A recombinant single B cell antibody against bluetongue virus in sheep

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a recombinant single B cell antibody against bluetongue virus in sheep. The present invention uses flow single cell sorting technology to obtain a single B cell antibody against bluetongue virus in peripheral blood lymphocytes of sheep immunized with inactivated bluetongue virus vaccine, obtains the light and heavy chain variable region sequences of the antibody by RT-PCR amplification, and constructs two recombinant light and heavy chain expression plasmids of the antibody with an expression vector containing the constant region of sheep IgG antibody, and successfully expresses the recombinant single B cell antibody against bluetongue virus in sheep after transfection of 293F cells; the single B cell antibody can react specifically with bluetongue virus in ELISA and cell immunofluorescence experiments. The invention avoids the problems of antibody heterogeneity, cell apoptosis and genetic instability encountered in traditional hybridoma antibody preparation technology, and lays the foundation for establishing a more accurate and sensitive bluetongue disease detection method.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant single B cell antibody against bluetongue virus in sheep. Background Art

[0002] Bluetongue is an infectious disease caused by bluetongue virus (BTV) infecting sheep, cattle, deer and other ruminants. It is an insect-borne disease mainly transmitted by blood-sucking midges. Sheep are the most susceptible to the disease. The clinical symptoms are mainly fever, facial edema and congestion, and oral and nasal mucosal ulcers or congestion. The World Organization for Animal Health (WOAH) lists it as an animal disease that must be reported, and my country lists it as a Class II animal disease. BTV is a double-stranded RNA virus belonging to the genus Orbivirus of the family Reoviridae. Its genome is composed of 10 RNA fragments of different sizes, encoding a total of 7 structural proteins and 4 non-structural proteins. There are many serotypes of BTV. At present, at least 29 serotypes have been found. There is no cross-reaction or very low cross-reaction between different serotypes. Early diagnosis and prevention are important means to control the epidemic of the disease.

[0003] The single B cell antibody preparation technology is to separate antigen-specific B cells from the immune body, then amplify the natural antibody sequence through single cell reverse transcription and PCR amplification technology, and finally express the antibody in eukaryotic cells. The outstanding feature of this method is that antigen-specific natural antibodies are obtained after high-throughput screening, which overcomes the difficulties of antibody heterogeneity, cell apoptosis and genetic instability encountered in the preparation of antibodies using traditional hybridoma technology. Sheep are naturally susceptible animals to BTV. Using them to prepare monoclonal antibodies against BTV can truly simulate the natural process of producing antibodies after the virus infects the body. Compared with antibodies obtained from mice, rabbits and other animals, this method is more conducive to obtaining sheep-derived antibodies with high specificity and high reactivity in natural conformations, providing support for the diagnosis and prevention of bluetongue disease. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a recombinant single B cell antibody against bluetongue virus in sheep. The recombinant single B cell antibody can react specifically with bluetongue virus; the expression of the antibody avoids the shortcomings of antibody heterogeneity, cell apoptosis and genetic instability during hybridoma cell antibody expression, laying a foundation for establishing a method for detecting bluetongue pathogens and antibodies using the recombinant antibody. Specifically, it includes the following contents:

[0005] In a first aspect, the present invention provides a recombinant single B cell antibody against bluetongue virus in sheep, wherein the amino acid sequence of the heavy chain variable region of the single B cell antibody is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0006] In a second aspect, the present invention provides a nucleic acid encoding the light chain variable region and heavy chain variable region of the single B cell antibody described in the first aspect above.

[0007] Preferably, the nucleic acid sequence encoding the heavy chain variable region of the single B cell antibody is as shown in SEQ ID NO.1, and the nucleic acid sequence encoding the light chain variable region of the single B cell antibody is as shown in SEQ ID NO.3.

[0008] In a third aspect, the present invention provides a primer set for amplifying the single B cell antibody described in the first aspect above, the primer set comprising:

[0009] Primers for amplifying the heavy chain variable region:

[0010] Sheep gamma chain F outer primer:ACCTCCTCTTTGTGCTCTCAG;

[0011] Sheep gamma chain R outer primer: TCCCCGCAGCAAGAAGTCA;

[0012] Sheep gamma chain F inner primer: GAAGCCCTCACAGACCCTCT;

[0013] Sheep gamma chain R inner primer: GTCAGAGGGTAGACTTTCGGGG;

[0014] And the primers for amplifying the light chain variable region include:

[0015] Sheep Lambda chain F outer primer:ATGGCCTGGTCCCCTCTG;

[0016] Sheep Lambda chain R outer primer: GGCGGGAACAGGGTGAC;

[0017] Sheep Lambda chain F inner primer:GTCCCY(C / T)GGGCCAGAS(C / G);

[0018] Sheep Lambda chain R inner primer: GGTGCGGACTTGGGC.

[0019] In a fourth aspect, the present invention provides a use of the recombinant single B cell antibody described in the first aspect in the in vitro detection of bluetongue virus for non-disease diagnosis purposes.

[0020] In a fifth aspect, the present invention provides a use of the recombinant single B cell antibody described in the first aspect in the preparation of a reagent, a test strip, or a kit for detecting bluetongue virus.

[0021] In a sixth aspect, the present invention provides a bluetongue virus ELISA detection kit, the kit comprising the recombinant single B cell antibody described in the first aspect above.

[0022] Preferably, the kit further comprises an ELISA plate, a blocking solution, a diluent, an enzyme-labeled secondary antibody, a washing solution, a color developer, and a stop solution.

[0023] In a seventh aspect, the present invention provides a method for preparing the recombinant single B cell antibody according to the first aspect, the method comprising the following steps:

[0024] (1) amplifying the light chain variable region and heavy chain variable region sequences encoding the single B cell antibody described in the first aspect by RT-PCR, and respectively constructing two recombinant light and heavy chain expression plasmids of the antibody with an expression vector containing a sheep IgG antibody constant region;

[0025] (2) The antibody was expressed in 293F suspension cells at a mass ratio of 3:1 of transfection reagent PEI to plasmid and a mass ratio of 2:1 of light chain recombinant plasmid to heavy chain recombinant plasmid.

[0026] Preferably, the light chain variable region and heavy chain variable region sequences of the single B cell antibody are obtained by amplifying the primer set described in the third aspect above.

[0027] Preferably, the antibody light chain recombinant plasmid is pcDNA3.4-sheep-CL, and its structure is: pcDNA3.4 backbone + NheI + variable region sequence + HpaI + sheep light chain constant region sequence (NCBI Reference Sequence: XM_060401348.1) + Myc-His tag sequence + AgeI;

[0028] The antibody heavy chain recombinant plasmid is pcDNA3.4-sheep-CH, and its structure is: pcDNA3.4 backbone + NotI + variable region sequence + NheI + sheep heavy chain constant region sequence (GenBank: X69797.1) + Myc-His tag sequence + AgeI.

[0029] In an eighth aspect, the present invention provides an immunoconjugate, comprising:

[0030] (i) the recombinant single B cell antibody described in the first aspect above;

[0031] (ii) and a conjugated moiety selected from the group consisting of a detectable label, a drug, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0032] The invention has the following beneficial effects: ① the invention provides a recombinant single B cell antibody against bluetongue virus in sheep, and through experiments such as immunoblotting, indirect ELISA, and cell immunofluorescence, it is identified that the single B cell antibody can specifically react with bluetongue virus; ② the invention provides a group of primers for amplifying single B cell antibodies in sheep, and a single B cell against bluetongue virus in sheep is amplified by flow cytometry single cell sorting technology, and the light and heavy chain variable region sequences of the antibody are amplified by RT-PCR, and the recombinant expression plasmid of the antibody is constructed with pcDNA3.4-sheep-CH and pcDNA3.4-sheep-CL antibody light and heavy chain expression vectors, and the recombinant single B cell antibody against bluetongue virus in sheep is successfully expressed after transfection; the expression of the antibody avoids the disadvantages of antibody heterogeneity, cell apoptosis, genetic instability, and the like in the process of hybridoma cell antibody expression, and lays a foundation for establishing a more accurate and sensitive bluetongue disease detection method by using the recombinant antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Results of flow cytometry sorting of sheep anti-BTV-1 single B cells;

[0034] Figure 2 PCR amplification results of sheep antibody variable region genes;

[0035] Figure 3 pcDNA3.4-sp-CL plasmid map;

[0036] Figure 4 pcDNA3.4-sp-CH plasmid map;

[0037] Figure 5 Western blot results of sheep recombinant single B cell antibody expression;

[0038] Figure 6 SDS-PAGE test results of sheep recombinant single B cell antibody purification;

[0039] Figure 7 Results of cellular immunofluorescence detection of sheep recombinant single B cell antibodies. DETAILED DESCRIPTION

[0040] The embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. In addition, if not explicitly stated, all reagents used in the following embodiments are commercially available, or can be synthesized according to text or known methods, and the reaction conditions not listed are also easily available to those skilled in the art.

[0041] Example

[0042] 1. Isolation of specific anti-BTV-1 single B cells from peripheral blood lymphocytes of immunized sheep

[0043] The inactivated BTV-1 vaccine emulsified with adjuvant ISA 201 was injected into the neck muscle of sheep. One million peripheral blood lymphocytes (PBMCs) were collected from 110# sheep peripheral blood lymphocytes by BD FACS AriaII flow sorter. Figure 1 As shown, in the IgM+ cell population (Sheep 110#), compared with the minus one control sample (Sheep 110#FMO), that is, BTV-1 antigen without biotin labeling, the proportion of IgM+BTV+B cells in PBMCs is about 1.3%; in the IgM- cell population, compared with FMO, the proportion of IgM-BTV+B cells in PBMCs is about 0.1%. In order to obtain antibodies with high affinity and high specificity, we excluded IgM+ cells and finally sorted CD21+IgM-BTV+B cells into a 96-well plate containing 10μL lysate.

[0044] 2. Amplification of light and heavy chain variable region sequences of sheep single B cell antibodies

[0045] The sorted 96-well plate was immediately centrifuged and 1 μL stop buffer was added to each well to terminate the lysis. Then 4 μL SuperScript IV VILO premix and 5 μL deionized water were added, and 20 μL reverse transcription system was prepared. Single B cell cDNA was obtained under the conditions of 25°C for 10 min, 42°C for 1 h, and 85°C for 5 min. Then, nested PCR was performed with the designed sheep antibody light chain and heavy chain primers (see Table 1). Single B cell cDNA was used as a template. MaxDNA polymerase was used to prepare a 25 μL reaction system, and amplification was performed according to the program of 98°C for 10 s, 57-58°C for 30 s, 72°C for 15 s, 35 cycles, and 72°C for 10 s. The size of the antibody light chain and heavy chain variable region sequences was then identified by agarose gel electrophoresis, such as Figure 2 As shown, an antibody light chain variable region of about 350 bp and an antibody heavy chain variable region of about 500 bp were obtained.

[0046] Table 1 Primers for amplifying the variable regions of sheep antibody light and heavy chains

[0047]

[0048] 3. Sequencing and analysis of light and heavy chain variable region sequences

[0049] The nested PCR products were sent to Qingke Biotechnology Company for sequencing, and the light chain and heavy chain variable region sequences of the single B cell antibody A9 were obtained by comparison and analysis of the sheep antibody sequences published on the NCBI and IMGT websites.

[0050] The amino acid sequence of the heavy chain variable region (A9-VH): QVRLQESGPSLVKPSQTLSLTCTVSGFSLTSNAV GWVRQAPGKVPEWLSGINTRRNKYYNSALKSRLSIDRDASKSQIYLSLSSVTTEDTAVYYCARDYDWSHEVYDYDFWGPGLLVTVSS (shown in SEQ ID NO. 2);

[0051] Gene sequence of heavy chain variable region (A9-VH): CAGGTGCGGCTGCAGGAGTCGGGACCCAGCCT GGTGAAGCCCTCACAGACCCTCTCCCTCACCTGCACGGTCTCTGGATTCTCATTAACCAGCAATGCTGTAGGCTGGGTCCGCCAGGCTCCAGGAAAGGTGCCGGAGTGGCTGAGTGGTATAAATACTAGAAGAAACAAGTACTATAACTCGGCCCTGAAATCCCGGC TCAGCATCGACAGGGACGCCTCCAAGAGCCAAATCTACCTGTCACTGAGTAGCGTGACAACTGAGGACACGGCCGTGTACTACTGTGCTAGAGATTATGATTGGAGTCATGAAGTTTATGACTACGACTTCTGGGGCCCAGGACTCCTGGTCACCGTCTCCTCA(SEQ ID NO.1 shown).

[0052] The amino acid sequence of the light chain variable region (A9-VL): QAVLTQPSSVSGSLGQRVSITCSGSFIGSEAAGW FQQVPGSGLRTVIYNDNKRPSGVPDRFSGSKSGNTATLTISSLQAEDEADYFCGTDAGGSNF GVFGSGTRLTVL (shown in SEQ ID NO.4);

[0053] Gene sequence of light chain variable region (A9-VL): CAGGCTGTGCTGACCCAGCCTTCCTCCGTGAGC GGCTCCCTGGGCCAGAGAGTGTCCATCACCTGCTCCGGCTCCTTCATCGGCAGCGAGGCCGCCGGATGGTTTCAGCAGGTGCCCGGCTCCGGCCTGAGGACAGTGATCTACAATGATAACAAGAGACCTTCCGGCGTGCCTGACAGG TTTAGCGGCAGCAAGAGCGGCAATACAGCCACCCTGACCATCAGCTCCCTGCAGGCCGAGGATGAGGCCGACTACTTTTGTGGCACCGATGCCGGCGGCAGCAATTTCGGCGTGTTTGGCAGCGGCACAAGGTTAACTGTGCTG(SE Q ID NO.3 shown).

[0054] 4. Construction of pcDNA3.4-sheep-CL and pcDNA3.4-sheep-CH expression vectors

[0055] The light chain and heavy chain recombinant expression vectors of single B cell antibody A9 were constructed. The specific structure is as follows:

[0056] The structure of the light chain expression vector pcDNA3.4-sheep-CL is as follows: pcDNA3.4 backbone + NheI + variable region sequence + HpaI + sheep light chain constant region sequence (NCBI Reference Sequence: XM_060401348.1) + Myc-His tag sequence + Age (such as Figure 3 shown);

[0057] Heavy chain expression vector pcDNA3.4-sheep-CH structure: pcDNA3.4 backbone + NotI + variable region sequence + NheI + sheep heavy chain constant region sequence (GenBank: X69797.1) + Myc-His tag sequence + AgeI (such as Figure 4 shown).

[0058] 5. Preparation and Expression of Single B Cell Antibodies

[0059] The mass ratio of transfection reagent PEI to the light and heavy chain recombinant expression vector of single B cell antibody A9 was 3:1 (the mass ratio of light chain recombinant expression vector to heavy chain recombinant expression vector was 2:1), and the recombinant antibody was expressed in 293F suspension cells. After 8 days of culture, the supernatant and cells were collected, and repeated freezing and thawing were repeated 3 times. Centrifugation at 8000xg for 5 minutes, the supernatant was collected and sampled for Western blot to verify the expression of the antibody. Mouse HRP-labeled anti-His monoclonal antibody (1:5000 dilution) was used, incubated at room temperature for 1 hour, washed 3 times with PBST, each time for 10 minutes, and finally observed by color development. The results are as follows Figure 5 As shown, a heavy chain of about 55 kDa and a light chain of about 25 kDa were obtained, indicating the successful expression of sheep recombinant single B cell antibody A9.

[0060] 6. Purification and Concentration of Single B Cell Antibodies

[0061] The 293F suspension cells and supernatant transfected with the single B cell antibody A9 recombinant expression plasmid described in 5 above were collected on the 8th day, a total of 25 mL was frozen and stored at -80°C, and after thawing, ultrasonicated for 5 to 10 minutes; then centrifuged at 12000 rpm for 15 minutes; and the supernatant was filtered with a 0.45 um filter.

[0062] Add 2mL Ni-NTA resin to a 10mL syringe, let stand to remove the preservation solution, and then balance the column with 10 times the volume of NPI-10. Then add the treated 10mL sample to the syringe containing 2ml resin and combine on a shaker at 4℃ overnight. Obtain the flow-through at a flow rate of 0.5-1mL / min, wash the sample with 30mL NPI-10 at a flow rate of 1mL / min to obtain washing solution 1, wash the impurities in the sample with 10 times the volume of NPI-20 and NPI-30 respectively, and collect washing solutions 2 and 3, add 1mL NPI-250 and let stand at room temperature for 5min, and elute the target protein at a flow rate of 0.5-1mL / min. Perform SDS-PAGE analysis on the liquid collected in each step and then concentrate it.

[0063] Boil the 8000-14000 dialysis bag in boiling water for 10 minutes, collect the eluted target protein into the treated dialysis bag, and then put it into 1×PBS and dialyze it on a shaker at 4℃ for more than 8 hours. Change PBS every 2-4 hours, and then put the dialysis bag into PEG 6000 dry powder and concentrate it to about 1 mL.

[0064] The results of SDS-PAGE were as follows: Figure 6 As shown, in the eluate, the recombinant antibody light chain (about 25 kDa) and heavy chain (about 55 kDa) bands are single, indicating that the sheep recombinant single B cell antibody A9 was successfully purified.

[0065] 7. Cellular Immunofluorescence Detection of Single B Cell Antibodies

[0066] BSR cells were plated on the slide one day in advance, and then infected with BTV-1, cultured at 37°C for 24h, the cell culture supernatant was discarded, fixed with 4% paraformaldehyde for 15min, washed 3 times with PBS, permeabilized with 0.5% Triton X-100 for 10min, washed 3 times with PBS, blocked with 3% BSA for 1h, and the recombinant single B cell antibody A9 expressed in 6 was added, incubated at 4°C overnight, then incubated with mouse anti-His antibody (1:5000 dilution) at room temperature for 1h, washed 3 times with PBS, and added with goat anti-mouse IgG antibody (Alexa Fluor 568) Incubate at room temperature in the dark for 1 h, wash 3 times with PBS, add Hoechst 33342 to stain the cell nucleus for 7 min, wash 3 times with PBS, and observe and collect images under a fluorescence microscope after sealing. The results are shown in Figure 7 As shown, there is red fluorescence in the cytoplasm of BSR infected with BTV-1, while the negative control has no fluorescence, indicating that the recombinant single B cell antibody A9 prepared by the present invention can bind to BTV in infected cells.

[0067] 8. Indirect ELISA detection of single B cell antibodies

[0068] Coating: Dilute the BTV-1 virus culture solution 100 times with CBS buffer (0.05 M carbonate-bicarbonate buffer pH 9.6), add 100 μL / well to the ELISA plate, and coat overnight at 4°C; wash the plate 5 times with PBST buffer;

[0069] Blocking: Block the ELISA plate with PBST buffer containing 5% skim milk powder, 100 μL / well, incubate at 37°C for 1 h; wash the plate 5 times with PBST buffer;

[0070] Detection: Single B cell antibody A9 was added to the ELISA plate, 100 μL / well, incubated at 37°C for 1 h, and the plate was washed 5 times with PBST buffer;

[0071] Add enzyme-labeled secondary antibody: add mouse HRP-labeled anti-His antibody diluted 1:40000 to the ELISA plate, 100 μL / well, incubate at 37°C for 1 hour; wash the plate 5 times with PBST buffer;

[0072] Color development: Use TMB color development solution in the dark, 100 μL / well, incubate at 37°C for 10 min; add stop solution 100 μL / well, read OD 450 The value of .

[0073] Judgment: S / N = sample OD value / negative control, S / N>2.1, judged as positive.

[0074] The results are shown in Table 2. The S / N value of the recombinant single B cell antibody A9 was greater than 2.1, and it was determined to be a positive sample, indicating that the recombinant single B cell antibody A9 prepared by the present invention can bind to BTV-1.

[0075] Table 2 Antibody indirect ELISA results

[0076] Antibody Recombinant single B cell antibody A9 BLANK <![CDATA[OD 450 ]]> 0.504 0.110 S / N 3.8 1

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant single B cell antibody against bluetongue virus in sheep, characterized in that: The amino acid sequence of the heavy chain variable region of the single B cell antibody is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

2. A nucleic acid, characterized in that The nucleic acid encodes the light chain variable region and the heavy chain variable region of the single B cell antibody according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleic acid sequence encoding the heavy chain variable region of the single B cell antibody is shown in SEQ ID NO.1, and the nucleic acid sequence encoding the light chain variable region of the single B cell antibody is shown in SEQ ID NO.

3.

4. A primer set for amplifying the single B cell antibody according to claim 1, characterized in that: The primer set comprises: Primers for amplifying the heavy chain variable region: Sheep gamma chain F outer primer:ACCTCCTCTTTGTGCTCTCAG; Sheep gamma chain R outer primer: TCCCCGCAGCAAGAAGTCA; Sheep gamma chain F inner primer: GAAGCCCTCACAGACCCTCT; Sheep gamma chain R inner primer: GTCAGAGGGTAGACTTTCGGGG; And the primers for amplifying the light chain variable region include: Sheep Lambda chain F outer primer:ATGGCCTGGTCCCCTCTG; Sheep Lambda chain R outer primer: GGCGGGAACAGGGTGAC; Sheep Lambda chain F inner primer:GTCCCYGGGCCAGAS; Sheep Lambda chain R inner primer: GGTGCGGACTTGGGC.

5. Use of the recombinant single B cell antibody according to claim 1 in in vitro detection of bluetongue virus for non-disease diagnosis purposes.

6. Use of the recombinant single B cell antibody according to claim 1 in preparing a reagent, a test strip or a kit for detecting bluetongue virus.

7. A bluetongue virus ELISA detection kit, characterized in that: The kit comprises the recombinant single B cell antibody of claim 1.

8. The ELISA test kit according to claim 7, characterized in that, The kit also includes an ELISA plate, a blocking solution, a diluent, an enzyme-labeled secondary antibody, a washing solution, a color developer, and a stop solution.

9. The method for preparing a recombinant single B cell antibody according to claim 1, characterized in that: The method comprises the following steps: (1) amplifying the light chain variable region and heavy chain variable region sequences encoding the single B cell antibody of claim 1 by RT-PCR, and respectively constructing the recombinant light and heavy chain expression plasmids of the antibody with expression vectors containing the constant region of sheep IgG antibody; (2) The antibody was expressed in 293F suspension cells at a mass ratio of 3:1 between the transfection reagent PEI and the plasmid and a mass ratio of 2:1 between the light chain expression plasmid and the heavy chain expression plasmid.

10. The preparation method according to claim 9, characterized in that: The light chain variable region and heavy chain variable region sequences of the single B cell antibody are amplified by the primer set of claim 4.

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