A single-chain antibody against LMBV and its application in preparation of quantum dot fluorescent immunochromatographic test strip

Abstract

The application discloses an anti-LMBV single-chain antibody and application thereof in preparation of a quantum dot fluorescent immunochromatography test strip, and belongs to the technical field of biological detection. The single-chain antibody comprises Ab-K1 and Ab-K2, the amino acid sequence of the Ab-K1 is shown as SEQ ID NO. 11, and the amino acid sequence of the Ab-K2 is shown as SEQ ID NO. 12. The single-chain antibody used in the application can accurately recognize and combine with iridovirus in a sample to be detected, so that the accuracy of detection is ensured. Meanwhile, the antibody labeled with fluorescent quantum dots has good fluorescent characteristics, so that the fluorescent signal is easy to observe and detect, and the specific surface area is relatively large, so that the antibody can be combined with more virus molecules, so that the sensitivity of detection is improved, the accuracy and sensitivity of detection are improved, the operation steps are simplified, and powerful technical support is provided for detection and prevention and control of iridovirus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a single-chain antibody against LMBV and application thereof in preparation of a quantum dot fluorescent immunochromatographic test strip. BACKGROUND

[0002] Largemouth bass (Micropterus salmoides), also known as California bass, is a eurythermic fish originally from the Mississippi River basin in North America. It has become an important freshwater aquaculture species in China due to its fast growth, low temperature tolerance, and delicious meat. In 2022, the aquaculture amount of largemouth bass in China reached 800,000 tons. However, in recent years, with the continuous increase of aquaculture density and the deterioration of the aquaculture environment, diseases have become a major bottleneck problem for the development of largemouth bass aquaculture industry, seriously affecting the development of the industry. Largemouth bass iridovirus disease (LMBVD) is one of the diseases that have attracted attention, and its pathogen is largemouth bass virus (LMBV). After largemouth bass is infected with iridovirus, the mortality rate can reach 70%, seriously threatening the largemouth bass aquaculture industry. Largemouth bass iridovirus disease has a long incubation period, fast outbreak speed, high mortality rate, and wide transmission range during aquaculture, so it is urgent to establish an efficient virus detection method.

[0003] The detection methods of LMBV mainly include isolation of pathogen, histological observation, polymerase chain reaction technology (RT-PCR), loop-mediated isothermal amplification technology (LAMP), etc. First, cell isolation of virus requires cell culture, and the detection cycle needs more than 14 days, which is complicated and time-consuming. Histological observation can only make a preliminary diagnosis and cannot make a definite diagnosis. Traditional colloidal gold detection method has low sensitivity, especially in the case of low virus concentration, it is difficult to accurately detect the presence of virus, which may lead to improper disease transmission and control during aquaculture. Second, the lack of strong specificity is also an important problem. LMBV and other viruses may have similar antigenic epitopes, leading to cross-reaction of antibodies during recognition, thereby affecting the accuracy of the detection results. In addition, although polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) have high accuracy, they usually require professional laboratory equipment and technical personnel, and the detection process is time-consuming, which is not suitable for rapid identification and control of the initial spread of the epidemic. LAMP has serious aerosol pollution problems, and its application is mainly for scientific research, and it is less used in actual sample quarantine. In addition, these methods have limitations in operability and convenience for on-site application, especially in situations that require rapid decision-making such as customs quarantine and sudden epidemic monitoring.

[0004] The immunochromatography technology relies on the interaction between specific antibodies and antigens to realize the qualitative and quantitative detection of antigens through chromatography. However, the traditional immunochromatography technology often faces challenges in the preparation of chromatography materials and antibody specificity, resulting in inaccurate detection results. The fluorescence labeling technology labels antibodies or antigens with fluorescent substances, and indicates the presence and quantity of target molecules through fluorescence signals. Although the fluorescence labeling technology can provide intuitive fluorescence signals, the stability of fluorescent substances, the luminous intensity and the quantitative analysis ability of fluorescence signals are important factors limiting its wide application. Although there are some reports on the application of the combination of the two technologies in the field of virus detection, most of the methods have problems such as low sensitivity, poor specificity or complex operation. Especially in the detection of largemouth bass iridovirus with complex characteristics, the current detection technology is often difficult to meet the requirements of rapid, efficient and accurate detection.

[0005] The selection of antibodies is the core of the preparation of immunochromatography test strips. At present, the antibodies used in commercial test strips are monoclonal antibodies. However, the high cost and long production cycle of monoclonal antibodies greatly limit their application in aquatic animals. These problems and defects limit the application effect of the prior art in LMBV detection. Therefore, it is an urgent need to develop a rapid, efficient, economical, specific and simple virus detection method. SUMMARY

[0006] The purpose of the present application is to provide a single-chain antibody against LMBV and its application in the preparation of quantum dot fluorescence immunochromatography test strips, so as to solve the problems existing in the prior art. The present application uses the means of fluorescence quantum dot immunochromatography technology, realizes the rapid and efficient detection of LMBV through the principle of high-specificity anti-large-mouth bass LMBV single-chain antibody combined with fluorescence quantum dot labeled antibody.

[0007] In order to achieve the above purpose, the present application provides the following solutions:

[0008] The present application provides a single-chain antibody against largemouth bass iridovirus, which comprises Ab-K1 or Ab-K2. The amino acid sequence of Ab-K1 is shown in SEQ ID NO. 11, and the amino acid sequence of Ab-K2 is shown in SEQ ID NO. 12.

[0009] The present application also provides a fluorescence quantum dot labeled antibody against largemouth bass iridovirus, which comprises the single-chain antibody.

[0010] The present application also provides the application of the single-chain antibody or the fluorescence quantum dot labeled antibody in the preparation of products for detecting largemouth bass iridovirus.

[0011] Further, the products include test strips and kits.

[0012] Further, the test strip comprises a quantum dot fluorescent immunochromatography test strip.

[0013] The application further provides a quantum dot fluorescent immunochromatography test strip for detecting largemouth bass iridovirus, comprising a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad, wherein the nitrocellulose membrane comprises a detection line and a quality control line, and the detection line comprises the fluorescent quantum dot labeled antibody.

[0014] Further, the quality control line comprises a goat anti-rabbit IgG antibody.

[0015] Further, when the quantum dot fluorescent immunochromatography test strip is used, the virus titer in the sample to be tested is greater than or equal to 10 2 TCID 50 / mL.

[0016] Further, the amount of the sample to be tested dropped on the quantum dot fluorescent immunochromatography test strip is 100 muL.

[0017] Further, the reaction time of the sample to be tested in the quantum dot fluorescent immunochromatography test strip is 20 min.

[0018] The application discloses the following technical effects:

[0019] The application adopts the spleen tissue of a New Zealand white rabbit infected with LMBV to construct a phage antibody library, and through 5 rounds of biological panning, two kinds of high-affinity single-chain antibody proteins Ab-K1 and Ab-K2 are screened by combining phage ELISA and gene sequencing technologies. When the dilution ratio of the two kinds of single-chain antibody proteins Ab-K1 and Ab-K2 is 1:3200, the affinity with LMBV virus particles is still maintained, and when the dilution ratio of the single-chain antibody protein Ab-K1 is 1:6400, the affinity with LMBV virus particles is still maintained.

[0020] The application realizes rapid and efficient detection of LMBV by means of the fluorescent quantum dot immunochromatography technology, through the principle that the high-specificity anti-largemouth bass LMBV single-chain antibody is combined with the fluorescent quantum dot labeled antibody. Specifically, the high-specificity antibody used in the application can accurately recognize and combine the iridovirus in the sample to be tested, thereby ensuring the accuracy of the detection. Meanwhile, the fluorescent quantum dot labeled antibody not only has good fluorescent properties, so that the fluorescent signal is easy to observe and detect, but also has a large specific surface area, and can be combined with more virus molecules, thereby improving the sensitivity of the detection. This process not only improves the accuracy and sensitivity of the detection, but also simplifies the operation steps, and provides strong technical support for the detection and prevention and control of the iridovirus. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The figure of antibody titer of serum of New Zealand white rabbits immunized with iridovirus, the data results are expressed as mean ± standard deviation;

[0023] Figure 2 The figure of light chain primer amplified antibody VL gene, wherein lane M1 is DL2000 Plus DNA Marker, lanes 1-4 are light chain genes VL (1-4);

[0024] Figure 3 The figure of heavy chain primer amplified antibody VH gene, wherein lane M1 is DL2000 Plus DNA Marker, lanes 1-6 are heavy chain genes VH (1-3);

[0025] Figure 4 The figure of splicing of scFv gene, wherein lane M1 is DL2000 Plus DNA Marker, lanes 1-6 are scFv genes scFv (1-6);

[0026] Figure 5 The figure of double enzyme digestion identification results of pCANTAB5E-scFv recombinant plasmid, wherein lane M1 is DL2000 Plus DNA Marker, lanes 1 and 2 are uncut recombinant plasmids, and lanes 3 and 4 are double enzyme cut recombinant plasmids;

[0027] Figure 6 The figure of verification of antibody library recombination rate, wherein lane M1 is DL2000 Plus DNA Marker, and lanes 1-18 are PCR of bacterial liquid of different monoclonal phages;

[0028] Figure 7 The figure of verification of antibody library recombination rate, wherein lane M1 is DL2000 Plus DNA Marker, and lanes 19-32 are PCR of bacterial liquid of different monoclonal phages;

[0029] Figure 8 The figure of Phage-ELISA antibody affinity detection, wherein the abscissa is 1-48 positive clones and a negative control; and the ordinate is OD 450 value;

[0030] Figure 9Figure 1 is a SDS-PAGE analysis chart of antibody protein Ab-K1, wherein lane M is a protein relative molecular mass standard, lane 1 is pET28a empty protein, lane 2 is purified Ab-K1 protein, and lane 3 is unpurified Ab-K1 protein;

[0031] Figure 10 Figure 2 is a SDS-PAGE analysis chart of antibody protein Ab-K2, wherein lane M is a protein relative molecular mass standard, lane 1 is pET28a empty protein, lane 2 is unpurified Ab-K2 protein, and lane 3 is purified Ab-K2 protein;

[0032] Figure 11 Figure 3 is an affinity analysis chart of antibody protein Ab-K1 and Ab-K2 respectively with LMBV, wherein K1 represents antibody protein Ab-K1, and K2 represents antibody protein Ab-K2;

[0033] Figure 12 Figure 4 is a schematic diagram of preparation of quantum dot microspheres;

[0034] Figure 13 Figure 5 is a schematic diagram of assembly and detection process of quantum dot flow immunoassay test strip;

[0035] Figure 14 Figure 6 is a preliminary detection result of test strip, a is the reaction under visible light, and b is the result under a fluorescence reader with excitation wavelength of 365 nm;

[0036] Figure 15 Figure 7 is a specific detection result of JY-Q@K1-based immunochromatography test strip, and N is a PBS control group;

[0037] Figure 16 Figure 8 is a sensitivity detection result of JY-Q@K1-based immunochromatography test strip, and N is a PBS control group;

[0038] Figure 17 Figure 9 is a clinical sample detection result of JY-Q@K1-based immunochromatography test strip, and N is a PBS control group. DETAILED DESCRIPTION

[0039] The various illustrative embodiments of the present application will now be described in detail in connection with the following figures. This description is made for the purpose of illustrating the general principles of the present application and is not meant to limit the present application in its application to such principles. Further, the description of the illustrative embodiments is intended to convey the concepts of the present application to one skilled in the art.

[0040] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise indicated, each intervening value by each intervening value, as well as any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these intervening values can independently be included or excluded in the range, and are also encompassed. These smaller ranges are thus each individually disclosed.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0042] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from the description and examples provided herein. The description and examples are illustrative only.

[0043] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0044] Example 1 Construction of Anti-LMBV Rabbit-derived Single-chain Antibody Library

[0045] 1. Preparation of polyclonal antibody

[0046] 1.1 Animal immunization

[0047] LMBV-FS001 type strain (LMBV-FS001 type strain is disclosed in Chinese patent with application number 202310757360.5 and invention name "mRNA vaccine for aquaculture and preparation method thereof") with virus concentration of 10 7 TCID 50 / mL was used as immunogen to immunize New Zealand white rabbits by multiple booster immunization, a total of four times. Complete Freund's adjuvant was emulsified with an equal amount of LMBV virus solution as immunogen for the first immunization, and incomplete Freund's adjuvant was emulsified with an equal amount of LMBV virus solution as immunogen for the booster immunization.

[0048] 1.2 Antibody titer detection

[0049] The serum specific antibody titer was determined by using LMBV as antigen, the collected serum as the serum to be tested, mouse anti-rabbit IgM monoclonal antibody as primary antibody, and the dilution ratio was 1:2000; HRP goat anti-mouse IgG monoclonal antibody as secondary antibody, and the dilution ratio was 1:4000, and the enzyme-linked immunosorbent assay was used to determine the antibody titer in the serum. After color development, the absorbance at a wavelength of 450 nm was determined by an enzyme-labeled instrument, and the results are shown in Figure 1 After the fourth booster immunization, the serum antibody titer reached the highest, which was more than 1:25,600, indicating that the polyclonal antibody with high antibody titer was prepared by using LMBV as immunogen, which could meet the needs of library construction.

[0050] 1.3 Collection of polyclonal antibody

[0051] After the immunization was completed, the New Zealand white rabbits were anesthetized with ether, and the heart blood was collected, and the serum was stored in a-20℃ refrigerator for standby. Then the spleen was collected and cut into small pieces, which were divided into 10 mL centrifuge tubes, immersed with Trizol, and stored in a-80℃ refrigerator for standby.

[0052] 2, Construction of single-chain antibody library

[0053] 2.1 Design of antibody variable region gene primers

[0054] The immunoglobulin related sequence information of New Zealand white rabbits was searched in NCBI and IMGT, and according to the relatively conservative gene framework region (FR region) in the antibody variable region and the degenerate primer design rules, the degenerate primers of antibody heavy chain VH and light chain VL were designed by primer design software Primer5.0, in which there were four upstream amplification primers and one downstream amplification primer for light chain; one upstream amplification primer and three downstream amplification primers for heavy chain, which were Sfi I-VL-FP (1-4), VL-RP0-Linker, and Linker-VH-FP0, VH-RP (1-3)-Not I, respectively. In order to splice the heavy chain and light chain of the antibody into a complete single-chain antibody structure, a flexible peptide linker was used for connection, and the amino acid sequence of the linker was GGGGSGGGGSGGGGS (SEQ ID NO. 1). According to the restriction enzyme cutting sites on the connecting vector pCANTAB-5E, Sfi I enzyme cutting sites and Not I enzyme cutting sites and protective bases were added to the upstream primer of the heavy chain and the downstream primer of the light chain. The specific sequences of the amplification primers are shown in Tables 1 and 2.

[0055] Table 1 Amplification primers of antibody light chain VL

[0056]

[0057] Note: The underlined part represents the enzyme cutting site, and the bold part represents the VL sequence.

[0058] Table 2 Antibody heavy chain VH amplification primer

[0059]

[0060] Note: The underlined part represents the enzyme digestion site, and the bold part represents the VH sequence.

[0061] 2.2 Extraction and reverse transcription of RNA

[0062] Take about 50 mg of spleen tissue and put it into a 1.5 mL centrifuge tube to extract RNA. Use the HiScript Q RT SuperMix for qPCR kit from Nanjing Novozyme Biotech Co., Ltd. to synthesize cDNA by two-step method. First, add 8.0 μL RNase free ddH2O, 4.0 μL 4×gDNA wiper Mix, and 4.0 μL template RNA to an RNase free centrifuge tube. Mix gently with a pipette and incubate at 42℃ for 2 min. Then add 5×qRT SuperMix II 4.0 μL to the reaction tube from the first step, mix gently, and incubate at 50℃ for 15 min and at 85℃ for 2 min. The cDNA obtained can be stored at -20℃ for later use.

[0063] 2.3 PCR amplification of light chain VL and heavy chain VH genes

[0064] Using the cDNA obtained by reverse transcription as a template, the designed primers were used to amplify the antibody light chain VL and heavy chain VH, respectively. The amplification products were named VL1, VL2, VL3, VL4 and VH1, VH2, VH3, respectively.

[0065] PCR amplification reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 36 cycles; 72℃ extension for 10 min. The PCR products were subjected to agarose gel electrophoresis identification, and the electrophoresis conditions were 120 V constant voltage for 20 min. The results are shown in Figure 2 and Figure 3 The size of the target band is between 250 bp and 500 bp, about 330 bp, which is consistent with the expected product size, indicating that the VL and VH gene amplification is successful.

[0066] 2.4 Splicing single-chain antibody gene

[0067] The amplified product from the previous step was recovered using a purification and recovery kit. The recovered heavy and light chain genes (VL1, VL2, VL3, VL4 and VH1, VH2, VH3) were used as templates and overlap extension PCR was performed using the light chain upstream and heavy chain downstream primers to splice the complete single-chain antibody scFv. The spliced ​​product was detected by nucleic acid electrophoresis and recovered by gel excision. The results of nucleic acid electrophoresis detection are as follows: Figure 4 As shown, the size of the scFv gene fragment ranges from 500 bp to 750 bp, with a size of approximately 700 bp, which is consistent with the expected theoretical product size of scFv splicing.

[0068] PCR amplification reaction conditions: Step 1: pre-denaturation at 95°C for 5 min; 7 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 30 s; and extension at 72°C for 5 min. Step 2: pre-denaturation at 95°C for 5 min; 21 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 30 s; and extension at 72°C for 5 min.

[0069] 3. Construction of single-chain antibody library

[0070] 3.1 Construction of recombinant plasmid pCantab5E-Ab

[0071] ScFv and pCANTAB 5E were first digested with NotI at 37°C for 10 h, and then digested with SfiI at 50°C for 5 h. After digestion, ligation was performed. The digestion products and ligation products were analyzed by agarose gel electrophoresis. The double digestion results showed that the recombinant plasmid pCANTAB 5E-ScFv was successfully constructed ( Figure 5 The digested product was purified and recovered using a gel recovery kit. The purified product was ligated overnight at 16°C and transformed into E. coli TG1 competent cells. The transformation was repeated 25 times to obtain a library capacity of 3.6×10 6 pfu / mL of the original bacterial solution of the LMBV primary antibody library.

[0072] 3.2 Construction and identification of phage libraries

[0073] Randomly pick 32 single colonies with good growth from the plate and place them in 500 μL 2×YT-A medium. After shaking and incubating, use them as templates for bacterial liquid PCR. Calculate the recombination rate of the antibody library as follows: Figure 6 and Figure 7 As shown, 30 of the 32 single colonies contained scFv genes, indicating positive antibodies; the PCR results of single colonies 26 and 27 showed no obvious bands, indicating that the recombination rate of the antibody library was 30 / 32≈93.75%.

[0074] The remaining culture after transformation was inoculated into 100 mL 2xYT-Amp liquid medium (AMP concentration 50 μg / mL), 1 / 5 volume of 25% glucose stock solution was added, and after 4 h of culture at 37°C, 180 rpm, helper phage M13K07 was added, and after 1 h of incubation at 37°C, 2 h of culture at 180 rpm, the supernatant was discarded by centrifugation at 5000 rpm for 15 min, and the precipitate was resuspended in 100 mL 2xYT-Amp-Kan liquid medium (AMP and KAN concentrations 50 μg / mL), and cultured overnight on a shaker, and then centrifuged at 10000 rpm for 20 min at 4°C, and the supernatant was transferred to a 50 mL centrifuge tube, 1 / 5 volume of PEG / NaCl was added, and the mixture was allowed to stand on ice for 1 h, and then centrifuged at 12000 rpm for 30 min at 4°C, and the supernatant was discarded, and the precipitate was resuspended in 150 μL PBS, and then collected by centrifugation for 10 min, and the supernatant was the primary phage single-chain antibody library, which was stored at 4°C for later use. After assembly by helper phage M13K07, the titer of the constructed phage scFv antibody library was 2.1x10 11 pfu / mL, which met the requirements for subsequent biopanning of the phage library, and biopanning should be performed immediately.

[0075] Example 2. Anti-LMBV single-chain antibody panning, identification, and prokaryotic expression

[0076] 1. Biopanning of the anti-LMBV phage display antibody library

[0077] 10 7 TCID 50 / mL of LMBV was diluted 10-fold, and 100 μL / well was added to the enzyme-labeled plate (three replicates each time), and the plate was coated overnight at 4°C; the plate was washed with PBS for 3 times, each time for 5 min, 100 μL / well of 5% skim milk powder was added to block the solid carrier, and the plate was blocked at 37°C for 2 h, and then washed with PBS, 100 μL of the phage library was added to each well, and the plate was incubated at 37°C for 2 h; after washing with PBST, 100 μL of Gly-HCl elution buffer was added, and the plate was incubated at 37°C with slow shaking at 60 rpm for 10 min, and then the eluate was transferred to a centrifuge tube, 100 μL of Tris-HCl with pH 7.4 was immediately added to neutralize the eluate (to maintain the infectivity of the phage); 500 μL of TG1 cultured overnight was added to the eluate, and the plate was incubated at 37°C for 4 h; the eluate was diluted and plated on a plate, the number of colonies was counted, and the remaining eluate was preserved and used for the next round of panning, and the operation was repeated for five rounds of panning, the phage output of the first round was 1.3x10 5 pfu / mL, and the phage output of the fifth round was 1.3x10 8pfu / mL, the specific phage output was greatly improved; after 5 rounds of panning, the phage output ratio increased from 4.6×10 -5 Increased to 4.8×10 -4 The enrichment factor was 104 times, which showed that specific phages binding to LMBV antigens were effectively enriched.

[0078] 2. Positive clone screening

[0079] After 5 rounds of panning, 48 different monoclonal colonies were randomly selected to prepare phage supernatants. The affinity of phage single-chain antibody to LMBV was determined by enzyme-linked immunosorbent assay (ELISA) using LMBV virus liquid as antigen, phage amplification product as test antibody, and anti-M13 monoclonal antibody as binding antibody at a dilution ratio of 1:2000. 450 A positive phage clone was defined as one with a value greater than 2 times that of the negative control (NC). Figure 8 As shown, among the 48 monoclonal colonies, 43 monoclonal colonies had an OD of 450 The ratio of the NC value to the NC value was greater than 2.0, indicating a positive clone. The positive rate of the recombinant phage was 43 / 48 = 89.58%.

[0080] Based on the affinity test results above, the 10 positive phages with the highest affinity were sequenced to detect the antibody gene insertion status of each monoclonal colony. Comparative analysis of the sequencing results revealed that four of the positive phages had identical antibody sequences, with an actual DNA size of 687 bp. This antibody was designated Ab-K1. Two of the positive phages had identical antibody sequences, with an actual DNA size of 714 bp. This antibody was designated Ab-K2. The results are shown in Table 3.

[0081] Table 3 Amino acid sequences of Ab-K1 and Ab-K2

[0082]

[0083] Note: The bold part in the table represents the linker sequence.

[0084] 3. Synthesis and prokaryotic expression of single-chain antibody gene recombinant expression plasmid

[0085] The six scFv strains obtained by bio-panning were expressed in prokaryotes. The target genes were ligated to the pMD19-T vector and amplified in large quantities. The amplified target genes were then ligated to the expression vector PET-28a and transformed into Escherichia coli for prokaryotic expression. The expressed ScFvs were mainly present in the bacteria in the form of inclusion bodies. After fragmentation, protein denaturation, nickel affinity column purification, renaturation, and lyophilization, the antibody recombinant proteins Ab-K1 and Ab-K2 were obtained. The SDS-PAGE results are shown in Figure 2. Figure 9 、 Figure 10 As shown in FIG. 2, the sizes of the antibody proteins Ab-K1 and Ab-K2 are both about 40 kDa, which is consistent with the expected result. The expressed proteins can be recognized by the anti-His monoclonal antibody, which indicates that the antibody proteins are successfully expressed in E. coli.

[0086] 4. Affinity detection of antibody proteins

[0087] The affinity of pET28a-scFv to LMBV virions was detected by ELISA. The freeze-dried protein powder was dissolved in PBS buffer to prepare a dilution mother liquor. The antibody dilution ratio was 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800. The antigen used in this method was LMBV virus liquid, the diluted antibody was purified protein, the primary antibody was mouse anti-6×His monoclonal antibody, the secondary antibody was HRP-labeled goat anti-mouse IgG, and finally TMB single-component color developing liquid was used for color development, and the OD value was measured at a wavelength of 450 nm. As shown in FIG. 3, with the increase of the dilution multiple, the affinity of the two antibody proteins to LMBV virions gradually decreased. When the dilution ratio was 1:3200, Ab-K2 still had affinity to LMBV, and when the dilution ratio was 1:6400, Ab-K1 still had affinity to LMBV. The overall affinity of Ab-K1 was also higher than that of Ab-K2. Therefore, Ab-K1 was selected as the antibody marker for the subsequent test strip. Figure 11

[0088] Example 3 Establishment and optimization of quantum dot immunochromatographic technology

[0089] 1. Antibody-labeled fluorescent quantum dot microspheres

[0090] The high-specificity anti-LMBV single-chain antibody obtained by screening was coupled with carboxylated quantum dots by activated ester method. 0.15 mg of quantum dot microspheres (QBs, Wuhan Jia Yuan Quantum Dot Technology Development Co., Ltd., with a fluorescence emission wavelength of 365 nm) were added to 0.75 mL of phosphate buffer solution PB (0.01 mol / L, pH 6.0), diluted to 0.1 mg / mL, and ultrasonicated for 3 min. A prepared 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, Sigma Company, USA) solution with a final concentration of 10 μg / mg was added, and the mixture was shaken at 37 °C and 120 r / min for 15 min to obtain carboxylated QBs (10 mg·mL -1 ). 0.25 g of K1 (Ab-K1) was added to 100 mL of deionized water to prepare a K1 solution. 0.2 mL of the prepared carboxylated QBs (10 mg·mL -1 ​) were dispersed in K1 solution for 30 min under ultrasonication. Finally, 1% BSA was added to the solution, and the solution was incubated at 37 °C for 1 h on a 120 r / min shaker. After the reaction was completed, the solution was centrifuged at 12000 r / min for 15 min, and the supernatant was discarded. The process was repeated twice to ensure that the QBs were completely adsorbed on the surface of K1. Finally, the antibody-labeled quantum dot microspheres (K1-QBs) were prepared by centrifugation (6000 rpm, 6 min) and resuspension in 10 mL ethanol. The preparation of quantum dot microspheres is shown in Figure 12 .

[0091] 2. Characterization of quantum dot microspheres

[0092] Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) were used to analyze and characterize the morphology and dispersity of the prepared JY-Q and JY-Q@K1 nanocomposites. Zeta potential analyzer was used to determine the zeta potential. The prepared JY-Q@K1 suspension was irradiated with visible light and ultraviolet light (365 nm), and the images were recorded to analyze the fluorescence properties.

[0093] 3. Preparation of immunochromatographic test strips

[0094] The immunochromatographic test strip is composed of four independent parts: a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent pad. Rabbit-derived anti-LMBV single-chain antibodies and goat anti-rabbit IgG antibodies were simultaneously sprayed onto the NC membrane to construct the test line and the control line, respectively, with a distribution rate of 0.1 μL / mm. The prepared NC membrane was dried in a constant-temperature drying oven at 37 °C for 2 h. Subsequently, the sample pad, the conjugate pad, the NC membrane, and the absorbent pad were assembled onto a plastic backing card, and the test strip detection process is shown in Figure 13 . Finally, the prepared test strip was placed in a plastic box and stored with a desiccant at room temperature. The fluorescence intensity on the test line was read with a fluorescence reader (excitation wavelength: 365 nm).

[0095] 4. Preliminary detection of test strip performance

[0096] The prepared test strip was inserted into a PBS buffer solution containing LMBV virus, and the solution moved towards the absorbent pad by capillary force. The LMBV in the solution rapidly combined with the antibody-modified JY-Q@K1 to form a JY-Q@K1-LMBV complex, and the antibody recognized and combined with the LMBV at the T line position of the NC membrane. The excess immunized JY-Q@K1 continued to move forward and was fixed on the control line of the test strip. After the assembled test strip was restored to room temperature, PBS diluent and virus with a concentration of 10 6 TCID 50The LMBV was detected by 100 μL of each LMBV at 100 μL, and finally, the fluorescence intensity on the detection line was read by a fluorescence reader (excitation wavelength was 365 nm), and the results are shown in Figure 14 The K1 antibody protein has detection activity (only the C line is colored in the control group, and the T and C lines are colored in the experimental group), and can be used for the preparation of LMBV immunochromatographic test strips for the detection of LMBV.

[0097] 5. Optimization of JY-Q@K1 test strip

[0098] In order to further optimize the quantum dot immunochromatography, factors such as NC membrane, buffer, optimal reaction time, and antibody spraying concentration were optimized. Under the optimized buffer system, the LMBV of largemouth bass iridovirus was gradiently diluted with the buffer, and the concentration range was 10 7 -10 CFU·mL -1 Then the test strip was inserted into 100 μL of sample buffer, and the antigen binding label was moved to the absorption pad by capillary action. After 10 min of chromatography reaction, the fluorescence signal on the detection line was recorded by a fluorescence reader (365 nm excitation), and each detection line was read three times.

[0099] 5.1 Specific detection

[0100] In order to evaluate the specificity of the detection method, 6 test strips were taken, and 5 common fish viruses preserved in the laboratory were used for investigation: infectious hematopoietic necrosis virus (IHNV), largemouth bass rhabdovirus (MSRV), carp spring virus (SVCV), grass carp reovirus (GCRV), and largemouth bass iridovirus (LMBV) virus samples. After culture, they were diluted to 10 6 TCID 50 / mL with PBS, 100 μL was added to each sample well, and the detection results were observed after standing for 20 min. The specificity of the prepared fluorescent LFIA strip was verified. The results are shown in Figure 15 It is shown that the LMBV detected by the test strip is positive, and the others are negative (only the LMBV two lines are colored, and the others are C line), so the test strip has good specificity.

[0101] 5.2 Sensitivity detection

[0102] The LMBV virus (concentration was 10 7 TCID 50 / mL) was diluted to 10 6 TCID50 / mL, 10 5 TCID 50 / mL, 10 4 TCID 50 / mL, 10 3TCID 50 / mL, 10 2 TCID 50 / mL, 10 TCID 50 / mL, seven samples from the same batch were detected for sensitivity. Before detection, the samples to be detected were equilibrated to room temperature, and 100 μL of each dilution sample was added to the sample pad of the test strip (the same batch of test strips were used to reduce error) using a pipette. After 20 min, the detection results were observed, and the results are shown in Table 1. Figure 16 Table 1 shows that the LMBV cell culture was negative when the concentration was 10 TCID 50 / mL; when the concentration was 10 6 TCID 50 / mL to 10 2 TCID 50 / mL, the detection result was positive. Therefore, the test strip can effectively detect LMBV when the virus concentration is ≥10 2 TCID 50 / mL.

[0103] 5.3 Stability detection

[0104] Sixteen LMBV quantum dot immunochromatographic test strips of the same batch were prepared, placed in a desiccant, and stored in a 4°C refrigerator. Four test strips were taken out after 1, 2, 3, and 6 months of storage, respectively. Three of them were detected using LMBV with a concentration of 10 6 TCID 50 / mL as the sample, and the other one was detected using PBS buffer as the sample (negative control). The stability of the test strip was determined by changes in the sensitivity of the test strip. The detection results showed that the four test strips with PBS buffer as the sample only had C-line coloration, and the T-line did not color, indicating that the test strip had no quality problems. The C-line and T-line of the twelve test strips with virus liquid as the sample both colored, indicating that the test strip had good reproducibility.

[0105] 5.4 Clinical sample detection

[0106] Twenty μL of LMBV with a virus concentration of 10 6 TCID 50 / mL was injected intraperitoneally into a 50-day-old largemouth bass, and the surface mucus, liver, gills, and intestinal tract of the largemouth bass were taken when the largemouth bass was dying. Tissue suspensions were prepared (1 mg of tissue was added to 100 μL of 0.01M PBS buffer to lyse the tissue), and the same batch of test strips was used to detect the virus in the tissue suspensions. The results were analyzed to analyze the universality of the test strip. The results are shown in Table 2. Figure 17 Table 2 shows that the intestinal tract tissue suspension of the largemouth bass infected with LMBV can detect LMBV.

[0107] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the present application made by those skilled in the art, without departing from the design spirit of the present application, should fall within the scope of the present application as defined by the claims.

Claims

1. A single-chain antibody against largemouth bass iridovirus, characterized in that: The single-chain antibody includes Ab-K1 or Ab-K2. The amino acid sequence of Ab-K1 is shown in SEQ ID NO.11, and the amino acid sequence of Ab-K2 is shown in SEQ ID NO.

12.

2. A fluorescent quantum dot-labeled antibody against largemouth bass iridovirus, characterized in that: The fluorescent quantum dot-labeled antibody comprises the single-chain antibody according to claim 1.

3. Use of the single-chain antibody according to claim 1 or the fluorescent quantum dot-labeled antibody according to claim 2 in the preparation of a product for detecting largemouth bass iridovirus.

4. The use according to claim 3, characterized in that The products include test strips and test kits.

5. The use according to claim 4, characterized in that The test strips include quantum dot fluorescence immunochromatography test strips.

6. A quantum dot fluorescent immunochromatographic test strip for detecting largemouth bass iridovirus, characterized in that: The method comprises a sample pad, a conjugate pad, a nitrocellulose membrane and an absorption pad. The nitrocellulose membrane comprises a detection line and a quality control line. The detection line comprises the fluorescent quantum dot-labeled antibody according to claim 2.

7. The quantum dot fluorescent immunochromatographic test strip according to claim 6, characterized in that: The quality control line includes goat anti-rabbit IgG antibody.

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