infection is considered to be the main cause of the outbreak of summer oyster mortality syndrome. Epidemiological investigations have found that in addition to causing large-scale deaths of oysters, OsHV-1 can infect and cause large-scale deaths of various economic shellfish including Chlamys farreri ( Japanese scallop ), Ruditapes philippinarum ( Razor clam ), Scapharca subcrenata ( Scapharca subcrenata ), and Scapharca broughtonii ( Asiatic blood clam ) within a short period of time. At present, large-scale shellfish death events caused by OsHV-1 infection have occurred frequently in the aquaculture sea area since the end of the last century.

[0003] Monitoring the early stage of OsHV-1 infection (latency or the initial stage of infection) is a crucial stage for early warning and prevention and control of this disease. Effective detection of the virus can effectively avoid economic losses caused by large-scale infection and death of cultured shellfish populations. However, the virus copy number is low at this stage. Due to the lack of nucleic acid reference materials to control the quality of the detection process and results, it is difficult for OsHV-1 monitoring institutions to accurately judge the virus load in samples using qPCR technology, resulting in incomparable results. Therefore, it is urgent to develop OsHV-1 nucleic acid reference materials with uniformity, stability, and specific quantity values to control the quality of the nucleic acid detection process and verify the accuracy of nucleic acid detection results. The existing reference materials have the following problems: (1) Nucleic acid reference materials prepared from whole virus particles are restricted in their development and application as reference materials due to problems such as unstable sources of preparation raw materials, high transportation and storage conditions, large uncertainties in reconstitution during use, and potential biological infectivity; (2) Synthetic virus target nucleic acid fragments, as nucleic acid reference materials, are easily degraded by physical, chemical, or biological factors; they have a simple structure and cannot participate in the virus lysis and nucleic acid extraction processes, and cannot achieve quality monitoring of the entire process of virus nucleic acid detection; plasmid DNA templates cannot be completely eliminated during the preparation of in vitro transcribed RNA, which easily leads to false positives in detection results, etc.; (3) Packaging virus target nucleic acid fragments with virus-like particles (VLPs) as nucleic acid reference materials, however, the enzymatic digestion reaction is limited by the enzymatic digestion efficiency, thus affecting the total yield of the final annealing product. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the present invention provides a reference material for loading oyster herpesvirus nucleic acid based on a cage-like structural protein.

[0005] The present invention also provides a preparation method of the above reference material.

[0006] The present invention further provides the application of the above reference material in the preparation of a kit for detecting oyster herpesvirus.

[0007] The technical solution adopted by the present invention to achieve the above purpose is as follows: The present invention provides a reference material for loading oyster herpesvirus nucleic acid based on a cage-like structural protein. The reference material is to change the charge property on the surface of the hollow inner cavity of the cage-like structure of ark clam ferritin from negative to positive. The change from negative to positive is: mutating the negatively charged amino acid residues on the surface of the hollow inner cavity of ark clam ferritin into positively charged amino acids to obtain positive-charge inner cavity ferritin nucleic acid fragments with different mutation sites Sb Fn(+)Δ1 or Sb Fn(+)Δ2, with codon-optimized Sb Fn(+)Δ1 or codon-optimizedSb Fn(+)Δ2 is used as a vector to carry the nucleic acid of ostreid herpesvirus.

[0008] Furthermore, the amino acid sequence of the ark shell ferritin is shown as SEQ ID NO.1.

[0009] Specifically: MAQTQPRQNYHVESEAGINKQINMELYASYVYQSMYMYFDRDDVALPGFAKYFKHNSEEEREHAEKLMKYQNKRGGRVVLQDIQKPDRDEWGSPLEAMQTTLALEKSVNQALLDLHKVADKHGDAQMMDFLEGEYLKEQVEAIKEISDHITNLKRVGTGLGEYMYDKETMSS.

[0010] Furthermore, the mutation sites of the ark shell ferritin are Glu-59, Glu-62, Glu-138, and Glu-145; the positively charged amino acids are lysine or arginine.

[0011] Furthermore, the sequence of the ostreid herpesvirus nucleic acid (SEQ ID NO.2) is as follows: GTCGCATCTTTGGATTTAACAATTGCCCCTGTCATCTTGAGGTATAGACAATCGCCAGAAAATTTCCCATGTTCAGTCCATGGGTTGTCAGTCGGATCCCAGT.

[0012] Furthermore, in step (1), the Sb The nucleotide sequence of Fn(+)Δ1 (SEQ ID NO.3) is: ATGGCTCAAACACAACCAAGACAAAACTACCATGTGGAAAGTGAGGCCGGAATCAACAAACAGATCAACATGGAGTTATATGCCAGTTATGTCTATCAGTCCATGTATATGTACTTTGACAGAGATGATGTAGCTCTGCCTGGTTTTGCCAAGTACTTCAAGCATAATTCCGAG AAG GAACGT CGA CATGCTGAGAAACTCATGAAGTACCAGAACAAGAGAGGTGGCAGAGTTGTCCTTCAAGACATTCAGAAACCTGACCGTGATGAATGGGGTTCTCCACTGGAAGCCATGCAGACTACATTAGCTCTGGAGAAAAGTGTTAACCAGGCACTTCTGGACCTGCACAAAGTTGCAGATAAACATGGTGATGCCCAGATGATGGATTTCCTTGAAGGAGAATACCTGAAGGAGCAGGTAGAAGCCATTAAGGAGATATCGGACCACATCACCAATCTGAAACGTGTGGGTACAGGTCTCGGAGAGTACATGTATGACAAGGAAACCATGAGCAGT; The codon-optimized Sb The nucleotide sequence of Fn(+)Δ1 (SEQ ID NO.4) is: ATGGCGCAGACCCAGCCGCGTCAGAACTACCACGTTGAAAGCGAAGCGGGTATCAACAAACAGATCAACATGGAACTGTACGCGAGCTACGTTTACCAGAGCATGTACATGTACTTCGATCGTGATGATGTTGCGCTGCCGGGTTTCGCGAAATACTTCAAACACAACAGCGAAAAAGAACGTCGTCACGCGGAAAAACTGATGAAATACCAGAACAAACGTGGTGGCCGTGTTGTTCTGCAGGATATCCAGAAACCGGATCGTGATGAATGGGGTAGCCCGCTGGAAGCGATGCAGACCACCCTGGCGCTGGAAAAAAGCGTTAACCAGGCGCTGCTGGATCTGCACAAAGTTGCGGATAAACACGGTGATGCGCAGATGATGGATTTCCTGGAAGGTGAATACCTGAAAGAACAGGTTGAAGCGATCAAAGAAATCAGCGATCACATCACCAACCTGAAACGTGTTGGCACCGGCCTGGGTGAATACATGTACGATAAAGAAACCATGAGCAGC; The Sb The nucleotide sequence of Fn(+)Δ2 (SEQ ID NO.5) is: ATGGCTCAAACACAACCAAGACAAAACTACCATGTGGAAAGTGAGGCCGGAATCAACAAACAGATCAACATGGAGTTATATGCCAGTTATGTCTATCAGTCCATGTATATGTACTTTGACAGAGATGATGTAGCTCTGCCTGGTTTTGCCAAGTACTTCAAGCATAATTCCGAG AAG GAACGT CGA CATGCTGAGAAACTCATGAAGTACCAGAACAAGAGAGGTGGCAGAGTTGTCCTTCAAGACATTCAGAAACCTGACCGTGATGAATGGGGTTCTCCACTGGAAGCCATGCAGACTACATTAGCTCTGGAGAAAAGTGTTAACCAGGCACTTCTGGACCTGCACAAAGTTGCAGATAAACATGGTGATGCCCAGATGATGGATTTCCTTGAAGGAGAATACCTGAAG AAG CAGGTAGAAGCCATTAAG AAG ATATCGGACCACATCACCAATCTGAAACGTGTGGGTACAGGTCTCGGAGAGTACATGTATGACAAGGAAACCATGAGCAGT; The codon-optimized Sb nucleotide sequence of Fn(+)Δ2 (SEQ ID NO.6) is: ATGGCGCAGACCCAGCCGCGTCAGAACTACCACGTTGAATCTGAAGCGGGCATCAACAAACAGATCAACATGGAACTGTACGCGAGCTACGTTTACCAGAGCATGTACATGTACTTCGATCGTGATGATGTTGCGCTGCCGGGCTTCGCGAAATACTTCAAACACAACAGCGAAAAAGAACGTCGTCACGCGGAAAAACTGATGAAATACCAGAACAAACGTGGCGGCCGTGTTGTTCTGCAGGATATCCAGAAACCGGATCGTGATGAATGGGGTAGCCCGCTGGAAGCGATGCAGACCACCCTGGCGCTGGAAAAAAGCGTTAACCAGGCGCTGCTGGACCTGCACAAAGTTGCGGATAAACACGGTGATGCGCAGATGATGGATTTCCTGGAAGGTGAATACCTGAAAAAACAGGTTGAAGCGATCAAGAAAATCTCTGATCACATCACCAACCTGAAACGTGTTGGTACCGGCCTGGGTGAATACATGTACGATAAAGAAACCATGAGCAGC; In the above nucleotide sequence, the italicized and underlined part is the mutation site.

[0013] The present invention also provides a method for preparing the above reference substance, comprising the following steps: (1) Connect the nucleic acid fragments of positively charged luminal ferritin with different mutation sites Sb Fn(+)Δ1 or Sb Fn(+)Δ2 to the pET-30a(+) vector (Novagen), and the recombinant expression vector is transformed into the expression competent cell Transetta(DE3) by heat shock, and positive clones are screened by colony PCR; (2) Induce the expression of the positive clones; centrifuge to collect the bacterial cells, resuspend them with lysis equilibration buffer and then perform sonication; centrifuge to collect the supernatant, heat the supernatant and then centrifuge again to collect the supernatant, filter it and then perform purification; wash it with washing buffer until OD 280 is stable; add elution buffer for elution; analyze and purify the purified recombinant protein using GE liquid chromatography, and collect the ferritin mutants according to the absorption peaks; (3) Use OsHV-1 qPCR detection primers to perform PCR amplification on the target fragment and purify it to obtain the OsHV-1 target DNA fragment; (4) Using the pH method to mediate the depolymerization / recombinant loading of the OsHV-1 target DNA fragment by the ferritin mutant: Depolymerize the ferritin mutant in a gradient acidic environment, and obtain an acidic depolymerization system after adjusting the pH; Add the OsHV-1 target DNA fragment to the acidic depolymerization system, and slowly adjust the pH to 7.6 for reassembly after incubation to complete the nucleic acid fragment loading; Remove the unloaded nucleic acid fragments to obtain the reference material.

[0014] Further, in step (2), the specific method of induction expression is: Inoculate the positive clone into an LB medium containing kanamycin resistance and culture it until OD 600 reaches 0.3 - 0.4, add IPTG with a final concentration of 0.1 mM for induction for 5 h; The heating is carried out at 72 °C for 15 min; The composition of the LB medium containing kanamycin resistance is: 1 g Tryptone, 0.5 g Yeast Extract, 1 g NaCl, 5 mg Kanamycin per 100 ml;.

[0015] Further, in step (3), the OsHV-1 qPCR detection primers are: BF (SEQ ID NO.7): GTCGCATCTTTGGATTTAACAA; B4 (SEQ ID NO.8): ACTGGGATCCGACTGACAAC.

[0016] Further, in step (4), the molar ratio of the OsHV-1 target DNA fragment to the ferritin mutant is 5:1; The gradient acidic environment is pH 7 - 2; Adjust the pH value to 4; The incubation is carried out at 4 °C for 1 h.

[0017] The present invention also provides the application of the above-mentioned reference material in the preparation of a kit for detecting oyster herpesvirus.

[0018] The present invention uses the hollow nanocage structure of ferritin to load DNA, and establishes a set of nucleic acid reference materials based on nanoscale particles - ferritin loading the OsHV-1 virus target DNA fragment and its standard preparation process. Ferritin, as an important iron storage protein in organisms, has a hollow cage structure formed by the self-assembly of 24 subunits, and its inner cavity diameter is about 8 nanometers, which can be used as an ideal space for material loading.

[0019] The beneficial effects of the present invention are: (1) Based on the positive charge modification of the negatively charged hollow inner cavity of ferritin, the present invention successfully screened ferritin mutants and loading methods that stably loaded the OsHV-1 nucleic acid detection target DNA fragment, and established a preparation process for the OsHV-1 nucleic acid reference material based on the ferritin nanocage structure. The creation of the OsHV-1 nucleic acid reference material can improve the accuracy and comparability of the detection results of this pathogen nucleic acid.

[0020] (2) The reference material provided by the present invention will be widely used in the fields of shellfish disease monitoring and early warning, risk assessment, etc., laying a foundation for the construction of a monitoring network for aquaculture shellfish diseases and promoting the green and healthy development of the shellfish aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 For Sb Design of the positively charged inner cavity mutant of Fn; (A) Sb Mutation sites, theoretical molecular weights, and pI values of the subunits of the Fn(+) mutant; (B) WT Sb Fn and Sb Sequences and structures of the subunits of the Fn(+) mutant; Figure 2 For Sb Construction and characterization of the positively charged inner cavity mutant of Fn; (A) Sb Schematic diagram of the preparation process of the Fn(+) mutant; (B - C) WT Sb Fn and Sb SDS-PAGE analysis of the Fn and Fn(+) mutants, where 1 - 4 represent whole bacteria, supernatant and precipitate after ultrasonic disruption, and protein after preliminary purification by affinity chromatography, respectively; (D) WT Sb Fn and Sb Native PAGE analysis of the Fn and Fn(+) mutants. (E) Purified WT Sb Fn and Sb Representative TEM images of the Fn and Fn(+) mutants; (F) Acid-mediated disassembly of WT Sb Fn and Sb Representative TEM images of the disassembly of the Fn(+) mutant; (G) Representative TEM images of the reassembled WT Sb Fn and Sb Representative TEM images of the Fn(+) mutant after reassembly; Figure 3 To evaluate the characteristics of different Sb Positively charged inner cavity mutants of Fn as nanocarriers for loading OsHV-1 nucleic acid; (A) Sb Schematic diagram of the process of loading OsHV-1 nucleic acid by the positively charged inner cavity mutant of Fn through pH-mediated decomposition / reassembly; (B) Evaluation of WT Sb Fn and SbThe loading rate of the Fn(+) mutant on OsHV-1 nucleic acid and the protein recovery rate (C) DNase I digestion of WT Sb Fn and Sb The Fn(+) mutant loaded with nucleic acid products and untreated WT Sb Fn and Sb The co-incubation products of the Fn(+) mutant nucleic acid and WT analyzed by fluorescence quantitative PCR Sb Fn and Sb The loading position of the Fn(+) mutant on OsHV-1 nucleic acid; (D) DNA@ Sb TEM characterization of Fn(+)Δ2; (E) Selected Sb Schematic diagram of the Fn(+) mutant. Specific implementation manners

[0022] The technical solutions of the present invention are further explained and illustrated below through specific embodiments.

[0023] Example 1 (I) Materials and methods 1. Construction of positively charged inner cavity ferritin mutants According to the analysis results of the cage structure of Scapharca broughtonii ferritin and the key negatively charged amino acid residue sites on the surface of the hollow inner cavity, the negatively charged amino acid residues (Glu-59, Glu-62, Glu-138, Glu-145) on the surface of the hollow inner cavity were replaced with the positively charged amino acids lysine (Lysine, Lys) or arginine (Arginine, Arg), and the spatial distribution analysis was carried out synchronously to avoid the steric hindrance effect, and positively charged inner cavity ferritin mutants with different mutation sites were designed.

[0024] 2. Expression and purification of positively charged inner cavity ferritin mutants The nucleic acid sequences of positively charged inner cavity ferritin with different mutation sites Sb Fn(+)Δ1 or Sb Fn(+)Δ2 was synthesized by Sangon Biotech and ligated to the pET-30a(+) vector (Novagen). The recombinant expression vector was transformed into the expression competent cell Transetta(DE3) by heat shock transformation (heat shock for 60 s in a 42°C metal bath), and positive clones were screened by colony PCR and sent to Sangon Biotech for sequencing to ensure that the target inserted gene fragment had no frameshift or deletion.

[0025] SbThe nucleotide sequence of Fn(+)Δ1 is: ATGGCTCAAACACAACCAAGACAAAACTACCATGTGGAAAGTGAGGCCGGAATCAACAAACAGATCAACATGGAGTTATATGCCAGTTATGTCTATCAGTCCATGTATATGTACTTTGACAGAGATGATGTAGCTCTGCCTGGTTTTGCCAAGTACTTCAAGCATAATTCCGAG AAG GAACGT CGA CATGCTGAGAAACTCATGAAGTACCAGAACAAGAGAGGTGGCAGAGTTGTCCTTCAAGACATTCAGAAACCTGACCGTGATGAATGGGGTTCTCCACTGGAAGCCATGCAGACTACATTAGCTCTGGAGAAAAGTGTTAACCAGGCACTTCTGGACCTGCACAAAGTTGCAGATAAACATGGTGATGCCCAGATGATGGATTTCCTTGAAGGAGAATACCTGAAGGAGCAGGTAGAAGCCATTAAGGAGATATCGGACCACATCACCAATCTGAAACGTGTGGGTACAGGTCTCGGAGAGTACATGTATGACAAGGAAACCATGAGCAGT; Sb The nucleotide sequence of Fn(+)Δ2 is: ATGGCTCAAACACAACCAAGACAAAACTACCATGTGGAAAGTGAGGCCGGAATCAACAAACAGATCAACATGGAGTTATATGCCAGTTATGTCTATCAGTCCATGTATATGTACTTTGACAGAGATGATGTAGCTCTGCCTGGTTTTGCCAAGTACTTCAAGCATAATTCCGAG AAG GAACGT CGA CATGCTGAGAAACTCATGAAGTACCAGAACAAGAGAGGTGGCAGAGTTGTCCTTCAAGACATTCAGAAACCTGACCGTGATGAATGGGGTTCTCCACTGGAAGCCATGCAGACTACATTAGCTCTGGAGAAAAGTGTTAACCAGGCACTTCTGGACCTGCACAAAGTTGCAGATAAACATGGTGATGCCCAGATGATGGATTTCCTTGAAGGAGAATACCTGAAG AAG CAGGTAGAAGCCATTAAG AAG ATATCGGACCACATCACCAATCTGAAACGTGTGGGTACAGGTCTCGGAGAGTACATGTATGACAAGGAAACCATGAGCAGT; After codon optimization Sb The sequence of Fn(+)Δ1 is: ATGGCGCAGACCCAGCCGCGTCAGAACTACCACGTTGAAAGCGAAGCGGGTATCAACAAACAGATCAACATGGAACTGTACGCGAGCTACGTTTACCAGAGCATGTACATGTACTTCGATCGTGATGATGTTGCGCTGCCGGGTTTCGCGAAATACTTCAAACACAACAGCGAAAAAGAACGTCGTCACGCGGAAAAACTGATGAAATACCAGAACAAACGTGGTGGCCGTGTTGTTCTGCAGGATATCCAGAAACCGGATCGTGATGAATGGGGTAGCCCGCTGGAAGCGATGCAGACCACCCTGGCGCTGGAAAAAAGCGTTAACCAGGCGCTGCTGGATCTGCACAAAGTTGCGGATAAACACGGTGATGCGCAGATGATGGATTTCCTGGAAGGTGAATACCTGAAAGAACAGGTTGAAGCGATCAAAGAAATCAGCGATCACATCACCAACCTGAAACGTGTTGGCACCGGCCTGGGTGAATACATGTACGATAAAGAAACCATGAGCAGC; After codon optimization Sb The sequence of Fn(+)Δ2 is: ATGGCGCAGACCCAGCCGCGTCAGAACTACCACGTTGAATCTGAAGCGGGCATCAACAAACAGATCAACATGGAACTGTACGCGAGCTACGTTTACCAGAGCATGTACATGTACTTCGATCGTGATGATGTTGCGCTGCCGGGCTTCGCGAAATACTTCAAACACAACAGCGAAAAAGAACGTCGTCACGCGGAAAAACTGATGAAATACCAGAACAAACGTGGCGGCCGTGTTGTTCTGCAGGATATCCAGAAACCGGATCGTGATGAATGGGGTAGCCCGCTGGAAGCGATGCAGACCACCCTGGCGCTGGAAAAAAGCGTTAACCAGGCGCTGCTGGACCTGCACAAAGTTGCGGATAAACACGGTGATGCGCAGATGATGGATTTCCTGGAAGGTGAATACCTGAAAAAACAGGTTGAAGCGATCAAGAAAATCTCTGATCACATCACCAACCTGAAACGTGTTGGTACCGGCCTGGGTGAATACATGTACGATAAAGAAACCATGAGCAGC。

[0026] Inoculate the positive clone into LB medium containing kanamycin resistance (1 g Tryptone, 0.5 g Yeast Extract, 1 g NaCl, 5 mg Kanamycin per 100 ml) and culture until OD 600 reaches 0.3 - 0.4, then add IPTG with a final concentration of 0.1 mM for induction expression. After 5 h of induction, centrifuge the cells at 8,000 g for 5 min at 4 °C to collect the cell pellet. Resuspend the cell pellet according to a volume ratio of 6:1 with lysis equilibrium buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and then perform sonication. Centrifuge at 12,000 g for 5 min to collect the supernatant, heat it at 72 °C for 15 min to remove impurity proteins. Centrifuge at 12,000 g for 10 min to collect the supernatant, filter it through a 0.45 μm filter membrane and then load it onto a High Affinity Ni-NTA affinity chromatography column (GenScript) for purification, controlling the flow rate at 1 mL / min; wash it with washing buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) until OD 280Stability; Elution was carried out by adding elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH 8.0). The purified recombinant protein was further analyzed and purified by Hiprep 16 / 60 sephacry S-200HR using GE liquid chromatography. The target protein was collected according to the absorption peak and detected by SDS-PAGE.

[0027] 3. Analysis of mutant structural integrity Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the molecular weight of the subunits of the positively charged lumen ferritin mutant, while native polyacrylamide gel electrophoresis (Native-PAGE) was used to analyze the assembly state of the mutant, and transmission electron microscopy (TEM) was used to observe the morphological characteristics of the positively charged lumen ferritin mutant.

[0028] The preparation method of the TEM negative staining sample is as follows: Drop 20 μL of the purified recombinant protein sample onto the copper mesh support film, and incubate it at room temperature for 5 min to allow the copper mesh to adsorb the protein sample. After adsorption, blot the excess liquid on the copper mesh with filter paper. Drop 20 μL of 2% phosphotungstic acid negative staining solution at pH 7.0 on the copper mesh for negative staining, and incubate it at room temperature for 5 min. After negative staining, blot the negative staining solution, and observe the morphology of the recombinant protein using a Hitachi transmission electron microscope.

[0029] 4. Analysis of the ability of mutants to load the target DNA fragment of OsHV-1 The target sequence of the OsHV-1 qPCR detection method formulated by WOAH was selected to analyze the DNA loading ability of the ferritin mutant. The target fragment was amplified by PCR using OsHV-1 qPCR detection primers (BF: GTCGCATCTTTGG ATTTAACAA / B4: ACTGGGATCCGACTGACAAC) and purified.

[0030] The pH method was used to mediate the depolymerization / reassembly of the ferritin mutant to load the OsHV-1 target DNA fragment (the molar ratio of the initial nucleic acid to ferritin was set to 5:1): Under a gradient acidic environment (pH 7-2), at intervals of 1-2, the ferritin was depolymerized, and the pH of the depolymerization system was slowly adjusted to 4 using 0.1 M Na2CO3 / NaHCO3 solution (pH 9.4). The nucleic acid fragment was added to the acidic depolymerization system, and after incubation for 1 h, the pH was slowly adjusted to 7.6 for reassembly to complete the loading of the nucleic acid fragment.

[0031] The unloaded nucleic acid fragments were removed using ultrafiltration to obtain the ferritin-loaded nucleic acid product. The nucleic acid and protein concentrations were measured, and the molar ratio of nucleic acid to ferritin was calculated to determine the nucleic acid loading capacity of the mutant. Considering that a high degree of mutation may increase the possibility of mutant structural instability, the best mutant was screened.

[0032] To determine the loading site of the OsHV-1 target DNA fragment in the ferritin nanocarrier, deoxyribonuclease I (DNase I) was incubated with the ferritin-loaded nucleic acid product after ultrafiltration and the co-incubation product of non-dissociated / recombinant ferritin nucleic acid after ultrafiltration at 37 °C for 20 min, and then the OsHV-1 target DNA copy number in the digested product was quantitatively analyzed by fluorescence quantitative PCR. The reagent used was THUNDERBIRD Probe qPCR Mix from Toyobo Co., Ltd., and the experiment was carried out using a Bio-Rad CFX connect fluorescence quantitative PCR instrument.

[0033] The reaction system is shown in Table 1.

[0034] Table 1

[0035] The PCR reaction conditions were as follows:

[0036] 5. Preparation, sub-packaging and reference value determination of the reference material The best mutant was screened according to the nucleic acid loading capacity and structural stability of the mutant as Sb Fn(+)Δ2, and Sb OsHV-1 reference material was prepared using Fn(+)Δ2 as the carrier. The packaging volume of the prepared OsHV-1 reference material was set at 200 μL / tube, which could ensure that most commercially available kits could extract this reference material 2 - 4 times. It was sub-packaged in 1.5 ml screw-cap rubber-sealed cryotubes free of Rnase / Dnase in a biosafety cabinet, with a total of 300 tubes sub-packaged. After sub-packaging, it was sealed with a sealing film and stored at -20 °C.

[0037] 6. Reference value determination of the reference material Fifteen reference materials were randomly selected, and the DNA of the reference material was extracted using a marine animal tissue genomic DNA extraction kit (Tiangen). The specific operation method was as follows: A) Add 50 μL of the reference material to a 1.5 mL enzyme-free centrifuge tube and mix well with 200 μL of GA buffer.

[0038] B) Add 20 μL of proteinase K, mix well and incubate at 56 °C for 10 min.

[0039] C) After the incubation is completed, add 200 μL of GB buffer, mix well, and incubate at 70 °C for 10 min; add 200 μL of absolute ethanol.

[0040] D) Add the obtained solution to the adsorption column and centrifuge at 12,000 rpm for 1 min.

[0041] E) Replace the collection tube, add 500 μL of GD buffer to the adsorption column, and centrifuge at 12,000 rpm for 1 min.

[0042] F) Replace the collection tube, add 600 μL of washing buffer to the adsorption column, and centrifuge at 12,000 rpm for 30 s, and wash twice in total.

[0043] G) After the washing is completed, centrifuge at 12,000 rpm for 2 min to remove the residual liquid.

[0044] H) Place the adsorption column into a new enzyme-free centrifuge tube, add 50 μL of elution buffer preheated to 70 °C to the adsorption column, let it stand for 2 min, and then centrifuge at 12,000 rpm for 2 min to collect the nucleic acid.

[0045] Use the above-mentioned OsHV-1 real-time fluorescence quantitative PCR detection method to determine the Ct value of the reference material, and calculate the copy number of each sample according to the standard curve constructed by the plasmid reference material. Detect and eliminate abnormal data using the Grubbs Criterion, and calculate the overall average value after eliminating abnormal data. Overall average level of experimental data: , ( is the experimental data, n is the number of detections), subtract each experimental value from the average value to screen the data. , if > λ(0.05, 15) * S, then this data is eliminated; continue to calculate the overall average value of the remaining data, and repeat the above method until the experimental data with large errors is completely eliminated. is the residual, which is the difference between the experimental value and the average value. is the experimental value, λ(α, n) selects the corresponding value according to the Grubbs critical value table. In this experiment, α = 5%; S is the variance, and the variance formula: . Uncertainty analysis is calculated according to the formula specified in GB / T15000.3-2008 , select the corresponding value according to the t-test critical value table, m is the number of samples, is the sample value, is the sample average value.

[0046] 7. Homogeneity Analysis of Reference Materials In accordance with the provisions of GB / T 15000.3-2008 "General Principles and Statistical Methods for the Certification of Reference Materials (3) Guidelines for the Work of Reference Samples", the homogeneity of the reference materials was evaluated by the method of repeated measurement in random order. Fifteen tubes were randomly selected from the 300 tubes of reference materials prepared and numbered sequentially as 1 # ~15 # . Three samples were taken from each tube, 50 μL each time. DNA extraction was carried out one by one, and the nucleic acid load of the mutant was determined by the OsHV-1 qPCR detection method to evaluate the homogeneity between and within sample units. After the result data was judged for normal distribution by the Shapiro-Wilk test, the homogeneity of the nucleic acid-loaded ferritin product was confirmed by the one-way analysis of variance (F test), and the minimum sampling amount was determined.

[0047] According to the requirements of GB / T 15000.3-2008 "General Principles and Statistical Methods for the Certification of Reference Materials (3) Guidelines for the Work of Reference Samples", under the conditions of α = 5% and degrees of freedom (9, 20), the critical value can be obtained by referring to the Frieman test table , compared with the [[ID=1④]] calculated by the formula , if it is, it is considered that there is no significant difference in the results between the inside of the bottle and the evaluation.

[0048] 8. Stability Analysis of Reference Materials In accordance with the provisions of GB / T 15000.3-2008 "General Principles and Statistical Methods for the Certification of Reference Materials (3) Guidelines for the Work of Reference Samples", the stability of the reference materials was evaluated by the method of repeated measurement in random order. The long-term stability at -20 °C and -80 °C and the short-term stability at 4 °C and 37 °C were evaluated by the model, where the time is , the quantity values of the reference materials at different time points are , is the intercept, is the slope. Based on the standard deviation of (S( b1 ))), the following judgment can be made by the t-test: If < t (0.95, n-2) *S( ) (n is the number of measurement time points), it indicates that there is no significant difference in the slope and no instability is detected. t (0.95, n-2) Select the corresponding value according to the table. The degrees of freedom of the straight line are n - 2 (in this experiment, there are two constraints, namely the slope and the intercept, and the confidence level is 95%).

[0049] Long-term stability determination: The reference material was stored at -20 °C and -80 °C to simulate the conventional freezing storage temperatures in most biological laboratories. The sampling frequency and sampling time points were set as shown in Table 1. The nucleic acid load of the mutant in each tube of sample was determined by the OsHV-1 qPCR assay, and the detection was repeated 3 times. The average value of the detection results was calculated to analyze the long-term stability.

[0050] Short-term stability determination: The reference material was stored at 4 °C and 37 °C to simulate the daily operation habits in the laboratory. For example, when in frequent use, the operator is accustomed to placing the reference material at 4 °C; 37 °C simulates the abnormal temperature of the cold chain to test the stability of the reference material under relatively high temperature conditions. The sampling frequency and sampling time points were set as shown in Table 2. The nucleic acid load of the mutant in each tube of sample was determined by the OsHV-1 qPCR assay, and the detection was repeated 3 times. The average value of the detection results was calculated to analyze the short-term stability.

[0051] The specific calculation process and experimental operations are as follows: The data was first analyzed by the Kolmogorov-Smirnov test for the normal distribution of the sample data. Then, based on the obtained data, the following statistical calculations were performed: slope , ( is the number of measurement days, is the copy number of the reference material, , are the average values of the detection days and the sample copy numbers respectively, and n is the number of detections); intercept ; standard deviation of the straight line ; slope uncertainty ; judgment whether it is less than t (0.95, n-2) *S( ), if it is less, it is determined that no instability is observed.

[0052] Table 2 Certified reference value data of the reference sample

[0053] (2) Experimental results and analysis 1. Design, construction, and characterization of the positively charged inner cavity mutant of Scapharca broughtonii ferritin As an iron storage protein, the inner cavity of Scapharca broughtonii ferritin ( Sb Fn) is usually negatively charged to bind iron ions. Therefore, based on the analysis of the Sb Fn nanocage structure, by means of bioengineering techniques, the negatively charged Glu and Asp at the iron nucleation sites in the inner cavity of Sb Fn were replaced with the positively charged lysine (Lys) or arginine (Arg), and 2 SbFn positively charged lumen mutants Sb Fn(+)Δ1 and Sb Fn(+)Δ2. Under the same conditions, due to the presence of a strong basic guanidine (pKa = 12.48) on the side chain of Arg (pI = 10.76), it has a higher positive charge intensity than Lys (pI = 9.74). However, the long side chain of Arg is prone to cause steric hindrance problems, resulting in structural instability. Therefore, when selecting positively charged amino acids for substitution, we comprehensively considered their effects on surrounding amino acids and analyzed their spatial distribution, thus deciding whether to use Arg or Lys ( Figure 1 A). Sb Fn(+)Δ1 and Sb The specific mutation sites, amino acid sequences, and structures of Fn(+)Δ2 are shown in Figure 1 B. The amino acid sequences of the target gene and the corresponding protein amino acid sequences of other species were subjected to multiple sequence alignment through the ClustalX multiple alignment program, and the results were obtained through ESPript 3.0 ( Sb Homology modeling models of Fn and Fn(+) subunits with different mutation degrees Sb . The mutation sites were displayed using PyMOL and highlighted in red.

[0054] WT Sb Fn and Sb The expression and purification processes of Fn(+)Δ1-2 recombinant proteins are shown in Figure 2 A. First, the whole cell supernatant after induced expression was centrifuged and then sonicated, and the supernatant was heated at 72 °C to remove miscellaneous proteins. Then, the protein was preliminarily purified by Ni-NTA affinity chromatography, and finally, high-purity recombinant proteins were obtained by size exclusion chromatography ( Figure 2 B). After successfully obtaining the purified Sb Fn(+)Δ1-2 recombinant proteins, the present invention systematically evaluated their structural morphology, depolymerization / reassembly ability, nucleic acid loading ability, and stability to screen out the best Sb Fn(+) mutants for subsequent research.

[0055] First, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify the Sb molecular weight (Mw) of the Fn(+) mutant subunits. The results showed that WT Sb Fn, Sb Fn(+)Δ1, and Sb the protein bands of the Fn(+)Δ2 subunits were located between 20 - 25 kD ( Figure 2 C), and their Mw was consistent with the theoretical Mw value. At the same time, non-denaturing PAGE analysis was also used to evaluate the Sb assembly state of Fn(+)Δ1-2, as shown in Figure 2As shown in D, Sb The protein band of Fn(+)Δ1-2 and WT Sb Fn is almost horizontal, indicating Sb The recombinant protein of Fn(+)Δ1-2 subunit was successfully self-assembled into a 24-mer structure identical to WT Sb Fn and maintained similar surface properties. In the morphological characterization by transmission electron microscopy (TEM), Sb Fn(+)Δ1-2 showed a clear spherical nanocage structure similar to WT Sb Fn, Sb FnΔ1-2 and WT Sb Fn had basically the same outer diameter measured under the electron microscope, about 14 ± 0.5 nm, indicating that the mutation of the negatively charged site did not affect Sb the tertiary structure of FnΔ1-2, and it could still form a 24-mer ( Figure 2 E). To explore its recombinant ability, Fn(+)Δ1-2 was depolymerized in a gradient acidic environment (pH 3.5 - 1.9), and the pH was slowly adjusted to 7.6 with 0.1 M carbonate solution (pH 9.4) for reassembly. Transmission electron microscopy observation confirmed that the acid treatment used in this study successfully depolymerized Sb Fn(+)Δ1-2 ( Sb F). At the same time, [[ID=I106]]Figure 2 the Fn(+)Δ1-2 subunit could reassemble into a complete nanocage structure under neutral conditions ( Sb G), Figure 2 and Fn(+)Δ1-2 maintained the same reversible self-assembly ability as WT Sb Fn. In summary, the above evaluation data verified that these rationally designed Sb Fn(+)Δ1-2 had excellent structural properties and self-assembly ability similar to WT Sb Fn. Sb Fn.

[0056] 2. Screening of the optimal Sb Fn(+) nanocarrier loaded with the OsHV-1 target DNA fragment After evaluating the basic properties of different Sb Fn(+) mutants, the present invention further studied their loading ability for the OsHV-1 target DNA fragment. The target sequence of the OsHV-1 qPCR detection method formulated by WOAH was selected to analyze the DNA loading ability of Sb Fn(+) mutants. As Figure 3 shown in A, the OsHV-1 target DNA fragment was loaded into the hollow inner cavity of the Sb Fn(+) nanocarrier by a pH-mediated depolymerization / reassembly method.

[0057] By calculating the molar ratio of OsHV-1 target DNA to WT Sb Fn and Sb Fn(+)Δ1-2 to determine the loading efficiency of WT Sb Fn and Sb Fn(+)Δ1-2. As expected, the positively charged mutation in the hollow lumen effectively improved the Sb affinity of Fn for OsHV-1 target DNA. Compared with WT Sb Fn, Sb the Fn(+) mutants all had a higher loading capacity for OsHV-1 target DNA ( p <0.05). The comparison of the loading efficiency of the Sb Fn(+) mutants showed that Sb the overall loading capacity of the Fn(+) mutants increased with the increase in the number of positively charged mutations. The loading efficiencies of Sb Fn(+)Δ1 and Sb Fn(+)Δ2 were 1.71 ± 0.06 and 2.47 ± 0.10 times that of WT Sb Fn, respectively. At the same time, Sb the Fn(+) mutants maintained a high protein recovery rate after the pH-mediated loading process of OsHV-1 target DNA, and the recovery efficiency was greater than 50% ( Figure 3 B).

[0058] In addition, considering the possibility of external adsorption of target nucleic acid fragments by the mutants, the present invention examined Sb the loading position of the OsHV-1 target DNA fragment in Fn(+)Δ1-2. As shown in Figure 3 C, after digesting the nucleic acid products loaded by WT Sb Fn and Sb Fn(+) and the nucleic acid co-incubation products of WT Sb Fn and Sb Fn(+) that were not depolymerized / recombined after ultrafiltration with deoxyribonuclease I (DNase I), the copy number of the OsHV-1 target DNA fragment was detected by fluorescence quantitative PCR. The results showed that after digestion, WT Sb Fn and Sb Fn(+)Δ1-2 were 1.69×10 4 、8.71×10 6 and 1.79×10 8 . At the same time, it was found that WT Sb Fn and Sb Fn(+)Δ1-2 could not load the OsHV-1 target DNA fragment without pH-mediated lumen exposure ( Figure 3 C), WT without depolymerization / recombination after ultrafiltrationSb Fn and Sb the copy number of the OsHV-1 target DNA fragment in the co-incubation product of Fn(+) nucleic acid is less than 1×10 1 , indicating that the OsHV-1 target DNA fragment binds to positively charged amino acids through electrostatic interaction and is loaded into the WT Sb Fn and Sb the hollow lumen of Fn(+). Finally, according to the loading efficiency, we selected Sb Fn(+)Δ2 as the best carrier for the OsHV-1 target DNA.

[0059] For the selected Sb Fn(+)Δ2 (E59K / E62R / E138K / E145K), TEM characterization was further carried out. Sb After loading the OsHV-1 target DNA fragment (DNA@ Sb Fn(+)Δ2), it still retained the complete cage structure, showing a circular monodisperse state, with a diameter that was basically uniform, about 14 nm ( Figure 3 D). At the same time, through surface electrostatic potential analysis and comparison with WT Sb Fn, it was found that Sb the electrostatic potential energy of the subunits and the inner cavity of Fn(+)Δ2 changed from negative to positive, while the outer surface hardly changed ( Figure 3 E). In summary, the above results confirmed that Sb Fn(+)Δ2 has the feasibility to be used as a carrier for loading the OsHV-1 target DNA fragment.

[0060] 3. Reference value determination of the reference material The result of the first-step determination was (0.93±0.12)×10 8 copies / μL, as shown in Table 3 in detail. This result was mainly used as a reference basis for the analysis of sample homogeneity and stability.

[0061] Table 3 Data of reference value determination of the reference sample

[0062] * The order of magnitude of the copy number and the unit are 10 8 copies / μL.

[0063] 4. Homogeneity evaluation of the reference material Homogeneity is one of the most fundamental properties of reference materials. To examine whether the reference samples of vegetation are homogeneous, a certain number of samples are usually randomly selected and measured under the same experimental conditions, so that the differences between samples are fully reflected by the inhomogeneity of the samples themselves. According to the provisions of GB / T 15000.3-2008 "General Principles and Statistical Methods for the Certification of Reference Materials (3)", the homogeneity of reference materials is evaluated by the method of repeated measurement in random order. After the Shapiro-Wilk test, the sample homogeneity data is normally distributed. Calculated from the formula Calculated , (2.04), it is determined that there is no significant difference in the results within and between bottles, confirming that the reference sample is homogeneous. The results are shown in detail in Tables 4 and 5.

[0064] Table 4 Homogeneity Analysis of Reference Sample

[0065]

[0066] * The copy number order of magnitude and the unit are 10 8 copies / μL.

[0067] Table 5 Results of Variance Analysis of Homogeneity Experiment of Reference Sample

[0068] 5. Stability Test of Reference Materials The sample test data is normally distributed after the Kolmogorov-Smirnov test. The stability data of the reference materials at 4 different temperatures are shown in Table 6, and the stability trend judgments under each temperature condition are shown in Table 7.

[0069] The long-term stability test focuses on the stability of reference materials under specific storage conditions. The qPCR test results of the reference materials stored at -20 °C and -80 °C for 2.3 and 3.3 months respectively show that the copy number ranges of OsHV-1 target DNA are 0.35×10 8 copy / μL to 2.13×10 8 copy / μL and 0.64×10 8 copy / μL to 2.25×10 8 copy / μL, with the average values of 1.18×10 8 copy / μL and 1.23×10 8 copy / μL. Further analysis of the long-term stability test data shows that the results <t (0.95, n-2) *S( ), indicating that there is no significant difference in slope and no instability is detected.

[0070] Short-term stability tests focus on the stability of reference materials under specific transportation and storage conditions. To simulate extreme cases of abnormal cold chain temperatures, it was selected to store for one week at 37 °C to test the stability of the reference standard. Additionally, to simulate daily laboratory operating habits, it was also selected to store for three weeks at 4 °C to test the stability of the reference standard.

[0071] The qPCR test results showed that the copy number ranges of OsHV-1 target DNA were respectively in the range of 0.43×10 8 copy / μL to 2.04×10 8 copy / μL and 0.38×10 8 copy / μL to 1.94×10 8 copy / μL, with an average of 1.26×10 8 copy / μL and 1.09×10 8 copy / μL. Further analysis of the long-term stability test data showed that the results <t (0.95, n-2) *S( ), indicating that there is no significant difference in slope and no instability is detected, as shown in Table 6 specifically.

[0072] Table 6 Stability analysis data of standard samples

[0073] * The order of magnitude of the copy number is in units of 10 8 copies / μL.

[0074] Table 7 Results of trend analysis of stability of standard samples

[0075] * The order of magnitude is in units of 10 8 copies / μL.

Claims

1. A reference material for loading oyster herpesvirus nucleic acid based on a cage-like structural protein, characterized in that, The reference material is to change the charge property on the surface of the hollow inner cavity of the cage structure of ark shell ferritin from negative to positive; the change from negative to positive is: mutating the negatively charged amino acid residues on the surface of the hollow inner cavity of ark shell ferritin into positively charged amino acids to obtain nucleic acid fragments of positively charged inner cavity ferritin with different mutation sites Sb Fn(+)Δ1 or Sb Fn(+)Δ2, with the codon-optimized Sb Fn(+)Δ1 or the codon-optimized Sb Fn(+)Δ2 as a vector to load the nucleic acid of ostreid herpesvirus.

2. According to the reference material described in claim 1, the amino acid sequence of the ark shell ferritin is as shown in SEQ ID NO.

1.

3. The reference material according to claim 1 or 2, characterized in that, The mutation sites of the ark shell ferritin are Glu-59, Glu-62, Glu-138, Glu-145; the positively charged amino acids are: lysine or arginine.

4. The reference material according to claim 1 or 2, characterized in that, The sequence of the oyster herpesvirus nucleic acid is: GTCGCATCTTTGGATTTAACAATTGCCCCTGTCATCTTGAGGTATAGACAATCGCCAGAAAATTTCCCATGTTCAGTCCATGGGTTGTCAGTCGGATCCCAGT.

5. The reference material according to claim 1, wherein In step (1), The said Sb The nucleotide sequence of Fn(+)Δ1 is: ATGGCTCAAACACAACCAAGACAAAACTACCATGTGGAAAGTGAGGCCGGAATCAACAAACAGATCAACATGGAGTTATATGCCAGTTATGTCTATCAGTCCATGTATATGTACTTTGACAGAGATGATGTAGCTCTGCCTGGTTTTGCCAAGTACTTCAAGCATAATTCCGAG AAG GAACGT CGA CATGCTGAGAAACTCATGAAGTACCAGAACAAGAGAGGTGGCAGAGTTGTCCTTCAAGACATTCAGAAACCTGACCGTGATGAATGGGGTTCTCCACTGGAAGCCATGCAGACTACATTAGCTCTGGAGAAAAGTGTTAACCAGGCACTTCTGGACCTGCACAAAGTTGCAGATAAACATGGTGATGCCCAGATGATGGATTTCCTTGAAGGAGAATACCTGAAGGAGCAGGTAGAAGCCATTAAGGAGATATCGGACCACATCACCAATCTGAAACGTGTGGGTACAGGTCTCGGAGAGTACATGTATGACAAGGAAACCATGAGCAGT; The codon-optimized Sb The nucleotide sequence of Fn(+)Δ1 is: ATGGCGCAGACCCAGCCGCGTCAGAACTACCACGTTGAAAGCGAAGCGGGTATCAACAAACAGATCAACATGGAACTGTACGCGAGCTACGTTTACCAGAGCATGTACATGTACTTCGATCGTGATGATGTTGCGCTGCCGGGTTTCGCGAAATACTTCAAACACAACAGCGAAAAAGAACGTCGTCACGCGGAAAAACTGATGAAATACCAGAACAAACGTGGTGGCCGTGTTGTTCTGCAGGATATCCAGAAACCGGATCGTGATGAATGGGGTAGCCCGCTGGAAGCGATGCAGACCACCCTGGCGCTGGAAAAAAGCGTTAACCAGGCGCTGCTGGATCTGCACAAAGTTGCGGATAAACACGGTGATGCGCAGATGATGGATTTCCTGGAAGGTGAATACCTGAAAGAACAGGTTGAAGCGATCAAAGAAATCAGCGATCACATCACCAACCTGAAACGTGTTGGCACCGGCCTGGGTGAATACATGTACGATAAAGAAACCATGAGCAGC; The said Sb The nucleotide sequence of Fn(+)Δ2 is as follows: ATGGCTCAAACACAACCAAGACAAAACTACCATGTGGAAAGTGAGGCCGGAATCAACAAACAGATCAACATGGAGTTATATGCCAGTTATGTCTATCAGTCCATGTATATGTACTTTGACAGAGATGATGTAGCTCTGCCTGGTTTTGCCAAGTACTTCAAGCATAATTCCGAG AAG GAACGT CGA CATGCTGAGAAACTCATGAAGTACCAGAACAAGAGAGGTGGCAGAGTTGTCCTTCAAGACATTCAGAAACCTGACCGTGATGAATGGGGTTCTCCACTGGAAGCCATGCAGACTACATTAGCTCTGGAGAAAAGTGTTAACCAGGCACTTCTGGACCTGCACAAAGTTGCAGATAAACATGGTGATGCCCAGATGATGGATTTCCTTGAAGGAGAATACCTGAAG AAG CAGGTAGAAGCCATTAAG AAG ATATCGGACCACATCACCAATCTGAAACGTGTGGGTACAGGTCTCGGAGAGTACATGTATGACAAGGAAACCATGAGCAGT; The codon-optimized Sb The nucleotide sequence of Fn(+)Δ2 is: ATGGCGCAGACCCAGCCGCGTCAGAACTACCACGTTGAATCTGAAGCGGGCATCAACAAACAGATCAACATGGAACTGTACGCGAGCTACGTTTACCAGAGCATGTACATGTACTTCGATCGTGATGATGTTGCGCTGCCGGGCTTCGCGAAATACTTCAAACACAACAGCGAAAAAGAACGTCGTCACGCGGAAAAACTGATGAAATACCAGAACAAACGTGGCGGCCGTGTTGTTCTGCAGGATATCCAGAAACCGGATCGTGATGAATGGGGTAGCCCGCTGGAAGCGATGCAGACCACCCTGGCGCTGGAAAAAAGCGTTAACCAGGCGCTGCTGGACCTGCACAAAGTTGCGGATAAACACGGTGATGCGCAGATGATGGATTTCCTGGAAGGTGAATACCTGAAAAAACAGGTTGAAGCGATCAAGAAAATCTCTGATCACATCACCAACCTGAAACGTGTTGGTACCGGCCTGGGTGAATACATGTACGATAAAGAAACCATGAGCAGC; In the nucleotide sequence, the italicized and underlined sites are mutation sites.

6. A preparation method of a reference material according to any one of claims 1-5, characterized in that, It includes the following steps: (1) Link the nucleic acid fragments of positively charged lumen ferritin with different mutation sites Sb Fn(+)Δ1 or Sb Fn(+)Δ2 to the pET-30a(+) vector. The recombinant expression vector is transformed into the expression competent cell Transetta DE3 by heat shock, and positive clones are screened by colony PCR; (2) Induce the expression of the positive clone; centrifuge to collect the bacterial cells, resuspend with the lysis equilibration buffer and then perform sonication; Centrifuge to collect the supernatant, heat the supernatant and then centrifuge again, collect the supernatant, filter and then perform purification; Wash with the washing buffer until OD 280 stable; Add the elution buffer for elution; analyze and purify the purified recombinant protein using GE liquid chromatography, and collect the ferritin mutant according to the absorption peak; (3) Use the OsHV-1 qPCR detection primers to perform PCR amplification of the target fragment and then purify it to obtain the OsHV-1 target DNA fragment; (4) Mediate the depolymerization / reassembly of the ferritin mutant to load the OsHV-1 target DNA fragment by the pH method: depolymerize the ferritin mutant in a gradient acidic environment, adjust the pH to obtain an acidic depolymerization system; add the OsHV-1 target DNA fragment into the acidic depolymerization system, incubate and then slowly adjust the pH to 7.6 for reassembly to complete the loading of the nucleic acid fragment; remove the unloaded nucleic acid fragments to obtain the reference material.

7. The preparation method according to claim 6, characterized in that, In step (2), the specific method for inducible expression is as follows: inoculate the positive clone into an LB medium containing kanamycin resistance and culture it until the OD 600 reaches 0.3 - 0.4, add IPTG with a final concentration of 0.1 mM for induction for 5 h; the heating is carried out at 72 °C for 15 min; the composition of the LB medium containing kanamycin resistance is: 1 g Tryptone, 0.5 g Yeast Extract, 1 g NaCl, 5 mg Kanamycin per 100 ml.

8. The preparation method according to claim 6, characterized in that, In step (3), the OsHV-1 qPCR detection primers are: BF: GTCGCATCTTTGGATTTAACAA; B4: ACTGGGATCCGACTGACAAC.

9. The preparation method according to claim 6, characterized in that, In step (4), the molar ratio of the OsHV-1 target DNA fragment to the ferritin mutant is 5:1; the gradient acidic environment is pH 7-2; the pH value is adjusted to 4; the incubation is carried out at 4 °C for 1 h.

10. Use of a reference material according to any one of claims 1-5 in the preparation of a kit for detecting ostreid herpesvirus.

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