CN114163527A - Nano antibody aiming at toxoplasma clavuligerus protein 5 and coding sequence and application thereof - Google Patents

Nano antibody aiming at toxoplasma clavuligerus protein 5 and coding sequence and application thereof Download PDF

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CN114163527A
CN114163527A CN202111294797.7A CN202111294797A CN114163527A CN 114163527 A CN114163527 A CN 114163527A CN 202111294797 A CN202111294797 A CN 202111294797A CN 114163527 A CN114163527 A CN 114163527A
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nano antibody
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toxoplasma gondii
toxoplasma
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CN114163527B (en
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郑斌
丁豪杰
付益修
卓洵辉
孔庆明
陆绍红
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Hangzhou Medical College
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C—CHEMISTRY; METALLURGY
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • 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 nano antibody aiming at toxoplasma clavulirus protein 5, a coding sequence and application thereof, wherein the VHH chain amino acid sequence of the nano antibody is shown as SEQ ID No. 10. The nanobody comprises two VHH chains. The nano antibody aiming at the toxoplasma gondii rod-shaped body protein 5 is prepared into a rod-shaped body protein 5 specific nano antibody library after the toxoplasma gondii rod-shaped body protein 5 antigen is immunized camel, and the phage display technology is utilized to screen the rod-shaped body protein 5 specific nano antibody with high affinity, so that the nano antibody has high water solubility and conformation stability, and the nano antibody of the toxoplasma gondii rod-shaped body protein 5 can be specifically combined with the toxoplasma gondii antigen, and can be used for preparing a toxoplasma gondii detection kit.

Description

Nano antibody aiming at toxoplasma clavuligerus protein 5 and coding sequence and application thereof
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a nano antibody aiming at toxoplasma clavulirus protein 5, and a coding sequence and application thereof.
Background
Toxoplasmosis (toxoplasmosis) is a zoonotic opportunistic parasitic disease caused by the human body parasitizing toxoplasma gondii, and is distributed worldwide. The toxoplasma infection of the patient with normal immune function is usually self-limited, has no obvious clinical manifestation, and is mostly in a recessive infection state. Infection of toxoplasma by patients with low immune function, such as AIDS patients and tumor patients, often leads to serious consequences, mainly manifested as encephalitis, retinochoroiditis or mental abnormality, and the like, and serious patients can cause death. Infection of a pregnant woman with toxoplasma can be spread vertically through the placenta, causing poor pregnancy outcome, or causing congenital toxoplasmosis in the infant. With the change of modern dietary habits and the increase of domestic pets, the infection rate of toxoplasmosis in China is on a rising trend year by year.
At present, the diagnosis methods of toxoplasmosis are mainly classified into etiology, immunology and molecular biology. The pathogenic detection needs long time and has poor sensitivity and specificity. The molecular diagnosis technology has the advantage of high sensitivity, but the molecular diagnosis technology is easy to form aerosol pollution and is difficult to remove, most laboratories in China cannot be strictly divided at present, and certain false positive exists. The immunological ELISA detection method is simple, convenient and feasible, has high sensitivity, is easy to standardize and automate, and is a good detection means with the widest application. However, the national drug administration approved toxoplasmosis detection reagents only for the detection of IgG and IgM antibodies. The antibody level measurement alone cannot fully reflect the toxoplasma infection condition, and is very likely to cause missed diagnosis of a part of patients with the existing diseases. Therefore, there is a need to develop a toxoplasma antigen detection method, which combines the detection results of toxoplasma IgG and IgM antibodies to determine the course of disease.
Toxoplasma claviform protein 5 (ROP 5) is secreted by the anterior secretory organ, and is located on the theca of Nanohong to interact with host cytoplasm, so that the immune response factor of host cells is disordered. ROP5 is an important virulence factor, and in mouse models, knockout of ROP5 results in loss of virulence in virulent strains of Toxoplasma gondii. In addition, research shows that ROP5 as Toxoplasma gondii secretory protein has good immunogenicity, immunoprotection and diagnostic value.
A specific antibody which is naturally deleted of heavy chains and still has biological activity in camelids (alpaca and camel) and cartilaginous fish bodies is called a single domain antibody, an antigen binding site VHH (nano antibody) of the single domain antibody has independent antigen recognition capacity, and the single domain antibody has the advantages of small volume, high solubility, strong specificity, capability of being expressed in bacteria in large quantity and the like, and the nano antibody has wider application prospect in the medical fields of diagnosis and treatment of diseases, drug development and the like. Therefore, the invention utilizes the technology and the nano antibody specific to the Toxoplasma gondii ROP5 antigen obtained by screening to detect Toxoplasma gondii infection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a nano antibody for toxoplasma gondii rod-shaped body protein 5, a coding sequence and application thereof, wherein the nano antibody for toxoplasma gondii rod-shaped body protein 5 can be specifically combined with toxoplasma gondii antigen and can be used for preparing a toxoplasma gondii detection kit.
The invention provides a VHH chain of a nano antibody aiming at toxoplasma gondii clavulans protein 5, which comprises three complementarity determining regions CDR1, CDR2 and CDR3, and is characterized in that the amino acid sequence of CDR1 is shown as SEQ ID No.15, the amino acid sequence of CDR2 is shown as SEQ ID No.16, and the amino acid sequence of CDR3 is shown as SEQ ID No. 17.
Specifically, the amino acid sequence of the VHH chain is shown as SEQ ID No. 10.
The invention also provides a nano antibody aiming at the toxoplasma clavulirus protein 5, which comprises two VHH chains.
The invention further provides genes encoding a VHH chain as described, or encoding a nanobody as described.
Preferably, the nucleotide sequence of the gene is shown in SEQ ID No. 5.
The invention also provides a recombinant expression vector containing the gene.
The invention also provides a gene engineering cell which is obtained by introducing the recombinant expression vector into a host cell.
Preferably, the host cell is an escherichia coli, yeast or CHO cell.
The invention also provides application of the nano antibody in preparation of a toxoplasma gondii detection kit.
The nano antibody aiming at the toxoplasma gondii claviform protein 5 is prepared into an ROP5 specific nano antibody library after the camel is immunized by the toxoplasma gondii ROP5 antigen, the high-affinity ROP5 specific nano antibody is screened by utilizing a phage display technology, the high-water solubility and the conformational stability are realized, and the toxoplasma gondii ROP5 nano antibody can be specifically combined with the toxoplasma gondii antigen, so that the toxoplasma gondii detection kit can be prepared.
Drawings
FIG. 1 is a SDS-PAGE result of expression and purification electrophoresis detection of recombinant ROP5, wherein M is protein Marker, and 1 is purified recombinant Toxoplasma gondii ROP 5.
FIG. 2 is a PCR identification positive rate chart of toxoplasma ROP5 nano antibody library colonies, wherein M is standard DNA Marker DL2000, and lanes 1-20 represent 20 colonies respectively.
FIG. 3 is a diagram of the phylogenetic tree analysis of 5 different clones of ROP5 nanometer antibody.
FIG. 4 is a schematic structural diagram of clone No. 32 plasmid PMECS-ROP5-32 of Toxoplasma gondii ROP5 nano antibody.
Fig. 5 is a diagram showing the results of the electrophoresis detection of the expression and purification of anti-ROP 5 nanobody, lane 1 shows the electrophoresis result of the Toxoplasma gondii ROP5 nanobody before purification, and lane 2 shows the electrophoresis result of the nanobody against Toxoplasma gondii ROP5 after purification.
Figure 6 is a ROP5 nm antibody specific ELISA assay.
Detailed Description
Example 1
Recombinant Toxoplasma gondii ROP5, the steps are as follows:
(1) extracting total RNA of Toxoplasma gondii by Trizol method, reverse transcribing to total cDNA, designing PCR amplification primer according to Toxoplasma gondii ROP5 gene sequence (GenBank number: DQ116423) on NCBI;
forward：5’-GGATCCTTGAAATTGGTGGAGCCTCT；
reverse：5’-CTCGAGTCAGAGGAACTCTGGCGTCT。
(2) performing PCR amplification to obtain an ROP5 target gene fragment with the fragment size of 885bp, and constructing a pET28a-ROP5 recombinant vector;
(3) the pET28a-ROP5 recombinant vector transforms competent cells E.coli BL21(DE3) by a heat shock method, stays overnight at 37 ℃, induces with IPTG, expresses recombinant protein, collects thalli, carries out ultrasonic disruption, purifies by a Ni-NTA nickel column, and verifies that the recombinant ROP5 obtains expression by SDS-PAGE (figure 1).
Example 2
Constructing a toxoplasma specific nano antibody library, comprising the following steps:
(1) firstly, Toxoplasma gondii ROP5 antigen is purified, then 1mg of Toxoplasma gondii ROP5 and Freund's adjuvant are mixed in equal volume, a healthy adult dromedarius camel (Camelus dromedarius) is immunized once a week and 5 times in total, and B cells are stimulated to express antigen-specific nano-antibody;
(2) after 5 times of immunization, extracting 100mL camel peripheral blood lymphocytes and extracting total RNA;
(3) synthesizing cDNA through reverse transcription, and amplifying total VHH by utilizing nested PCR;
(4) digesting 20 mu g of phage display vector PMECS and 10 mu g of VHH by using restriction enzymes PstI and NotI and connecting the two fragments to obtain a connecting product;
(5) the ligation product is electrically transformed into competent Escherichia coli TG1, a Toxoplasma gondii ROP5 nano antibody library is constructed and the library capacity is determined, wherein the size of the library capacity is 2.6 multiplied by 109(Table 1).
(6) 20 single colonies were randomly picked from the plates counted from the library colonies and colony PCR was performed to identify the insertion rate of the VHH gene. The colony PCR results are shown in FIG. 2, and the 20 single colonies in the experimental group amplified a band of 500-750bp, with a 100% positive rate, from which it can be seen that the actual library capacity of the initial library is 2.6X 109. Further packaging into foodThe titer of the phage nano antibody library constructed by plaque counting is 1.83 multiplied by 10 by applying a double-layer agar plate method to the thallus nano antibody library13pfu/mL (Table 1).
TABLE 1
Library capacity Positive rate Phage library titer
2.6×109 20/20 1.83×1013pfu/mL
Example 3
Screening Toxoplasma gondii ROP5 specific nano-antibody, the steps are as follows:
(1) 100 μ L of this was dissolved in 100mM NaHCO3Toxoplasma gondii ROP5 coating solution (20 μ g/mL) with pH8.2, placing on CORNING ELISA plate at 4 deg.C overnight;
(2) adding 100 μ L of 3% skimmed milk the next day, sealing at room temperature for 2 hr;
(3) after 2h, 100. mu.L of 10 was added11pfu contains phage of toxoplasma ROP5 nanometer antibody library, and acts for 1h at room temperature;
(4) washing 10 times with 0.05% PBS + Tween-20 in the first round of panning/20-25 times in the second round of panning to remove the phage bound non-specifically;
(5) phages specifically bound to Toxoplasma gondii ROP5 were dissociated with 100mM TEA (triethylamine) and infected with E.coli TG1 in logarithmic growth phase, cultured at 37 ℃ for 1h, phages were generated and purified for the next round of screening, and the same screening procedure was repeated for 2-3 rounds to gradually obtain enrichment, with the results shown in Table 2.
TABLE 2
Figure BDA0003336155020000041
Figure BDA0003336155020000051
Example 4
Screening of specific single positive clones by phage enzyme-linked immunosorbent assay (ELISA) was carried out as follows:
(1) from the plates containing the phagemids after the 3-4 rounds of selection described above, 40 single colonies were picked and inoculated into TB medium containing 100. mu.g/mL ampicillin, grown to a logarithmic phase, followed by incubation with IPTG at a final concentration of 1mM overnight at 28 ℃.
(2) Obtaining a crude antibody by using an osmosis method, transferring the antibody into an ELISA plate coated by an antigen, and standing for 1h at room temperature;
(3) unbound antibody was washed away with PBST, mouse anti-His antibody (Abcam) was added, and left at room temperature for 1 h;
(4) unbound antibody was washed away with PBST and Goat Anti-Mouse IgG-HRP (Goat Anti Mouse HRP labeled antibody, Abcam) was added;
(5) washing away unbound antibody with PBST, adding TMB color developing solution, and reading OD on enzyme labeling instrument450A value;
(6) when the OD value of the sample hole is more than 2.1 times of the OD value of the control hole, the sample hole is judged to be a positive cloning hole;
(7) the bacteria of the positive cloning wells are transferred and shaken in a TB culture medium containing 100 mug/mL ampicillin, and plasmids are extracted for sequencing;
(8) the amino acid sequence of the VHH chain, consisting of the framework region FR and the complementarity determining region CDR. From the sequencing results, Vector was applied
Figure BDA0003336155020000052
And
Figure BDA0003336155020000053
the software analyzed individual clones, and identified strains with identical CDR1, CDR2, and CDR3 sequences as identical clones, while sequences that differ as different clones. 5 different clone strains (table 3) are obtained in total, the nucleotide sequences are shown as SEQ ID NO. 1-5, and the amino acid sequences are shown as SEQ ID NO. 6-10. Further evolutionary tree analysis was performed to preliminarily determine the epitope heterology against which the positive clones were directed (fig. 3).
TABLE 3
Cloning OD450(coating ROP5) OD450(not coated)
11 1.361 0.095
16 1.541 0.102
24 1.690 0.096
28 1.518 0.095
32 1.873 0.090
(9) A total of 5 different clones were obtained from 40 positive clones randomly selected as indicated by phage ELISA results. As shown in Table 3, among them, clone No. 32 recognized the best antigen. The gene sequence of the clone No. 32 ROP5 nano antibody is shown in SEQ ID No.5, the amino acid sequence of the VHH chain of the nano antibody is shown in SEQ ID No.10, each nano antibody molecule has a VHH chain, the amino acid sequence of the VHH chain consists of 4 framework regions FR and 3 complementarity determining regions CDR, the framework regions FR comprise FR1 shown in SEQ ID No.11, FR2 shown in SEQ ID No.12, FR3 shown in SEQ ID No.13 and FR4 shown in SEQ ID No. 14; the complementarity determining region CDR includes CDR1 shown in SEQ ID No.15, CDR2 shown in SEQ ID No.16 and CDR3 shown in SEQ ID No. 17.
Example 5
Expressing and purifying a No. 32 clone strain aiming at ROP5 nano antibody in escherichia coli WK 6:
(1) the plasmid pMECS-ROP5-32 (shown in FIG. 4, constructed in example 2, ROP5 indicates the name of the antigen, and 32 indicates the number of the clone) of clone No. 32, which had been previously optimized for antigen recognition, was electrically transformed into E.coli WK6, spread on LB plate containing ampicillin and glucose, and cultured overnight at 37 ℃;
(2) selecting a single colony, inoculating the single colony in 5mL LB culture solution containing ampicillin, and carrying out shake culture at 37 ℃ overnight;
(3) inoculating 1mL of overnight strain into 330mL of TB culture solution, carrying out shake culture at 37 ℃, adding 1mM IPTG (isopropyl thiogalactoside) with final concentration when the OD value reaches 0.6-1.0, and carrying out shake culture at 28 ℃ overnight;
(4) centrifuging and collecting bacteria;
(5) obtaining antibody crude extract by using an osmosis 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
The enzyme-linked immunosorbent assay is carried out by using the screened specific Toxoplasma gondii ROP5 nano antibody, and the steps are as follows:
(1) in order to detect whether the screened specific Toxoplasma gondii ROP5 nano antibody can recognize Toxoplasma gondii antigen, the whole Toxoplasma gondii antigen (2 mug/mL) is added into a 96-hole enzyme label plate, 100 mug/hole, and the temperature is kept overnight at 4 ℃;
(2) after washing the plate for 3 times by PBST, sealing the ELISA plate by 3% skimmed milk powder, and acting for 1h at 37 ℃; washing the plate 3 times with PBST, adding ROP5 nanometer antibody with dilution of 1:100, and reacting at 37 deg.C for 2 h; after PBST washing for 3 times, adding a Horse Radish Peroxidase (HRP) marked mouse anti-His monoclonal antibody (Abcam) for acting for 1 h; after PBST washing plates 3 times, TMB substrate solution is added for detection. And using Schistosoma japonicum (Sj), Schistosoma mansoni (Sm), Plasmodium falciparum (Pf), Trypanosoma evansi (Te) and Cryptosporidium parvum (Cp) whole-worm antigen as a comparison, and using no coating antigen as a negative control.
(3) As shown in FIG. 6, ROP5 nano-antibody can specifically recognize toxoplasma gondii whole worm antigen, but has no reaction with control group schistosoma japonicum, Schistosoma mansoni, Plasmodium falciparum, trypanosoma evansi and Cryptosporidium parvum whole worm antigen.
(4) The reagent for detecting toxoplasma gondii antigen can be further developed by applying the toxoplasma gondii ROP5 nano antibody.
Sequence listing
<110> Hangzhou college of medicine
<120> nano antibody aiming at toxoplasma clavulirus protein 5, and coding sequence and application thereof
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gaaatgacca gcctgaaacc tgaggacacg gccatgtatt actgcgcggc aggggaacca 300
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His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Asn Tyr Thr Val Ser Asn Asn
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Val Ile Gly Ser Ala Gly Ser Ser Phe Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Thr Tyr Leu
65 70 75 80
Glu Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Gly Glu Pro Gly Ser Phe Ala Trp Trp Trp Leu Leu Arg Ser Arg
100 105 110
Glu Phe Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 7
<211> 113
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
<400> 7
His Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Arg Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Ser
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val
35 40 45
Ser Ile Ile Tyr Ser Ser Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Leu Asp Ser Leu Lys Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Arg
85 90 95
Glu Tyr Ile Ser Gly Gln Arg Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser
<210> 8
<211> 127
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
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His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Asn Tyr Thr Val Ser Asn Asn
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Val Ile Gly Ser Ser Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu
65 70 75 80
Glu Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Gly Glu Pro Gly Thr Phe Ala Trp Trp Trp Leu Leu Arg Ser Arg
100 105 110
Glu Phe Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 9
<211> 127
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
<400> 9
His Val Gln Leu Val Glu Ser Gly Gly Gly Pro Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Asn Tyr Thr Val Ser Asn Asn
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Val Ile Gly Ser Ser Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu
65 70 75 80
Glu Met Thr Ser Leu Lys Pro Gln Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Gly Glu Pro Gly Ser Phe Ala Trp Trp Trp Leu Leu Arg Ser Arg
100 105 110
Glu Phe Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 10
<211> 127
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
<400> 10
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Lys Tyr Thr Val Ser Asn Asn
20 25 30
Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Val Ile Gly Ser Ser Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu
65 70 75 80
Glu Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Gly Glu Pro Gly Ser Phe Ala Trp Trp Trp Leu Leu Arg Ser Arg
100 105 110
Glu Phe Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 11
<211> 25
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
<400> 11
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 12
<211> 15
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
<400> 12
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Val
1 5 10 15
<210> 13
<211> 38
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
<400> 13
Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Ala Tyr Leu Glu Met Thr Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 14
<211> 11
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
<400> 14
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 15
<211> 10
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
<400> 15
Lys Tyr Thr Val Ser Asn Asn Tyr Met Gly
1 5 10
<210> 16
<211> 7
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
<400> 16
Ile Gly Ser Ser Gly Ser Thr
1 5
<210> 17
<211> 21
<212> PRT
<213> dromedarius camel (Camelus dromedarius)
<400> 17
Ala Ala Gly Glu Pro Gly Ser Phe Ala Trp Trp Trp Leu Leu Arg Ser
1 5 10 15
Arg Glu Phe Ala Tyr
20
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggatccttga aattggtgga gcctct 26
<210> 19
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ctcgagtcag aggaactctg gcgtct 26

Claims (9)

1. A VHH chain of a nanobody against Toxoplasma gondii clavulans protein 5, comprising three complementarity determining regions CDR1, CDR2 and CDR3, characterized in that the amino acid sequence of CDR1 is shown as SEQ ID No.15, the amino acid sequence of CDR2 is shown as SEQ ID No.16, and the amino acid sequence of CDR3 is shown as SEQ ID No. 17.
2. The VHH chain according to claim 1, characterized in that the amino acid sequence is according to SEQ ID No. 10.
3. A nanobody against toxoplasma clavulirus protein 5 comprising two VHH chains according to claim 2.
4. A gene encoding a VHH chain according to claim 1 or 2 or a nanobody according to claim 3.
5. The gene of claim 4, wherein the nucleotide sequence is as shown in SEQ ID No. 5.
6. A recombinant expression vector comprising the gene of 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.coli, yeast, or CHO cell.
9. Use of the nanobody of claim 3 in the preparation of toxoplasma gondii detection kits.
CN202111294797.7A 2021-11-03 2021-11-03 Nanometer antibody for toxoplasma rod protein 5, and coding sequence and application thereof Active CN114163527B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
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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