CN113717284B - Hepatitis A virus specific nano antibody and application thereof - Google Patents

Hepatitis A virus specific nano antibody and application thereof Download PDF

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CN113717284B
CN113717284B CN202111042354.9A CN202111042354A CN113717284B CN 113717284 B CN113717284 B CN 113717284B CN 202111042354 A CN202111042354 A CN 202111042354A CN 113717284 B CN113717284 B CN 113717284B
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叶华跃
彭福中
吴宏斌
王永智
雷高新
施小明
曹蕾
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Jiangsu Huachuang Medicine Research And Development Platform Management Co ltd
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Abstract

The invention discloses a hepatitis A virus specific nano antibody, the amino acid sequence of which is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8. The nano antibody has specific recognition and binding capacity to hepatitis A virus, can be used for research and development of hepatitis A virus detection reagents and preparation of anti-hepatitis A virus medicines, overcomes the defects of the existing hepatitis A virus detection technology, and has wide clinical application prospects.

Description

Hepatitis A virus specific nano antibody and application thereof
Technical Field
The invention relates to a hepatitis A virus specific nano antibody and application thereof, belonging to the technical field of biological medicine.
Background
Viruses belonging to the genus hepadnaviridae, the family picornaviridae, and the genus hepadnaviridae are causative agents of hepatitis a (hereinafter referred to as "hepatitis a"). Vaccination with a hepatitis a vaccine is the most effective measure for preventing and controlling hepatitis a epidemics. The titer detection of the hepatitis A inactivated vaccine mostly adopts an in-vivo efficacy method, but in-vivo efficacy test needs to observe the toxic attack protection effect or detect an antibody after immunizing animals, and has the defects of long operation time, large fluctuation of experimental results and the like and the limitation of animal ethics. With the technological progress and the improvement of the requirements for operation convenience, detection result accuracy, repeatability and the like, in-vitro potency detection methods such as a cell method, an immune method, biochemistry, physicochemical and the like are gradually established, and in-vitro immunological methods are used for replacing in-vivo potency detection for inactivated hepatitis A vaccines in Chinese pharmacopoeia. The titer detection of the inactivated hepatitis A vaccine is in vitro efficacy detection by taking virus titer (viable count) as an index, and a traditional enzyme-linked immunosorbent assay (ELISA) is adopted, but the preparation of the related monoclonal antibody has great challenges and great instability is brought to the detection method. Therefore, the search for a substitute antibody for the conventional antibody is urgently needed to overcome the defects caused by the titer detection of the inactivated hepatitis A vaccine based on the monoclonal antibody.
In 1989, the belgian immunologist Hamers-Casterman, when isolating antibodies in camel serum, accidentally discovered a heavy chain antibody naturally devoid of light chain, comprising only 1 heavy chain variable region (VHH) with structural stability and antigen-binding activity comparable to that of the original heavy chain antibody, and 2 conventional CH2 and CH3 regions, having a molecular mass of only 1/10 that of a monoclonal antibody, the smallest antibody unit obtained so far which is structurally stable and has antigen-binding activity, and hence also called nanobody. Compared with the traditional monoclonal antibody fragment, the nano antibody has extremely high solubility, is not easy to aggregate and precipitate, has very high stability, can keep the antigen binding activity under the denaturing conditions of high temperature, strong acid, strong alkali and the like, and has the characteristics of strong specificity and weak immunogenicity. At present, the nano antibody is widely applied to the basic scientific research fields of developing therapeutic antibody drugs, diagnostic reagents (colloidal gold method, enzyme-linked immunosorbent assay, electrochemiluminescence method), affinity purification matrixes, immunological research and the like.
Disclosure of Invention
The invention aims to provide a hepatitis A virus specific nano antibody and discloses a coding sequence thereof aiming at the defects of a hepatitis A virus detection technology in the prior art.
Technical scheme
A hepatitis A virus specific nano antibody has an amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
The inventor adopts hepatitis A virus immune inner Mongolian Alalashan bactrian camel to extract the total RNA of peripheral blood lymphocytes and reversely transcribe the total RNA into cDNA, then amplifies target genes by a nested PCR technology, constructs a high-quality phage display library by using a phage display technology, performs affinity panning on the library, and finally screens to obtain the hepatitis A virus nano antibody with better specificity.
The invention provides application of the nano antibody in preparation of a reagent for detecting hepatitis A virus.
The invention provides application of the nano antibody in preparing a medicament for preventing or treating hepatitis A virus infection.
A kit for detecting hepatitis A virus comprises the hepatitis A virus specific nano antibody.
The invention has the beneficial effects that:
the invention provides a hepatitis A virus specific nano antibody which has specific recognition and binding capacity to hepatitis A virus. The nano antibody can be used for research and development of a hepatitis A virus detection reagent and preparation of an anti-hepatitis A virus medicine, and has a wide clinical application prospect.
Drawings
FIG. 1 is an electrophoresis diagram of PCR amplification products of hepatitis A virus specific nanobody genes;
FIG. 2 is a SDS-PAGE detection of nanobodies HAV-a, HAV-b, HAV-c, HAV-e, HAV-g, HAV-h, HAV-j, HAV-k;
FIG. 3 is SEC-HPLC check result of purity of Nanobody HAV-a;
FIG. 4 is SEC-HPLC check result of purity of Nanobody HAV-b;
FIG. 5 is SEC-HPLC detection results of purity of Nanobody HAV-c;
FIG. 6 is SEC-HPLC detection results of purity of Nanobody HAV-e;
FIG. 7 is SEC-HPLC detection results of purity of Nanobody HAV-g;
FIG. 8 is SEC-HPLC detection results of purity of Nanobody HAV-h;
FIG. 9 is SEC-HPLC check result of purity of Nanobody HAV-j;
FIG. 10 shows SEC-HPLC detection results of purity of Nanobody HAV-k.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below by combining the attached drawings and the detailed implementation modes.
Example 1
Construction of a Nanobody library against hepatitis A Virus:
(1) A Bactrian camel was immunized once per week with 0.5ml of inactivated hepatitis A virus vaccine (purchased from Eimenghuai biopharmaceuticals, jiangsu, ltd.) for a total of 7 consecutive immunizations. Bactrian camel venous blood was collected at week 8 at 100ml. The Ficoll reagent was prepared as follows: 1 adding venous blood, centrifuging for 30 minutes under the condition of 400g, then taking out a middle bactrian camel white lymphocyte layer by using a dropper, and extracting Total RNA by using a Total RNA kit of precious bioengineering (Dalian) Co., ltd;
(2) Total RNA is subjected to reverse transcription by adopting Takara reverse transcription kit of Takara Shuzo Co., ltd to obtain cDNA.
(3) Taking cDNA as a template and F/R sequence as a primer, wherein the upstream primer F1:5'-CTGGGTGGTCCTGGCTGCTCTT-3', downstream primer R1:5'-GCGGTACGTGTGTTGAACTGTT-3', amplifying a VHH target gene (heavy chain antibody variable region) by a nested PCR technology, respectively digesting pMECS carrier and VHH fragment by restriction enzyme, and then linking the digested fragment with the carrier to obtain a recombinant carrier. And recovering the gel to obtain a target fragment PCR product of 750 bp.
(4) The recombinant vector is electrically transformed into an electrotransformation competent cell TG1 to obtain a recombinant TG1 cell, an HAV nano antibody library is constructed, and the library capacity is 1 multiplied by 10 9 . And (3) carrying out colony PCR detection, wherein the result shows that the insertion rate of the constructed library is more than 80%, and the VHH is proved to be effectively inserted into the vector.
Example 2
Screening a nano antibody aiming at the hepatitis A virus;
1) Culturing 500ul of recombinant TG1 cells in a2 XTY culture medium, adding 100ul of helper phage to infect the TG1 cells during the culture, and culturing overnight to amplify the phage;
2) Precipitating the phage by using PEG the next day, and centrifugally collecting the amplified phage;
3) Coupling 800ug of neutral avidin protein diluted on an enzyme label plate, standing overnight at 4 ℃, and simultaneously setting a negative control hole;
4) Adding 200ul of biotin-labeled hepatitis A virus capsid protein the next day, incubating for 2 hours at room temperature, and adding 100ul of PBS into a negative control hole;
5) Adding 100ul of 5% skimmed milk, and sealing at room temperature for 2 hr;
6) After the sealing is finished, adding 500ul of the amplified phage library, and acting for 2h at room temperature;
7) Washing for 5 times by using PBS, and washing away the unbound phage;
8) Dissociating the phage specifically bound with the capsid protein of hepatitis A virus by trypsin with the concentration of 50mg/mL, infecting Escherichia coli TG1 cells in logarithmic growth phase, culturing at 37 ℃ for 2h, collecting phage for next round of screening, and repeating the screening process for 3 rounds. The P/N values from 2.2 to about 10000 in the first round and the I/E values from 13605 to about 13 in the first round proved that the library had a very significant enrichment for the hepatitis A virus capsid protein.
Screening to obtain 8 strains of HAV nano antibodies with better specificity, respectively marking as HAV-a, HAV-b, HAV-c, HAV-e, HAV-g, HAV-h, HAV-j and HAV-k, sequencing to obtain nucleotide sequences, and converting the nucleotide sequences into amino acid sequences which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
Example 3
Expression and purification of the hepatitis A virus specific nano antibody:
(1) HAV-a, HAV-b, HAV-c, HAV-e, HAV-g, HAV-h, HAV-j, HAV-k) heavy chain variable regions were synthesized in their respective whole genes, PCR was performed using these as templates, the resulting PCR products were subjected to gel electrophoresis, and then the corresponding bands of interest were excised, and the PCR products were recovered according to Axygen instructions.
FIG. 1 is an electrophoresis diagram of PCR amplification products of hepatitis A virus-specific nanobody genes, in which lanes 1 to 8 are HAV-a, HAV-b, HAV-c, HAV-e, HAV-g, HAV-h, HAV-j, HAV-k,7 is M: and (4) protein molecule standard. As can be seen, the clone target product to be recovered has a significant band around 750 bp.
(2) Constructing a recombination reaction system which comprises the following steps: recovering a target PCR product by glue: 4ul, linearized vector: 3.5ul, recombinase: 2.5ul, reaction conditions: water bath at 50 deg.C for 25min, standing for 2-3min to reduce temperature, performing transformation and bacterial liquid coating experiment, and incubating overnight at 37 deg.C.
Picking single colony in the overnight plate, performing colony PCR by using a CMV-F/SEQ-R primer, performing electrophoresis for positive cloning, randomly selecting 4 positive bacteria, sequencing the positive bacteria liquid by using the CMV-F/SEQ-R primer, selecting the positive bacteria liquid of the clone with correct sequencing, extracting low endotoxin plasmid after amplification culture, and performing sequencing verification. The correct expression plasmid was obtained by sequencing verification. Plasmid concentrations are shown in table 1:
TABLE 1
Name of plasmid Plasmid concentration ng/ul Name of plasmid Plasmid concentration ng/ul
HAV-a 605 HAV-g 971
HAV-b 885 HAV-h 1145
HAV-c 683 HAV-j 1204
HAV-e 814 HAV-k 1065
(3) Transient transfection of suspension HEK293F cells expressed foreign proteins:
1) Cells were acclimated to OPM-293CD05 medium and cultured at 0.3x10 6 cells/ml density inoculation, continuous passage at least twice, and then transfection experiment.
2) Before transfection, samples were taken and cell density was counted at 1-1.5X 10 6 cells were seeded in fresh pre-warmed medium at a cell/ml density;
3) Taking a 15ml/50ml centrifuge tube, diluting 50ug of plasmid DNA and 200ul of PEI respectively by using Opti-MEMI medium, and slightly reversing and mixing uniformly;
4) The diluted plasmid was sterilized by filtration through a 0.22um filter;
5) Incubating the diluted PEI reagent for 5min at room temperature, adding the diluted PEI reagent into the diluted DNA, and uniformly mixing;
6) Incubating the PEI/plasmid DNA complex for 15min at room temperature, then gently adding the PEI/plasmid DNA complex into the prepared cell suspension, and gently swirling the shake flask in the adding process;
7) The cells were subjected to 8% CO at 37 ℃ C 2 The cells were subjected to shake culture at 120rpm in humidified air, expression culture was carried out for one week, and OPM-CHO PFF05 feed with an initial volume of 5% was added at 24 hours and 96 hours after transfection
8) After one week of expression culture, the cells were centrifuged at 8000rpm for 5min, and the supernatant was collected and the cell density was measured. The test results are shown in table 2:
TABLE 2
Figure GDA0003302739810000051
Figure GDA0003302739810000061
(4) Purification and purity detection of nano antibody
(1) Collecting cell supernatant in a 50ml centrifuge tube, balancing, centrifuging at 7000g and 4 ℃ for 30 minutes, collecting supernatant in a 250ml collection bottle;
(2) adding 1ml of Ni Sepharose into each bottle, mixing uniformly, and incubating for more than 1h in a chromatographic cabinet shaking table;
(3) adding the incubated supernatant into a chromatographic column, and washing with 20mM imidazole for more than 30 column volumes;
(4) blocking the lower outlet, adding 1-1.5 column volumes of 500mM, and shaking-bed incubating at 4 deg.C for more than 10 min;
(5) and (3) elution: eluting in an EP tube or a centrifuge tube, adding half of the original eluent, eluting and collecting;
(6) preparing a sample: 10ul 4 Xprotein SDS PAGE padding Buffer +30ul purified sample, mixing, metal bath at 100 ℃ for 5min, and instantaneous separation for 30s;
(7) 20ul of sample run SDS-PAGE identification: voltage 141V,55min, placing the glue in pure water, and heating in a microwave oven for 5min at high temperature. Taking out and replacing with R250 staining solution, and heating in microwave oven for 5min; taking out and replacing with tap water, heating in a microwave oven for 5min at high temperature, and repeating the steps for three times;
(8) cutting the dialysis bag into a proper size, cleaning the dialysis bag under ultrapure water, and checking whether the dialysis bag leaks; diluting PBS to 1 × PBS, and placing in a chromatography cabinet for precooling;
(9) clamping one end of a dialysis bag by a dialysis clamp, adding a dialyzed protein sample, clamping the other end of the dialysis bag, checking whether the dialyzed protein sample leaks or not, putting the sample in 3LPBS for dialysis for more than 1h, sucking out the protein sample, and subpackaging and storing at-80 ℃.
And (3) purity detection:
1. the purity of the prepared nano antibodies HAV-a, HAV-b, HAV-c, HAV-e, HAV-g, HAV-h, HAV-j and HAV-k is detected by SDS-PAGE, and the SDS-PAGE detection result of the nano antibodies is shown in figure 2, wherein M is a protein molecular standard, lanes 1-8 are nano antibodies HAV-a, HAV-b, HAV-c, HAV-e, HAV-g, HAV-h, HAV-j and HAV-k in sequence, and it can be seen that the target protein with the molecular weight of about 40kDa is obtained, the size is the same as the predicted protein, no obvious hybrid band exists, the purity of the protein obtained by being carried into computer analysis software is more than 95%, and the protein purity is higher.
2. Detecting the purity of the prepared nano antibodies HAV-a, HAV-b, HAV-c, HAV-e, HAV-g, HAV-h, HAV-j and HAV-k by SEC-HPLC, wherein the specific method is as follows:
experimental materials: high performance liquid chromatograph, gel chromatographic column, deionized water and mobile phase (Na) 2 HPO 4 ·12H 2 0、NaH 2 PO 4 ·2H 2 O、NaCl)；
SEC experiments are carried out by using a high performance liquid chromatograph LC-20AT and a gel chromatographic column, and the experimental conditions are as follows: flow rate: 1ml/min; sample introduction amount: 20 mu l of the mixture; column temperature: 35 ℃; detection wavelength: 214nm,280nm; collecting time: and 15min. The water was replaced with the mobile phase and the flow rate was slowly increased to 1.000ml/min until the baseline leveled off. 50ul of antibody is taken to the sample injection bottle with the corresponding number, the sample injection bottle is placed at the corresponding position of the instrument, the sample injection time is 15min, the data is analyzed and processed and stored, the mobile phase is replaced by deionized water, and the sample is washed for 1.5h.
SEC-HPLC examination of the purity of the nanobodies HAV-a, HAV-b, HAV-c, HAV-e, HAV-g, HAV-h, HAV-j, HAV-k is shown in FIGS. 3-10, and it can be seen that the purity of the nanobody HAV-a is 99.23%, the purity of the nanobody HAV-b is 65.29%, the purity of the nanobody HAV-c is 95.46%, the purity of the nanobody HAV-e is 100%, the purity of the nanobody HAV-g is 97.47%, the purity of the nanobody HAV-h is 100%, the purity of the nanobody HAV-j is 67.68%, and the purity of the nanobody HAV-k is 98.57%, as determined by SEC-HPLC.
Example 4
Specific assay
The test method comprises the following steps: the specificity of hepatitis A virus nanobodies (HAV-a, HAV-b, HAV-c, HAV-e, HAV-g, HAV-h, HAV-j, HAV-k) was determined by matrix titration with hepatitis B antigen as a control. Taking an 8 x 12 96-hole standard ELISA plate, taking 8 as a column and 12 as a row, wherein the first column is used as a blank control group, the second to fourth columns are coated with hepatitis A antigen, and the fifth to seventh columns are coated with hepatitis B antigen; coating: add 100ul (10 ug/ml) of the corresponding antigen coating solution to each well in the above order, wherein only the coating buffer is added in the first column, and incubate at 4 ℃ overnight; and (3) sealing: after washing the ELISA plate for 3 times, 300. Mu.l of blocking solution was added to each well, and the mixture was allowed to stand at 37 ℃ for 2 hours; combining: after washing, 100 mul of enzyme-labeled nano antibody is added into each hole, wherein HAV-a enzyme-labeled antibody is added into A2-A7; adding HAV-B enzyme labeled antibody into B2-B7; adding HAV-C enzyme labeled antibody into C2-C7; adding HAV-e enzyme labeled antibody into the D2-D7; E2-E7 is added with HAV-g enzyme-labeled antibody; adding HAV-h enzyme-labeled antibody into F2-F7; adding HAV-j enzyme-labeled antibody into G2-G7; H2-H7 is added to HAV-k enzyme labeled antibody. Incubating for 1 hour at 37 ℃; color development: after washing, adding 100 mul of TMB solution into each hole, and incubating for 20 minutes at 37 ℃ in a dark place; and (4) terminating: after washing, 50. Mu.l of stop solution was added to each well; reading: setting the microplate reader at 450nm, performing measurement reading on each hole, and subtracting a blank group from an experimental group to obtain a mean value OD value; and (3) comparison: according to the specificity requirement in the Chinese biological product regulation, when ODnm is more than or equal to the average ODnm value +3SD, the test result is judged to be positive. When ODnm is less than the average ODnm value +3SD, the result is judged to be negative. According to the literature, the average ODnm value of the hepatitis A antigen detection kit is 0.139, the standard deviation SD is 0.0128, namely when the ODnm is more than or equal to 0.177, the kit is judged to be positive. When ODnm is less than 0.177, the result is judged to be negative. The results of the hepatitis A antigen specificity experiment are shown in Table 3, and the results of the hepatitis B antigen specificity experiment are shown in Table 4:
TABLE 3 hepatitis A antigen specificity test results
Figure GDA0003302739810000081
TABLE 4 hepatitis B antigen specificity test results
Figure GDA0003302739810000082
As can be seen from tables 3 and 4, the wells coated with hepatitis B antigen all have OD values less than the negative and positive critical values and are all negative, and the wells coated with hepatitis A antigen have OD values much greater than the negative and positive critical values, which indicates that the hepatitis A virus nanobody has high specificity and strong ability to recognize hepatitis A antigen, and eight kinds of nanobody OD values are compared according to Table 3, wherein the OD value of HAV-g is the highest, the OD value of HAV-j is the lowest, and the specificity of the nanobody of HAV-g subtype is the highest among all subtypes.
A sequence table:
SEQ ID NO.1
amino acid sequence of hepatitis A virus nano antibody HAV-a
ESGGGSVQAGGSLRLSCAASGYTHSSYCMGWFRQAPGKERQGVAAISPGGSFKYYADSVKGRFTISRDNAKNTVYLQMNSLQPEDTAMYYCAAAPFACDGDWYSGRWNNWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.2
Amino acid sequence of hepatitis A virus nano antibody HAV-b
ESGGGSVQAGGSLRLSCAASGSTYTIAWFRQAPGKEREGIAAIYTLAGGTYYADSVKGRFTISRDNAKNTLSLQMNSLKPEDTAMYYCAAAIGAGYGRNWLLSERWYNYWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.3
Amino acid sequence of hepatitis A virus nano antibody HAV-c
ESGGGSVQAGGSLRLSCAASGYTYSSSCMGWFRQAPGKEREGVATIDSFGNTKYADSVKGRFTISKDNAKNTLYLQMNSLKPEDTAMYYCTADMDGRWGGYCVRSAEDFAYWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.4
Amino acid sequence of hepatitis A virus nano antibody HAV-e
ESGGGSVQAGGSLRLSCTASGSTYSRRYIGWFRQAPGKEREGVATISTGGGFRSYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAAGSNVDRRWSLLSRDYNYWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.5
Amino acid sequence of hepatitis A virus nano antibody HAV-g
ESGGGSVQAGGSLRLSCAASGYTWSSTCMGWFRQAPGKERQGVAAISPGGSFKYYADSVKGRFTISRDNAKNTVYLQMNSLQPEDTAMYYCAAAPFACDGDWYSGRWNNWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.6
Amino acid sequence of hepatitis A virus nano antibody HAV-h
ESGGGTVQAGGSLRLSCAASGYTYSSACMGWFRQAPGKEREGVATIGSDGSTSYTDSVKGRFTISKDNVHNNLYLQMNSLKPEDTAMYYCAATITTVTMCRRRDGRLFGHWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.7
Amino acid sequence of hepatitis A virus nano antibody HAV-j
ESGGGSVQAGGSLRLSCAVSGFPYSTYYMGWFRQAPGKEREGVAAVYPGVGLKRYADSVKGRFTISQDDAKNTLYLQMNSLKPEDTALYYCAVDQAVSLGWELSPGRYRYWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO.8
Amino acid sequence of hepatitis A virus nano antibody HAV-k
ESGGGSAQAGGSLRLSCAASGNSYSRNCMGWFRQAPGKERQGVAAISPGGSFKYYADSVKGRFTISRDNAKNTVYLQMNSLQPEDTAMYYCAAAPLACDGDWYSGRWNNWGQGTQVTVSSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Sequence listing
<110> Jiangsu Huachuang medicine research and development platform management Limited company
<120> hepatitis A virus specific nano antibody and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 352
<212> PRT
<213> Hepatitis A virus specific Nanobody (Hepatitis a virus specific nano antibody)
<400> 1
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr His Ser Ser Tyr Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Gln Gly Val Ala Ala Ile Ser Pro
35 40 45
Gly Gly Ser Phe Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Gln Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Pro Phe
85 90 95
Ala Cys Asp Gly Asp Trp Tyr Ser Gly Arg Trp Asn Asn Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ala Asp Lys Thr
115 120 125
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
130 135 140
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
145 150 155 160
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
165 170 175
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
180 185 190
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
195 200 205
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
210 215 220
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
225 230 235 240
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
245 250 255
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
260 265 270
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
275 280 285
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
290 295 300
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
305 310 315 320
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
325 330 335
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 2
<211> 352
<212> PRT
<213> Hepatitis A virus specific Nanobody (Hepatitis a virus specific nano antibody)
<400> 2
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Ser Thr Tyr Thr Ile Ala Trp Phe Arg Gln Ala
20 25 30
Pro Gly Lys Glu Arg Glu Gly Ile Ala Ala Ile Tyr Thr Leu Ala Gly
35 40 45
Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
50 55 60
Asp Asn Ala Lys Asn Thr Leu Ser Leu Gln Met Asn Ser Leu Lys Pro
65 70 75 80
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Ile Gly Ala Gly Tyr
85 90 95
Gly Arg Asn Trp Leu Leu Ser Glu Arg Trp Tyr Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ala Asp Lys Thr
115 120 125
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
130 135 140
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
145 150 155 160
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
165 170 175
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
180 185 190
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
195 200 205
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
210 215 220
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
225 230 235 240
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
245 250 255
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
260 265 270
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
275 280 285
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
290 295 300
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
305 310 315 320
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
325 330 335
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 3
<211> 354
<212> PRT
<213> Hepatitis A virus specific Nanobody (Hepatitis a virus specific nano antibody)
<400> 3
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr Ile Asp Ser
35 40 45
Phe Gly Asn Thr Lys Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Thr Ala Asp Met Asp Gly
85 90 95
Arg Trp Gly Gly Tyr Cys Val Arg Ser Ala Glu Asp Phe Ala Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ala Asp
115 120 125
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
130 135 140
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
145 150 155 160
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
165 170 175
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
180 185 190
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
195 200 205
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
210 215 220
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
225 230 235 240
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
245 250 255
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
260 265 270
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
275 280 285
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
290 295 300
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
305 310 315 320
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
325 330 335
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
340 345 350
Gly Lys
<210> 4
<211> 353
<212> PRT
<213> Hepatitis A virus specific Nanobody (Hepatitis a virus specific nano antibody)
<400> 4
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Thr Ala Ser Gly Ser Thr Tyr Ser Arg Arg Tyr Ile Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr Ile Ser Thr
35 40 45
Gly Gly Gly Phe Arg Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Ser Asn
85 90 95
Val Asp Arg Arg Trp Ser Leu Leu Ser Arg Asp Tyr Asn Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ala Asp Lys
115 120 125
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
130 135 140
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
145 150 155 160
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
165 170 175
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
180 185 190
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
195 200 205
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
210 215 220
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
225 230 235 240
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
245 250 255
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
260 265 270
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
275 280 285
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
290 295 300
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
305 310 315 320
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
325 330 335
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
340 345 350
Lys
<210> 5
<211> 352
<212> PRT
<213> Hepatitis A virus specific Nanobody (Hepatitis a virus specific nano antibody)
<400> 5
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Trp Ser Ser Thr Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Gln Gly Val Ala Ala Ile Ser Pro
35 40 45
Gly Gly Ser Phe Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Gln Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Pro Phe
85 90 95
Ala Cys Asp Gly Asp Trp Tyr Ser Gly Arg Trp Asn Asn Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ala Asp Lys Thr
115 120 125
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
130 135 140
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
145 150 155 160
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
165 170 175
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
180 185 190
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
195 200 205
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
210 215 220
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
225 230 235 240
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
245 250 255
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
260 265 270
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
275 280 285
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
290 295 300
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
305 310 315 320
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
325 330 335
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350
<210> 6
<211> 353
<212> PRT
<213> Hepatitis A virus specific Nanobody (Hepatitis a virus specific nano antibody)
<400> 6
Glu Ser Gly Gly Gly Thr Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ala Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr Ile Gly Ser
35 40 45
Asp Gly Ser Thr Ser Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Lys Asp Asn Val His Asn Asn Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Thr Ile Thr Thr
85 90 95
Val Thr Met Cys Arg Arg Arg Asp Gly Arg Leu Phe Gly His Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ala Asp Lys
115 120 125
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
130 135 140
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
145 150 155 160
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
165 170 175
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
180 185 190
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
195 200 205
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
210 215 220
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
225 230 235 240
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
245 250 255
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
260 265 270
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
275 280 285
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
290 295 300
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
305 310 315 320
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
325 330 335
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
340 345 350
Lys
<210> 7
<211> 353
<212> PRT
<213> Hepatitis A virus specific Nanobody (Hepatitis a virus specific nano antibody)
<400> 7
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Val Ser Gly Phe Pro Tyr Ser Thr Tyr Tyr Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Val Tyr Pro
35 40 45
Gly Val Gly Leu Lys Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asp Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Val Asp Gln Ala
85 90 95
Val Ser Leu Gly Trp Glu Leu Ser Pro Gly Arg Tyr Arg Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ala Asp Lys
115 120 125
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
130 135 140
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
145 150 155 160
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
165 170 175
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
180 185 190
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
195 200 205
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
210 215 220
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
225 230 235 240
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
245 250 255
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
260 265 270
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
275 280 285
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
290 295 300
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
305 310 315 320
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
325 330 335
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
340 345 350
Lys
<210> 8
<211> 352
<212> PRT
<213> Hepatitis A virus specific Nanobody (Hepatitis a virus specific nano antibody)
<400> 8
Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Asn Ser Tyr Ser Arg Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Gln Gly Val Ala Ala Ile Ser Pro
35 40 45
Gly Gly Ser Phe Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Gln Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Pro Leu
85 90 95
Ala Cys Asp Gly Asp Trp Tyr Ser Gly Arg Trp Asn Asn Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ala Asp Lys Thr
115 120 125
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
130 135 140
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
145 150 155 160
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
165 170 175
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
180 185 190
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
195 200 205
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
210 215 220
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
225 230 235 240
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
245 250 255
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
260 265 270
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
275 280 285
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
290 295 300
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
305 310 315 320
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
325 330 335
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345 350

Claims (3)

1. A hepatitis A virus specific nano antibody is characterized in that the amino acid sequence of the nano antibody is shown as SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO. 8.
2. Use of the nanobody of claim 1 in the preparation of a reagent for the detection of hepatitis a virus.
3. A kit for detecting hepatitis a virus, comprising the hepatitis a virus-specific nanobody of claim 1.
CN202111042354.9A 2021-09-07 2021-09-07 Hepatitis A virus specific nano antibody and application thereof Active CN113717284B (en)

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