CN114133451B - Nanometer antibody for toxoplasma virulence factor ROP18 and coding sequence and application thereof - Google Patents
Nanometer antibody for toxoplasma virulence factor ROP18 and coding sequence and application thereof Download PDFInfo
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- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
- G01N2333/45—Toxoplasma
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a nanometer antibody aiming at toxoplasma virulence factor ROP18, a coding sequence and application thereof, wherein the VHH chain amino acid sequence of the nanometer antibody is shown as SEQ ID No. 18. The nanobody includes two VHH chains. According to the invention, the nanometer antibody of the toxoplasma virulence factor ROP18 is prepared by immunizing a camel with the toxoplasma virulence factor ROP18 antigen to prepare a nanometer antibody library specific to the toxoplasma virulence factor ROP18, and the phage display technology is utilized to screen the nanometer antibody specific to the toxoplasma virulence factor ROP18 with high affinity, so that the nanometer antibody has high water solubility and conformational stability, and the toxoplasma virulence factor ROP18 nanometer antibody can be specifically combined with the toxoplasma antigen, so that the nanometer antibody can be used for preparing a toxoplasma detecting kit.
Description
Technical Field
The invention relates to the field of bioengineering nanotechnology, in particular to a nanobody aiming at toxoplasma virulence factor ROP18, and a coding sequence and application thereof.
Background
Toxoplasma (Toxoplasma gondii) is an obligate cell parasitic protozoa, has a broad host population and is prevalent worldwide, causing parasitic diseases in both humans and animals. For patients with AIDS, organ transplantation, malignant tumor, etc., with impaired or suppressed immune function, toxoplasma gondii is used as an opportunistic infective agent, which can cause death of patients. Toxoplasmosis is not only an important medical disease, but also an important biological factor affecting the prenatal and postnatal care of humans. The pregnant women can be infected with toxoplasmosis, about half of them can be infected with maternal and fetal vertical infection, abortion, dead fetus, congenital defect or malformation (morphological malformation, functional mental retardation) caused by fetal infant, etc.
At present, the diagnosis method of toxoplasmosis is mainly divided into an etiology method, an immunology method and a molecular biology method. The etiology diagnosis method is simple, the result is reliable, but the detection rate is low, the time is consumed, and the diagnosis is easy to be missed. The molecular diagnosis technology has the advantages of sensitivity, specificity, rapid detection and the like, but the molecular diagnosis technology has high equipment requirement, aerosol pollution is easy to form and is difficult to remove, most of laboratories in China can not be strictly partitioned at present, and certain false positives exist. The immunological diagnosis ELISA is the technique with the widest application for diagnosing toxoplasma infection at present, and has the advantages of simplicity, easiness in implementation, high sensitivity, easiness in standardization and automation. Currently, toxoplasmosis detection reagents approved by the national drug administration are only IgG and IgM antibody detection. The measurement of the antibody level alone cannot fully reflect the toxoplasma infection, and is highly likely to cause missed diagnosis of a part of patients suffering from the symptoms. Therefore, development of toxoplasma antigen detection method, combined with toxoplasma IgG and IgM antibody detection result, is needed to achieve the purpose of judging the course of disease.
Toxoplasma rod protein 18 (roptry protein 18, rop 18) is an essential virulence protein of toxoplasma in the process of infection, and participates in the recognition, adhesion and invasion of toxoplasma to host cells. ROP8 has been shown to be a key virulence factor for toxoplasma gondii as a serine threonine kinase. In addition, the research shows that ROP18 as toxoplasma secretion protein has excellent immunogenicity, immunoprotection and diagnosis value.
A specific antibody which is a natural heavy chain lacking in camel bodies and cartilage fish bodies and still has biological activity is called a single domain antibody, and an antigen binding site VHH (nanobody) of the single domain antibody has independent antigen recognition capability, and has the advantages of small molecular weight, simple structure, stable physicochemical properties and the like. Can bind some hidden antigen epitopes in the aspect of antigen-antibody binding, and is particularly suitable for targets which are difficult to obtain antibodies. The nano antibody has wider application prospect in the medical fields of disease diagnosis and treatment, drug development and the like. Therefore, the invention utilizes the technology, and utilizes the screened toxoplasma ROP18 antigen specific nano antibody to detect toxoplasma infection.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art, and provides a nanometer antibody for toxoplasma virulence factor ROP18, a coding sequence and application thereof, wherein the toxoplasma virulence factor ROP18 nanometer antibody can be specifically combined with toxoplasma antigen, and can be used for preparing a toxoplasma detection kit.
The invention provides a VHH chain of a nano antibody aiming at toxoplasma virulence factor ROP18, which comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of the CDR1 is shown as SEQ ID No.23, the amino acid sequence of the CDR2 is shown as SEQ ID No.24, and the amino acid sequence of the CDR3 is shown as SEQ ID No. 25.
Specifically, the amino acid sequence of the VHH chain is shown as SEQ ID No. 18.
The invention also provides a nano antibody aiming at toxoplasma virulence factor ROP18, which comprises two VHH chains.
The invention further provides genes encoding the VHH chains, or encoding the nanobodies.
Preferably, the nucleotide sequence of the gene is shown as SEQ ID No. 9.
The invention also provides a recombinant expression vector comprising the gene.
The invention also provides a genetically engineered cell, which is obtained by introducing the recombinant expression vector into a host cell.
Preferably, the host cell is an E.coli, yeast or CHO cell.
The invention also provides application of the nano antibody in preparation of toxoplasma detection kit.
The invention prepares a nanometer antibody library specific to the toxoplasma virulence factor ROP18 by immunizing a camel with the toxoplasma virulence factor ROP18 antigen, screens the nanometer antibody specific to the toxoplasma virulence factor ROP18 with high affinity by using a phage display technology, has high water solubility and conformational stability, can be specifically combined with the toxoplasma virulence factor ROP18 antigen, and can be used for preparing a toxoplasma detection kit.
Drawings
FIG. 1 is a diagram showing the results of SDS-PAGE verification of expression and purification electrophoresis detection of recombinant virulence factor ROP18, wherein M is a protein Marker, and 1 is purified recombinant toxoplasma virulence factor ROP18.
FIG. 2 shows the PCR identification positive rate of toxoplasma virulence factor ROP18 nanobody library colony, wherein M is standard DNAMaroker DL2000, and lanes 1-20 represent 20 colonies respectively.
FIG. 3 is a graph of a phylogenetic tree analysis of 9 different clones of virulence factor nanobodies.
FIG. 4 is a schematic diagram of the construction of plasmid PMECS-ROP18-22, clone No.22, directed against toxoplasma virulence factor ROP18.
FIG. 5 is a diagram showing the results of detection of toxoplasma gondii virulence factor ROP18 nanobody expression and purification electrophoresis, lane 1 shows the results of toxoplasma gondii virulence factor ROP18 nanobody electrophoresis before purification, and lane 2 shows the results of toxoplasma gondii virulence factor ROP18 nanobody electrophoresis after purification.
FIG. 6 is a diagram of virulence factor ROP18 nanobody-specific ELISA assay.
Detailed Description
Example 1
Recombinant toxoplasma virulence factor ROP18, the steps are as follows:
(1) Extracting toxoplasma gondii total RNA by Trizol method, performing reverse transcription to obtain total cDNA, designing PCR amplification primers according to toxoplasma gondii virulence factor gene sequence (GenBank number: JX 045330.1) on NCBI, wherein the upstream and downstream primers are respectively:
upstream: :5' -CTAGCTAGCATGTTTTCGGTACAGCGGC;
downstream: :5' -CGCGGATCCTTATTCTGTGTGGAGATGTTCC.
(2) PCR amplification is carried out to obtain a ROP18 target gene fragment with the fragment size of 1665bp, and a pET28a-ROP18 recombinant vector is constructed.
(3) The pET28a-ROP18 recombinant vector is used for transforming competent cells E.coli BL21 (DE 3) through a heat shock method, is induced by IPTG overnight at 37 ℃, expresses recombinant proteins, collects thalli, and verifies that recombinant virulence factor ROP18 is expressed through SDS-PAGE after ultrasonic disruption.
(4) The purified fusion protein is purified by a Ni-NTA nickel column, and a Millipore ultrafiltration tube with the molecular weight cut-off of 10kDa is used for concentrating the expressed fusion protein, so that the concentration of the fusion protein is improved, and the concentration of the recombinant protein reaches 2mg/mL (figure 1) through detection by a Coomassie brilliant blue binding method.
Example 2
Constructing toxoplasma virulence factor specific nano antibody library, and the steps are as follows:
(1) Firstly purifying toxoplasma virulence factor antigen, then mixing 2mg toxoplasma virulence factor with Freund's adjuvant in equal volume (except for first use of complete Freund's adjuvant and the rest of complete Freund's adjuvant), immunizing a healthy adult dromedary (Camelus dromedarius), immunizing 6 times a week, and stimulating B cells to express antigen-specific nanobody;
(2) After 6 immunizations are finished, 100mL camel peripheral blood lymphocytes are extracted and total RNA is extracted;
(3) Reverse transcription to cDNA and amplifying the total VHH by nest PCR;
(4) Cleavage of 20. Mu.g of phage display vector PMECS and 10. Mu.g of VHH with restriction enzyme PstI and NotI and ligation of the two fragments;
(5) Electrotransferring the connected product into competent colibacillus TG1, constructing toxoplasma virulence factor nano antibody library and measuring the volume of the library, wherein the volume of the library is 3.12X10% 9 (Table 1).
(6) 20 single colonies were randomly picked from the plates of library colony counts and colony PCR was performed to identify the insertion rate of the VHH gene. As shown in FIG. 2, 20 single colonies in the experimental group amplified a 500-750bp band with a positive rate of 80%, and thus the actual library capacity of the initial library was 3.9X10 9 . Further packaging to obtain phage nanometer antibody library, and applying double-layer agar plateThe titer of the constructed phage nanobody library was calculated to be 2.11X10 by plaque counting 13 pfu/mL (Table 1).
TABLE 1
| Library capacity | Positive rate | Phage library titers |
| 3.12×10 9 | 16/20 | 2.11×10 13 pfu/mL |
Example 3
The method for screening toxoplasma virulence factor specific nano antibody comprises the following steps:
(1) mu.L of the extract was dissolved in 100mM NaHCO 3 Toxoplasma virulence factor coating solution (20 mug/mL) with pH of 8.2 is placed on a NUNC enzyme label plate at 4 ℃ overnight;
(2) The next day 100 μl of 3% skim milk was added and blocked at room temperature for 2h;
(3) After 2h, 100. Mu.L of 10 was added 11 The pfu contains phage of toxoplasma virulence factor nanobody library and acts for 1h at room temperature;
(4) The first round of panning was performed 10 times with 0.05% pbs + tween-20; the second round for 20-25 times, remove nonspecifically bound phage;
(5) Phages specifically binding to toxoplasma virulence factors were dissociated with triethylamine (100 mM) and infected with E.coli TG1 in the logarithmic growth phase, cultured at 37℃for 1h, and phages were generated and purified for the next round of screening, and the same screening process was repeated for 2-3 rounds to gradually obtain enrichment, and the results are shown in Table 2.
TABLE 2
Example 4
Screening specific single positive clones by phage enzyme-linked immunosorbent assay (ELISA) as follows:
(1) From the above 3-4 rounds of screening of phagemid-containing dishes, 40 individual colonies were picked and inoculated into TB medium containing 100. Mu.g/mL ampicillin, grown to the log phase, and then incubated overnight at 28℃with 1mM final IPTG.
(2) Crude extracted antibodies are obtained by a permeation method, and the antibodies are transferred into an ELISA plate coated with antigen and placed for 1h at room temperature;
(3) Washing off unbound antibody with PBST, adding mouse anti-His anti-antibody (mouse anti-HIS antibody, abcam), and standing at room temperature for 1 hr;
(4) Unbound antibody was washed off with PBST and Goat Anti-Mouse IgG-HRP (Goat Anti-Mouse HRP labeled antibody, abcam) was added.
(5) Washing unbound antibody with PBST, adding TMB color developing solution, and reading OD on enzyme labeling instrument 450 Values.
(6) When the OD value of the sample well was 2.1 times or more greater than that of the control well, positive clone wells were judged (Table 3).
TABLE 3 Table 3
(7) The positive clone well was transferred to TB medium containing 100. Mu.g/mL ampicillin, and the plasmid was extracted for sequencing.
(8) The amino acid sequence of the VHH chain is determined by the framework region FR and complementarityRegion CDR composition. Based on sequencing results, vector was appliedAnd->The software analyzed each clone, and identified the same strain as CDR1, CDR2 and CDR3 sequences as the same clone, and different sequences as different clones. 9 different clone strains (Table 4) are obtained, the nucleotide sequence is shown as SEQ ID NO. 1-9, and the amino acid sequence is shown as SEQ ID NO. 9-18. Further treelia analysis was performed to preliminarily determine epitope dissimilarity for positive clones (fig. 3).
TABLE 4 Table 4
(9) Out of 40 positive clones randomly selected, 9 different clones were obtained in total according to phage ELISA results. As shown in Table 3, clone No.22 identified the best antigen. The gene sequence of the 22 # Long Zhu anti-virulence factor nanobody is shown as SEQ ID No.9, the amino acid sequence of the VHH chain of the nanobody is shown as SEQ ID No.18, the amino acid sequence of the VHH chain consists of 4 framework regions FR and 3 complementarity determining regions CDR, and the framework regions FR comprise FR1 shown as SEQ ID No.19, FR2 shown as SEQ ID No.20, FR3 shown as SEQ ID No.21 and FR4 shown as SEQ ID No. 22; the complementarity determining region CDR includes CDR1 shown in SEQ ID No.23, CDR2 shown in SEQ ID No.24, CDR3 shown in SEQ ID No. 25.
Example 5
Clone No.22 of anti-virulence factor nanobody was expressed in e.coli WK6 and purified:
(1) The previously optimal 22-clone plasmid pMECS-ROP18-22 (shown in FIG. 4, constructed in example 2, ROP18 representing the name of the antigen, and 22 representing the clone number) for antigen recognition was electrotransformed into E.coli WK6, and spread on LB plates containing ampicillin and glucose, and cultured overnight at 37 ℃;
(2) Single colony is selected and inoculated in 5mL LB culture solution containing ampicillin, and shake culture is carried out at 37 ℃ for overnight;
(3) Inoculating 1mL of overnight strain into 330mL of TB culture solution, performing shake culture at 37 ℃, adding 1mM IPTG to the final concentration when the OD value reaches 0.6-1.0, and performing shake culture at 28 ℃ overnight;
(4) Centrifuging and collecting bacteria;
(5) Obtaining an antibody crude extract by using a permeation method;
(6) The nano antibody with the purity of more than 90 percent can be prepared by nickel column ion affinity chromatography, and the result is shown in figure 5.
Example 6
Affinity test of anti-virulence factor nanobody clone No.22 nanobody:
(1) The affinity between the nanobody and the antigen is tested by using a Biacore T100 protein interaction analysis system instrument and using a template method carried by the instrument (wherein the sample injection condition is set to 60s,30 mu L/min, the dissociation time is 600s, and the regeneration condition is 30s,30 mu L/min).
(2) The signal condition of the 2-1 channel is observed at any time. The affinity test procedure takes approximately 200min.
(3) Binding dissociation curves for several concentration gradients were selected and all curves were fitted using a 1:1 binding pattern to obtain the affinity values and important parameters such as binding and dissociation constants (see Table 5). E in the numerical values represents scientific counting methods, for example 2.716E-09 represents 2.716 ×10 -9 The affinity value of toxoplasma virulence factor nano antibody No.22 clone is up to 2.716E-09.
TABLE 5
| Sample numbering | Binding constant | Dissociation constant | Affinity for |
| 22 | 1.214E+04 | 3.045E-0.5 | 2.716E-09 |
Example 7
The specific toxoplasma virulence factor nano antibody obtained by screening is used for enzyme-linked immunosorbent assay, and the steps are as follows:
(1) To detect whether the screened specific toxoplasma virulence factor nanobody can recognize toxoplasma antigen, toxoplasma total antigen (2 mug/mL) is added into a 96-well ELISA plate, 100 mug/well, and the temperature is 4 ℃ overnight;
(2) After washing the plate 3 times by PBST, sealing the ELISA plate by 3% skimmed milk powder, and acting Ih at 37 ℃; washing the plate with PBST for 3 times, adding virulence factor nanobody with dilution of 1:100, and acting at 37 ℃ for 2 hours; after PBST plates were washed 3 times, horseradish peroxidase (HRP) labeled murine anti-His monoclonal antibody (Abcam) was added to act Ih; after washing the plates 3 times with PBST, TMB substrate solution was added for detection. And negative controls were made with schistosoma japonicum (Schistosoma japonicum, sj), cercaria mansoni (Sparganum mansoni, sm), plasmodium falciparum (Plasmodium falciparum, pf), trypanosoma evanescens (Trypanosoma evansi, te), cryptosporidium parvum (Cryptosporidium parvum, cp) whole worm antigens.
(3) As shown in fig. 6, the virulence factor nanobody can specifically recognize toxoplasma gondii whole worm antigen, but does not react with the schistosoma japonicum, cercaria mansoni, plasmodium falciparum, trypanosoma evans and cryptosporidium parvum whole worm antigens of the control group.
(4) The toxoplasma virulence factor nano antibody can be used for further developing a reagent for detecting toxoplasma antigen.
Sequence listing
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tacgcagact ccgtgaaggg ccgattcacc atctccagag acaacgccaa gagcactctg 240
tatttgcaca tgaacagcct gaaacctgat gacactgcca tgtactcctg tgcggcgggt 300
cggcctgctc cgtcgctcgg ttataagtgc atgtataact ctcgacacaa ggggacccag 360
gtcaccgtct cctca 375
<210> 8
<211> 375
<212> DNA
<213> dromedarion (Camelus dromedarius)
<400> 8
caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgcgactc 60
tcctgtgtag cctctcgata cacctacacc tacagtgcct actgcttggg ctggttccgc 120
caggctccag ggaaggagcg cgaggaaatc gcgattattg atagtgatgg tggcgctcgc 180
tacgcagact ccgtgaaggg ccgattcacc atctccagag acaacgccaa gagcactctg 240
tatttgcaca tgaacagcct gaaacctgat gacactgcca tgtactcctg tgcggcgggt 300
cggcctgctc cgtcgctcgg ttataagtgc atgtataact ctcgacacaa ggggaccctg 360
gtcaccgtct cctca 375
<210> 9
<211> 375
<212> DNA
<213> dromedarion (Camelus dromedarius)
<400> 9
caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgtag cctctcgata cacctacacc tacagtacct actgcttggg ctggttccgc 120
caggctccag ggaaggagcg cgaggaaatc gcgattattg atagtgatgg tggcgctcgc 180
tacgcagact ccgtgaaggg ccgattcacc atctccagag acaacgccaa gagcactctg 240
tatttgcaca tgaacagcct gaaacctgat gacactgcca tgtactcctg tgcggcgggt 300
cggcctgctc cgtcgctcgg ttataagtgc atgtataact ctcgacacaa ggggaccctg 360
gtcactgtct cctca 375
<210> 10
<211> 125
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 10
His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Tyr Thr Tyr Ser
20 25 30
Ala Tyr Cys Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Glu Ile Ala Ile Ile Asp Ser Asp Gly Gly Ala Arg Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu
65 70 75 80
Tyr Leu His Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Met Tyr Ser
85 90 95
Cys Ala Ala Gly Arg Pro Ala Pro Ser Leu Gly Tyr Lys Cys Met Tyr
100 105 110
Asn Ser Arg His Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 11
<211> 125
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 11
His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Tyr Thr Tyr Ser
20 25 30
Ala Tyr Cys Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Glu Ile Ala Ile Ile Asp Ser Asp Gly Gly Ala Arg Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu
65 70 75 80
Tyr Leu His Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Met Tyr Ser
85 90 95
Cys Ala Ala Gly Arg Pro Ala Pro Ser Leu Gly Tyr Lys Cys Met Tyr
100 105 110
Asn Ser Arg His Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 12
<211> 125
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 12
His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Tyr Thr Tyr Ser
20 25 30
Thr Tyr Cys Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Glu Ile Ala Ile Ile Asp Ser Asp Gly Gly Ala Arg Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu
65 70 75 80
Tyr Leu His Met Asn Ser Leu Asn Pro Asp Asp Thr Ala Met Tyr Ser
85 90 95
Cys Ala Ala Gly Arg Pro Ala Pro Ser Tyr Gly Tyr Lys Cys Val Tyr
100 105 110
Asn Ser Arg His Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 13
<211> 125
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 13
His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gln
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Tyr Thr Tyr Ser
20 25 30
Ala Tyr Cys Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Glu Ile Ala Ile Ile Asp Ser Asp Gly Gly Ala Arg Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu
65 70 75 80
Tyr Leu His Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Met Tyr Ser
85 90 95
Cys Ala Ala Gly Arg Pro Ala Pro Ser Leu Gly Tyr Lys Cys Met Tyr
100 105 110
Asn Ser Arg His Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 14
<211> 125
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 14
His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Tyr Thr Tyr Ser
20 25 30
Thr Tyr Cys Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Glu Ile Ala Ile Ile Asp Ser Asp Gly Gly Ala Arg Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu
65 70 75 80
Tyr Leu His Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Met Tyr Ser
85 90 95
Cys Ala Ala Gly Arg Pro Ala Pro Ser Leu Gly Tyr Lys Cys Met Tyr
100 105 110
Asn Ser Arg His Lys Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 15
<211> 125
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 15
His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Tyr Thr Tyr Ser
20 25 30
Thr Tyr Cys Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Glu Ile Ala Ile Ile Asp Ser Asp Gly Gly Ala Arg Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu
65 70 75 80
Tyr Leu His Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Met Tyr Ser
85 90 95
Cys Ala Ala Gly Arg Pro Ala Pro Ser Tyr Gly Tyr Lys Cys Met Tyr
100 105 110
Asn Ser Arg His Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 16
<211> 125
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 16
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Tyr Thr Tyr Ser
20 25 30
Ala Tyr Cys Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Glu Ile Ala Ile Ile Asp Ser Asp Gly Gly Ala Arg Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu
65 70 75 80
Tyr Leu His Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Met Tyr Ser
85 90 95
Cys Ala Ala Gly Arg Pro Ala Pro Ser Leu Gly Tyr Lys Cys Met Tyr
100 105 110
Asn Ser Arg His Lys Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 17
<211> 125
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 17
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Tyr Thr Tyr Ser
20 25 30
Ala Tyr Cys Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Glu Ile Ala Ile Ile Asp Ser Asp Gly Gly Ala Arg Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu
65 70 75 80
Tyr Leu His Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Met Tyr Ser
85 90 95
Cys Ala Ala Gly Arg Pro Ala Pro Ser Leu Gly Tyr Lys Cys Met Tyr
100 105 110
Asn Ser Arg His Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 18
<211> 125
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 18
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Tyr Thr Tyr Ser
20 25 30
Thr Tyr Cys Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Glu Ile Ala Ile Ile Asp Ser Asp Gly Gly Ala Arg Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu
65 70 75 80
Tyr Leu His Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Met Tyr Ser
85 90 95
Cys Ala Ala Gly Arg Pro Ala Pro Ser Leu Gly Tyr Lys Cys Met Tyr
100 105 110
Asn Ser Arg His Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 19
<211> 25
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 19
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser
20 25
<210> 20
<211> 12
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 20
Arg Tyr Thr Tyr Thr Tyr Ser Thr Tyr Cys Leu Gly
1 5 10
<210> 21
<211> 15
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 21
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Glu Ile Ala Ile
1 5 10 15
<210> 22
<211> 7
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 22
Ile Asp Ser Asp Gly Gly Ala
1 5
<210> 23
<211> 38
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 23
Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Ser Thr Leu Tyr Leu His Met Asn Ser Leu Lys Pro Asp Asp
20 25 30
Thr Ala Met Tyr Ser Cys
35
<210> 24
<211> 18
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 24
Ala Ala Gly Arg Pro Ala Pro Ser Leu Gly Tyr Lys Cys Met Tyr Asn
1 5 10 15
Ser Arg
<210> 25
<211> 10
<212> PRT
<213> dromedarion (Camelus dromedarius)
<400> 25
His Lys Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 26
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ctagctagca tgttttcggt acagcggc 28
<210> 27
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
cgcggatcct tattctgtgt ggagatgttc c 31
Claims (9)
1. A nanobody against toxoplasma virulence factor ROP18, the VHH chain of which comprises three complementarity determining regions CDR1, CDR2 and CDR3, characterized in that the amino acid sequence of CDR1 is shown in SEQ ID No.23, the amino acid sequence of CDR2 is shown in SEQ ID No.24, and the amino acid sequence of CDR3 is shown in SEQ ID No. 25.
2. The nanobody of claim 1, wherein the amino acid sequence of the VHH chain is set forth in SEQ ID No. 18.
3. The nanobody of claim 2, comprising two of said VHH chains.
4. A gene encoding the nanobody of any of claims 1-3.
5. The gene according to claim 4, wherein the nucleotide sequence is shown in SEQ ID No. 9.
6. A recombinant expression vector comprising the gene according to claim 5.
7. A genetically engineered cell obtained by introducing the recombinant expression vector of claim 6 into a host cell.
8. The genetically engineered cell of claim 7, wherein the host cell is an e.
9. Use of the nanobody according to any one of claims 1 to 3 for the preparation of a toxoplasma detection kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111294817.0A CN114133451B (en) | 2021-11-03 | 2021-11-03 | Nanometer antibody for toxoplasma virulence factor ROP18 and coding sequence and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111294817.0A CN114133451B (en) | 2021-11-03 | 2021-11-03 | Nanometer antibody for toxoplasma virulence factor ROP18 and coding sequence and application thereof |
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| CN114133451B true CN114133451B (en) | 2023-07-25 |
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| CN (1) | CN114133451B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108484766A (en) * | 2018-03-23 | 2018-09-04 | 浙江省医学科学院 | The nano antibody and its encoding gene of a kind of resisting toxoplasmosis Thioredoxin and application |
| CN108503707A (en) * | 2018-03-23 | 2018-09-07 | 浙江省医学科学院 | The nano antibody and its encoding gene of a kind of resisting toxoplasmosis SAG1 and application |
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2021
- 2021-11-03 CN CN202111294817.0A patent/CN114133451B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108484766A (en) * | 2018-03-23 | 2018-09-04 | 浙江省医学科学院 | The nano antibody and its encoding gene of a kind of resisting toxoplasmosis Thioredoxin and application |
| CN108503707A (en) * | 2018-03-23 | 2018-09-07 | 浙江省医学科学院 | The nano antibody and its encoding gene of a kind of resisting toxoplasmosis SAG1 and application |
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|---|---|
| CN114133451A (en) | 2022-03-04 |
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