A single-domain antibody of decapod iridescent virus 1 and a preparation method and application thereof

Abstract

The application discloses a single-domain antibody of decapod iridovirus 1 and a preparation method and application thereof, amino acid sequences of the single-domain antibody are shown in SEQ ID NO:1 or SEQ ID NO:2, and gene sequences are shown in SEQ ID NO:3 or SEQ ID NO:4; meanwhile, the application also provides a preparation method of the single-domain antibody of decapod iridovirus 1, adult healthy striped bonnet sharks are taken as immunization objects, and immunization is carried out by adopting DIV1 as an antigen; total RNA is extracted by separating peripheral blood lymphocytes of the immunized striped bonnet shark, and then the total RNA is reversely transcribed into cDNA; the cDNA is taken as a template, a vNAR fragment of the striped bonnet shark is amplified, and the vNAR fragment is connected with a carrier to construct a phage library; positive clones recognizing the DIV1 are selected from the phage library; an expression vector is constructed, the single-domain antibody of the DIV1 is induced to express, and finally the single-domain antibody of the decapod iridovirus 1 is obtained; and the single-domain antibody of the decapod iridovirus 1 is used for detecting the decapod iridovirus 1.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular immunology and pathogenic biology, and particularly relates to a single-domain antibody of Decapod iridescent virus 1 (DIV1) and a preparation method and application thereof. BACKGROUND

[0002] Shrimp iridovirus disease is an acute and infectious disease, and its pathogen is Decapod iridescent virus 1 (DIV1) of a new genus, Decapod iridovirus, of the iridovirus family. DIV1 is a cytoplasmic virus with linear double-stranded DNA, and the virus appears iridescence under the irradiation of oblique light, and thus is called iridovirus. The mortality rate of DIV1 to crustaceans can be as high as 100%. After the infection of DIV1, the shrimp presents the phenomena of lighter color of hepatopancreas, empty stomach and soft shell, and can cause large-area death in a short period. In recent years, Decapod iridovirus disease has broken out in most coastal shrimp breeding areas in China, and the harm spreads to numerous varieties such as Litopenaeus vannamei, Chinese shrimp, white shrimp, Japanese marsh shrimp and Macrobrachium rosenbergii, causing economic losses of hundreds of millions of US dollars every year, and seriously endangering the healthy development of the shrimp breeding industry.

[0003] At present, there is no effective prevention and control measure for Decapod iridescent virus 1, and the early diagnosis of the disease is an urgent problem to be solved. The existing methods for identifying DIV1 infection mainly include PCR detection and in situ hybridization; a Chinese patent with the publication number CN114717358A discloses a “dual-PCR detection method and kit for simultaneously detecting shrimp hepatocystis and Decapod iridescent virus 1”, wherein the specific disclosed detection method does not need to further clone, sequence and sequence align the detected pathogenic gene fragment, and can detect whether the sample contains one or both of the two pathogens in one experiment; a Chinese patent with the publication number CN111218530A discloses “primer set and kit for detecting Decapod iridescent virus 1 by fluorescence quantitative PCR”, wherein the specific disclosure includes designing specific primers and TaqMan-MGB probes and kits according to the conservative sequence of the MCP gene of DIV1, preparing a recombinant plasmid standard pMD18-T-MCPDIV1, and establishing a TaqMan-MGB probe fluorescence quantitative PCR method for detecting DIV1. However, the above-mentioned molecular biology detection method has a high technical requirement, depends on the establishment of a basic laboratory, and is difficult to realize “pool-side detection”, and thus is difficult to be popularized and applied in breeding production.

[0004] Developing monoclonal antibodies against the virus and utilizing immunological detection techniques to create on-site detection products are effective methods for early diagnosis of Decapoda iridovirus DIV1. In recent years, immunological detection has made significant progress in shrimp pathogen detection, enabling early detection of several major shrimp diseases, such as white spot syndrome and acute hepatopancreatic necrosis disease. Currently, there are no reports on the development of monoclonal antibodies against shrimp iridovirus DIV1. Summary of the Invention

[0005] To meet the aquaculture needs of "pondside testing" and solve the problem of complex detection of Decapoda iridovirus 1 in existing technologies, this invention creatively proposes a single-domain antibody for Decapoda iridovirus 1, its preparation method, and its application. The single-domain antibody prepared by this invention can specifically recognize DIV1 virus, with good sensitivity and accuracy, providing new ideas for establishing early diagnostic technology for shrimp iridovirus disease and research on prevention and treatment drugs.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides a single-domain antibody against Decapod Iridovirus 1, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0008] Furthermore, the gene sequence of the single-domain antibody is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0009] This invention also provides a method for preparing a single-domain antibody against decapod iridovirus 1, specifically comprising the following steps:

[0010] (1) Adult healthy striped bamboo sharks were used as immunization subjects, and DIV1 was used as the antigen for immunization;

[0011] (2) After immunization, peripheral blood lymphocytes of immunized sharks were isolated, total RNA was extracted and reverse transcribed into cDNA; using cDNA as a template, the vNAR fragment of the striped bamboo shark was amplified, and the vNAR gene fragment was enzymatically ligated with the vector to construct a phage library.

[0012] (3) Using phage display technology, positive clones of DIV1 were identified from the library: Two single-domain antibodies were obtained by screening, enriching and identifying the phage library, and named D02 and D13, single-domain antibodies of Decapoda iridovirus 1; the gene sequences of D02 and D13 antibodies were obtained by sequencing.

[0013] (4) Constructing an expression vector and inducing expression of DIV1 single-domain antibody: The gene sequences of D02 and D13 were cloned into PET30a to obtain an expression vector. The expression vector was transformed into E. coli Shuffle T7 strain, and after expansion culture, IPTG was added to induce expression. The antibody protein was collected and purified.

[0014] The present invention also provides the application of a single-domain antibody against Decapod Iridovirus 1 in the detection of Decapod Iridovirus 1.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] 1. This invention prepares single-domain antibodies against DIV1 virus, filling the current gap in monoclonal antibodies against DIV1 virus. Western blotting and mass spectrometry analysis confirm that the prepared D02 and D13 single-domain antibodies specifically recognize DIV1 virus, and both are specific to the structural protein YbiA of DIV1 virus. Enzyme-linked immunosorbent assay (ELISA) shows that the detection limits of the D02 and D13 single-domain antibodies against the virus reach 10-1 respectively. 2 copies / mL and 10 copies / mL.

[0017] 2. This invention applies the single-domain antibody of Decapoda iridovirus 1 to the early rapid detection of DIV1 pathogen, laying a solid foundation for establishing a monoclonal antibody diagnostic method for DIV1 pathogen, studying the pathogenesis, infection route and epidemic pattern of iridovirus disease, and blocking viral invasion to achieve prevention and control of shrimp iridovirus disease. Attached Figure Description

[0018] Figure 1 A schematic diagram of the SDS-PAGE gel electrophoresis results of the single-domain antibody prepared according to the method of the present invention;

[0019] Figure 2 This is a schematic diagram showing the results of Western Blot detection of the specific recognition of DIV1 virus by single-domain antibodies.

[0020] Figure 3 This is a schematic diagram showing the results of detecting the sensitivity of single-domain antibodies against DIV1 virus using ELISA. Detailed Implementation

[0021] To make the content of this invention easier to understand, the technical solutions of this invention will be further described below in conjunction with specific embodiments and accompanying drawings, but this invention is not limited thereto.

[0022] Example 1

[0023] A method for preparing a single-domain antibody against decapod iridovirus 1, specifically including the following steps:

[0024] (1) Adult healthy striped bamboo sharks were used as immunization subjects, and purified DIV1 was used as the antigen for immunization, according to 5 x 10 6 The dose is 1 copies / kg, administered via subcutaneous injection at multiple sites, for a total of 6 immunizations, with an interval of 14 days between each immunization.

[0025] (2) After immunization, peripheral blood lymphocytes from immunized sharks were isolated, and total RNA was extracted using the QIAGEN RNA extraction kit and the TAKRA PrimeScript RNA extraction kit. TM The II 1st Strand cDNA Synthesis Kit reverse transcribes RNA into cDNA; specific primers for amplifying the vNAR gene of the striped bamboo shark, as designed by us, are used;

[0026] Upstream primer: CGTGGCCCAGGCGGCCGGGCCCCCTGGTTACCAAATGT;

[0027] Downstream primer: CGTGGCCCAGGCGGCCGGGCCCTTTGCCAGGTTTCACAGTCAG;

[0028] The vNAR gene fragment was amplified using the obtained cDNA as a template. The purified vNAR gene fragment and the phage vector pComb3XSS were digested with Sfi I restriction endonuclease and then ligated to construct a recombinant phage vector. The constructed recombinant phage vector was transformed into XL1-Blue competent cells via electroporation to obtain a primary antibody library. An appropriate amount of the primary antibody library was inoculated into 100 mL of 2YTG medium (containing 2% glucose, 100 μg / mL ampicillin, and 50 μg / mL tetracycline) and cultured at 37°C with shaking at 250 rpm until the OD600 reached 0.5. 10 12 PFU VCSM13 helper phage was incubated at room temperature for 15 min, then cultured at 37°C with shaking at 150 rpm for 1 h. The cells were collected by centrifugation at 4000 rpm for 20 min and then incubated with 200 mL of 2YTG. amp+tet+kanaThe culture medium (containing 2% glucose, 100 μg / mL, 50 μg / mL tetracycline, and 50 μg / mL kanamycin) was resuspended and cultured at 37°C with shaking at 250 rpm for 16 h. The bacterial culture was transferred to a sterile centrifuge tube and centrifuged at 10,000 rpm at 4°C for 20 min. The supernatant was collected, and PEG / NaCl solution was added to the supernatant at 1 / 5 volume. The phage was precipitated by incubating on ice at 4°C for 1 h. The precipitate was collected by centrifuging at 10,000 rpm at 4°C for 20 min, and dissolved in 10 mL of PBS to obtain the phage antibody library.

[0029] (3) Using phage display technology, positive clones of DIV1 were identified from the library: The DIV1 phage antibody was screened and enriched in three rounds. The method of the first round of screening and enrichment is as follows:

[0030] 31. Make the number of copies 1x10 7 Copies of DIV1 virus particles were dissolved in 2 mL of PBS, transferred to immunotubes, and coated overnight at 4°C.

[0031] 32. Discard the coating solution, wash 3 times with PBS, add 3 mL of 2% BSA solution, and slowly shake at room temperature for 2 hours to block.

[0032] 33. Wash the immunotube three times with PBS, add 1 mL of the phage antibody obtained in the previous step, and incubate at room temperature with gentle shaking for 2 hours;

[0033] 34. After washing the immunoassay tube 10 times with PBST, add 2 mL of Glycine HCl (pH=2.0) to the immunoassay tube, shake slowly at room temperature for 15 min, then add 0.2 mL of Tris HCl (pH=9.0) to adjust the pH to about 7.5, then add 5 mL of XL1-Blue bacterial culture (OD600=0.6), transfer to a 50 mL centrifuge tube, and incubate at 37℃ with shaking for 1 h;

[0034] 35. Add a copy number of 10. 12 Copies of VCSM13 helper phage were incubated at room temperature for 30 min, then shaken at 150 rpm and 37°C for 1 h. The precipitate was then collected by centrifugation at 4000 rpm for 20 min and resuspended in 100 mL of 2×YT solution. amp+tet+kana Culture medium, shaken at 37°C for 16 h at 250 rpm;

[0035] 36. After precipitating the phage using the PEG / NaCl method, the precipitate was collected by centrifugation at 10,000 rpm and 4°C, and then dissolved in 5 mL of PBS.

[0036] Subsequently, the second and third rounds of screening and enrichment were the same as the first round, and a total of three rounds of screening and enrichment were performed before screening for positive recombinant antibodies. Positive recombinant antibodies were screened using Phage ELISA, and the steps are as follows:

[0037] 37. Take a 72-well culture plate and add 0.4 mL of 2×YTG to each well. amp+tet+kana Culture medium;

[0038] 38. Randomly select single antibody colonies from the tertiary antibody library (i.e., the DIV1 antibody library obtained after three rounds of screening), inoculate them into the above culture plate, label it Master Plate, and incubate overnight at 250 rpm and 37°C with shaking.

[0039] 39. Take another 72-well culture plate and take 0.4 mL of a solution containing 1x10⁻⁶ cells / well. 10 pfu VCSM13 helper phage 2YTG amp+tet+kana Add culture medium to each well; take 50 μL of culture medium from each well of the Master Plate and add it to the corresponding well, labeling it P1 plate. Incubate at 150 rpm and 37°C with shaking for 2 h; centrifuge at 4000 rpm for 20 min, discard the supernatant, and add 0.4 mL of 2×YT to each well. amp+tet+kan Culture medium, 37℃, 250rpm, shake and culture overnight; centrifuge at 4000rpm for 20min, collect the supernatant and store at 4℃ for later use. This completes the preparation of phage recombinant antibody.

[0040] 40. Make the number of copies 5x10 5 DIV1 copies were used to coat 96-well microplates and incubated overnight at 4°C. The coating solution was discarded, and the plates were washed three times with PBS. 2% BSA was added, and the plates were blocked by gentle shaking at room temperature for 2 hours. The prepared phage recombinant antibody was added accordingly, and the plates were incubated by gentle shaking at room temperature for 2 hours. The plates were washed six times with PBST, and the enzyme-labeled secondary antibody Anti-M13-HRP (diluted with PBST at a ratio of 1:5000) was added. The plates were incubated by gentle shaking at room temperature for 1 hour. The plates were washed six times with PBST, and 100 μL of TBM chromogenic solution was added to each well. After incubation in the dark for 10 minutes, 100 μL of 2M H2SO4 was added to each well to stop the chromogenic reaction. The OD450 value was read using a microplate reader. Positive clones were screened based on the OD450 value of the experimental group being more than three times higher than that of the negative control group. Sequencing analysis was performed, and after excluding duplicates, two positive clones were obtained and named single-domain antibodies D02 and D13.

[0041] The gene sequence of D02 (SEQ ID NO:3) is as follows:

[0042] ATGAATATTTTCTTGTTTTCGTGCCTTTTAGCCTGGTTACCAAATGTCTTCACTGCATGGGCTGACCAAACACCGACAACGACAACAAAAGAGGCAGGCGAATCACTGACCCTCCATTGCGCCCTAAGAAATTCCCCCTGTGGATTGGATAGCGCGTACTGGTATTTCACCAAAAAGGGCGCAACAAGGAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAGTTGACAAGTCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATTACTGTACAATCCGCGCCTACTGTCTCGCTGGATGGACCTATTATGGAGGAGGCGGCACCATTCTGACTGTGAAACCTGGCAAA;

[0043] The amino acid sequence of D02 (SEQ ID NO: 1) is: MNIFLFSCLLAWLPNVFTAWADQTPTTTTKEAGESLTLHCALRNSPCGLDSAYWYFTKKGATRKESLSNGGRYAETVDKSSKSFSLRISDLRVEDSGTYYCTIRAYCLAGWTYYGGGGTILTVKPGK;

[0044] The gene sequence of D13 (SEQ ID NO: 4) is:

[0045] ATGAATATTTTTCTTGTTTTCGTGCCTTTTAGCCTGGTTACCAAATGTCTTACTCAACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTGTGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAG CTTATCAACTGGCGGACGATACTCGGACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGCGTATACGCTGGGAACAGCTGGGCAGTACTGTGCGATGCGGGATTATGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGGCAAA;

[0046] The amino acid sequence of D13 (SEQ ID NO:2) is as follows:

[0047] MNIFLFSCLLAWLPNVFTQRVEQTPTTTTKEAGESLTINCVLKGSSCALGSTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYTLGTAGQYCAMRDYEGGGTILTVKPGK;

[0048] (4) Construction of expression vectors and induction of expression of DIV1 single-domain antibodies: Based on the sequencing results above, primers were designed to amplify the vNAR gene fragments of single-domain antibodies D02 and D13, and cloned into the Pet30a expression vector using homologous recombination. The recombinant expression vector was transformed into E. coli Shuffle T7 competent cells. After confirming the positive strain, it was inoculated into LB medium containing kanamycin resistance and cultured at 200 rpm until the OD600 reached 0.6-1.0. IPTG solution with a final concentration of 0.5 mM was added, and expression was induced at 18°C ​​and 200 rpm for 18 h. After the induction of expression, the bacterial cells were collected by centrifugation, the bacterial cells were broken by sonication, the supernatant was collected by centrifugation, and the antibody protein was purified by conventional His-Tag affinity chromatography. The purified recombinant antibody protein of single-domain antibodies D02 and D13 had a purity >90%. The SDS-PAGE electrophoresis results are as follows. Figure 1 As shown.

[0049] Example 2 Performance Detection of Single-Domain Antibodies D02 and D13

[0050] 1. Western blot detection of antibody specificity for DIV1 virus recognition

[0051] With a copy number of 1x10 6 The DIV1 virus particles were used as the sample. After SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane using a semi-dry transfer method. The membrane was blocked with 5% skim milk powder and incubated at room temperature with shaking for 2 hours. Affinity-purified D02 and D13 antibodies (0.2 μg / mL) were added, and the membrane was incubated at room temperature with shaking for 2 hours. After washing 6 times with TBST, HRP-conjugated mouse anti-striped bamboo shark IgNAR secondary antibody was added, and the membrane was incubated at room temperature with shaking for 1.5 hours. After washing 5 times with TBST, equal volumes of ECL chemiluminescence solutions A and B were mixed and poured onto the membrane. The membrane was then exposed and developed using a chemiluminescent gel imaging system (BIORAD ChemiDoc XRS). The experimental results are as follows: Figure 2 As shown, there are obvious specific bands between 70-100kDa, indicating that the single-domain antibodies D02 and D13 prepared in the examples can specifically recognize DIV1 virus.

[0052] 2. Mass spectrometry analysis to identify the structural proteins of the DIV1 virus bound by the antibody.

[0053] With a copy number of 1x10 6 The DIV1 virus particles were used as the sample. After SDS-PAGE electrophoresis, the electrophoresis gel was stained with Coomassie Brilliant Blue. A single electrophoretic band with a molecular weight of 70kDa to 100kDa was cut off from the gel with a clean scalpel and placed in an EP tube for mass spectrometry identification by Shanghai Aipticon Biotechnology Co., Ltd. The mass spectrometry results showed that both single-domain antibodies D02 and D13 specifically recognized the DIV1 virus structural protein Swarming motility protein YbiA.

[0054] 3. Detection sensitivity of the single-domain antibody prepared by ELISA against DIV1 virus.

[0055] DIV1 virus was serially diluted to a titer of 10. 8 copies / mL, 10 7 copies / mL, 10 6 copies / mL, 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2Coated microplates were prepared with 10 copies / mL and 10 copies / mL, 0.1 mL per well, and incubated overnight at 4°C. The next day, the coating liquid was discarded, and the plates were washed with PBS. 2% BSA was added, and the plates were blocked at room temperature for 2 hours. After washing three times with PBS, affinity-purified single-domain antibodies D02 and D13 (antibody concentration 0.1 μg / mL) were added, and the plates were incubated at room temperature with shaking for 2 hours. After washing six times with PBST, HRP-conjugated mouse anti-striped bamboo shark IgNAR secondary antibody was added, and the plates were incubated at room temperature with shaking for 1.5 hours. After washing with PBST, 100 μL of TBM chromogenic substrate was added to each well, and the plates were incubated in the dark for 10 minutes. 100 μL of 2M H2SO4 was added to each well to stop the chromogenic reaction. The OD450 value was read using a microplate reader. The binding was determined by the experimental group having an OD450 value more than three times higher than the negative control group. The experimental results are as follows: Figure 3 As shown, the detection sensitivity of the D02 antibody against DIV1 virus reaches 10. 2 The D13 antibody has a detection sensitivity of 10 copies / mL against DIV1 virus.

[0056] Application of the single-domain antibody prepared in Example 3 in the detection of shrimp iridovirus pathogen

[0057] The single-domain antibody prepared according to Example 1 was used in the detection of DIV1 virus in shrimp. Gill tissue from infected shrimp challenged with DIV1 virus was taken, and PBS was added at a ratio of 0.5 g / mL. The mixture was ground in an ice bath for 5 min, allowed to stand at room temperature for 5 min, and then 3 μL of the supernatant was spotted onto a 0.22 μm NC membrane (experimental group, negative control group, and blank control group were set up; the experimental group and negative control group were spotted with the gill tissue homogenate of infected shrimp, and the blank control group was spotted with PBS). After drying at room temperature, the membrane was placed in a 96-well plate; 2% BSA was added to each well. The membrane was blocked at room temperature for 2 hours. After washing with PBS, the experimental group and the blank control group were respectively added with affinity-purified single-domain antibodies D02 and D13 (antibody concentration of 0.1 μg / mL), and the negative control group was added with unrelated antibody (antibody concentration of 0.1 μg / mL). The membranes were incubated at room temperature with gentle shaking for 2 hours. After washing with PBST 6 times, HRP-conjugated mouse anti-striped bamboo shark IgNAR secondary antibody was added, and the membranes were incubated at room temperature with gentle shaking for 1.5 hours. TBM chromogenic substrate was added to each well until the color (blue) was clear. Each well was washed with 2M H2SO4, and the color development results were observed. The results showed that the experimental group showed clear purple-red dots on the NC membrane, while the negative control group and the blank control group did not show any color on the NC membrane.

[0058] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A single-domain antibody against a decapod iridovirus 1, characterized in that: The amino acid sequence of the single-domain antibody is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. The use of a single-domain antibody against Decapod Iridovirus 1 as described in claim 1 in the preparation of products for detecting Decapod Iridovirus 1.

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