CN114133451A - Nano antibody aiming at toxoplasma gondii virulence factor ROP18 as well as coding sequence and application thereof - Google Patents

Nano antibody aiming at toxoplasma gondii virulence factor ROP18 as well as coding sequence and application thereof Download PDF

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CN114133451A
CN114133451A CN202111294817.0A CN202111294817A CN114133451A CN 114133451 A CN114133451 A CN 114133451A CN 202111294817 A CN202111294817 A CN 202111294817A CN 114133451 A CN114133451 A CN 114133451A
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toxoplasma gondii
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郑斌
丁豪杰
付益修
卓洵辉
丁建祖
楼涤
孔庆明
陆绍红
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Hangzhou Medical College
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Abstract

The invention discloses a nano antibody aiming at toxoplasma gondii virulence factor ROP18, a coding sequence and application thereof, wherein the VHH chain amino acid sequence of the nano antibody is shown as SEQ ID No. 18. The nanobody comprises two VHH chains. The nano antibody aiming at toxoplasma gondii virulence factor ROP18 is prepared into a virulence factor ROP18 specific nano antibody library after the toxoplasma gondii virulence factor ROP18 antigen is immunized with camel, a phage display technology is utilized to screen the high-affinity virulence factor ROP18 specific nano antibody, the nano antibody has high water solubility and conformation stability, and the toxoplasma gondii virulence factor ROP18 nano antibody 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 gondii virulence factor ROP18 as well as coding sequence and application thereof
Technical Field
The invention relates to the field of bioengineering nanotechnology, in particular to a nano antibody aiming at toxoplasma gondii virulence factor ROP18, and a coding sequence and application thereof.
Background
Toxoplasma gondii (Toxoplasma gondii) is an obligate parasitic protozoan with a wide host population and is prevalent worldwide and can cause zoonotic parasitosis. Toxoplasma gondii is an opportunistic infection factor for patients with AIDS, organ transplantation, malignant tumor, etc. with impaired or suppressed immune function, and can cause death. Toxoplasmosis is not only an important medical disease, but also an important biological factor affecting the good prenatal and postnatal care of human beings. Whether pregnant women are infected with toxoplasma, about half of the pregnant women can have vertical maternal-fetal infection, abortion, dead fetus and congenital defects or malformations (morphological malformations, functional mental retardation) of fetus and infants, and the like.
At present, the diagnosis methods of toxoplasmosis are mainly classified into etiology, immunology and molecular biology. The etiology diagnosis method is simple, the result is reliable, but the detection rate is low, the time is consumed, and the missed diagnosis is easy. The molecular diagnosis technology has the advantages of sensitivity, specificity, rapid detection and the like, but the molecular diagnosis technology has high equipment requirement, is easy to form aerosol pollution and difficult to remove, most laboratories in China cannot be strictly partitioned at present, and certain false positive exists. The immunological diagnosis ELISA is the most widely applied technology for diagnosing toxoplasma infection at present, and has the advantages of simplicity, convenience, practicability, high sensitivity, and easy standardization and automation. At present, only IgG and IgM antibodies are approved as toxoplasmosis detection reagents by the national drug administration. 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 18 (ROP 18) is essential for toxoplasma in infection process and participates in recognition, adhesion and invasion of host cells by toxoplasma. ROP8, a serine threonine kinase, has been shown to be a key virulence factor for toxoplasma. In addition, research shows that ROP18 as Toxoplasma gondii secretory protein has good immunogenicity, immunoprotection and diagnostic value.
A specific antibody which is naturally deficient in heavy chain and still has biological activity in camelids (alpaca and camel) and cartilaginous fish is called a single domain antibody, and an antigen binding site VHH (nano antibody) of the single domain antibody has independent antigen recognition capacity, and has the advantages of small molecular weight, simple structure, stable physicochemical property and the like. The antibody can bind with some hidden antigen epitopes in the aspect of antigen-antibody binding, and is particularly suitable for targets of relatively unavailable 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 the nano antibody specific to the Toxoplasma gondii ROP18 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 virulence factor ROP18, a coding sequence and application thereof, wherein the nano antibody for toxoplasma gondii virulence factor ROP18 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 virulence factor ROP18, 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.23, the amino acid sequence of CDR2 is shown as SEQ ID No.24, and the amino acid sequence of 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 nanobody aiming at toxoplasma gondii virulence factor ROP18, 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. 9.
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 toxoplasma gondii virulence factor ROP18 is prepared into a virulence factor ROP18 specific nano antibody library after the toxoplasma gondii virulence factor ROP18 antigen is immunized with camel, a phage display technology is utilized to screen the high-affinity virulence factor ROP18 specific nano antibody, the nano antibody has high water solubility and conformation stability, and the nano antibody of the toxoplasma gondii virulence factor ROP18 can be specifically combined with the toxoplasma gondii antigen, so that the nano antibody can be used for preparing a toxoplasma gondii detection kit.
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FIG. 1 is a SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test result for verifying the expression and purification electrophoresis detection of a recombinant virulence factor ROP18, wherein M is a protein Marker, and 1 is a purified recombinant toxoplasma virulence factor ROP 18.
FIG. 2 shows the positive rate of colony PCR identification of toxoplasma gondii virulence factor ROP18 nano antibody library, wherein M is standard DNA Marker DL2000, and lanes 1-20 represent 20 colonies respectively.
FIG. 3 is a phylogenetic tree analysis diagram of 9 different clones of virulence factor nanobody.
FIG. 4 is a structural diagram of clone No.22 plasmid PMECS-ROP18-22 against toxoplasma gondii ROP 18.
FIG. 5 is a diagram showing the results of detection of toxoplasma toxigenic factor ROP18 nano antibody expression and purification electrophoresis, Lane 1 is the result of toxoplasma toxigenic factor ROP18 nano antibody electrophoresis before purification, and Lane 2 is the result of nano antibody electrophoresis of toxoplasma toxigenic factor ROP18 after purification.
FIG. 6 is a virulence factor ROP18 nanobody specific ELISA assay.
Detailed Description
Example 1
A recombinant toxoplasma virulence factor ROP18 is prepared by the following steps:
(1) toxoplasma total RNA is obtained by Trizol method, reverse transcription is carried out to obtain total cDNA, PCR amplification primers are designed according to toxoplasma virulence factor gene sequence (GenBank number: JX045330.1) on NCBI, and the upstream and downstream primers are respectively:
the upstream: 5' -CTAGCTAGCATGTTTTCGGTACAGCGGC;
the following steps: 5' -CGCGGATCCTTATTCTGTGTGGAGATGTTCC.
(2) The 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 transforms competent cells E.coli BL21(DE3) by a heat shock method, stays overnight at 37 ℃, induces with IPTG, expresses recombinant protein, collects thalli, and verifies that the recombinant virulence factor ROP18 obtains expression by SDS-PAGE after ultrasonication.
(4) The fusion protein is purified by Ni-NTA nickel column, the purified fusion protein uses Millipore ultrafiltration tube with cut-off molecular weight of 10KDa to concentrate the expressed fusion protein, the concentration of the fusion protein is increased, and the concentration of the recombinant protein reaches 2mg/mL (figure 1) through detection of Coomassie brilliant blue binding method.
Example 2
Constructing a toxoplasma gondii virulence factor specific nano antibody library, comprising the following steps of:
(1) firstly, toxoplasma virulence factor antigen is purified, then 2mg of toxoplasma virulence factor is mixed with Freund's adjuvant in equal volume (except that complete Freund's adjuvant is used for the first time and Freund's incomplete adjuvant is used for the rest several times), a healthy adult dromedarius (Camelus) is immunized once a week for 6 times in total, and B cells are stimulated to express antigen-specific nano-antibodies;
(2) after 6 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;
(5) electrically transferring the connection product to competent Escherichia coli TG1, constructing toxoplasma gondii virulence factor nano antibody library, and determining the library capacity, wherein the size of the library capacity is 3.12 × 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 positive rate of 80%, from which it can be seen that the actual library capacity of the initial library is 3.9X 109. Further packaging into phage nano antibody library, applying double-layer agar plate method, calculating by plaque count to obtain phage nano antibody library with titer of 2.11 × 1013pfu/mL (Table 1).
TABLE 1
Library capacity Positive rate Phage library titer
3.12×109 16/20 2.11×1013pfu/mL
Example 3
Screening the Toxoplasma gondii virulence factor specific nano antibody, the steps are as follows:
(1) 100 μ L of this was dissolved in 100mM NaHCO3Toxoplasma gondii virulence factor coating solution (20 mug/mL) with pH of 8.2 is placed on a NUNC enzyme label plate at 4 ℃ for 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 gondii virulence factor nano antibody library, and acts for 1h at room temperature;
(4) the first round of panning was washed 10 times with 0.05% PBS + tween-20; the second round is 20-25 times, and the phage which are not specifically bound are removed;
(5) the phage specifically bound to toxoplasma gondii virulence factor was dissociated with triethylamine (100mM) and infected with E.coli TG1 in logarithmic growth phase, cultured at 37 ℃ for 1h, phage was generated and purified for the next round of screening, the same screening process was repeated for 2-3 rounds, and enrichment was achieved step by step, with the results shown in Table 2.
TABLE 2
Figure BDA0003336154170000051
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 instrument450The value is obtained.
(6) When the OD value of the sample well is more than 2.1 times greater than that of the control well, the sample well is judged to be a positive clone well (Table 3).
TABLE 3
Figure BDA0003336154170000061
Figure BDA0003336154170000071
(7) The positive clone well was transferred to TB medium containing 100. mu.g/mL ampicillin, and the plasmid was extracted and sequenced.
(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 BDA0003336154170000072
And
Figure BDA0003336154170000073
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. Totally obtaining 9 different clone strains (table 4), wherein the nucleotide sequences are shown as SEQ ID NO. 1-9, and the amino acid sequences are shown as SEQ ID NO. 9-18. Further evolutionary tree analysis was performed to preliminarily determine the epitope heterology against which the positive clones were directed (fig. 3).
TABLE 4
Figure BDA0003336154170000074
Figure BDA0003336154170000081
(9) A total of 9 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.22 recognized the best antigen. The gene sequence of the clone No.22 anti-virulence factor nano antibody is shown as SEQ ID No.9, the amino acid sequence of the VHH chain of the nano antibody 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, 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 and CDR3 shown in SEQ ID No. 25.
Example 5
The anti-virulence factor nano antibody No.22 clone is expressed and purified in Escherichia coli WK 6:
(1) the plasmid pMECS-ROP18-22 (shown in FIG. 4, constructed in example 2, ROP18 indicates the name of the antigen, 22 indicates the number of the clone) of clone No.22, 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
Anti-virulence factor nanobody clone No.22 nanobody affinity test:
(1) the affinity between the nano antibody and the antigen is tested by using a Biacore T100 protein interaction analysis system instrument and a template method carried by the instrument (wherein the sample injection condition is set to be 60s and 30 mu L/min; the dissociation time is set to be 600s, and the regeneration condition is set to be 30s and 30 mu L/min).
(2) The signal condition of the 2-1 channel is observed at any time. The affinity testing process takes approximately 200 min.
(3) The binding and dissociation curves with several appropriate concentration gradients were selected and all curves were fitted using a 1:1 binding (binding) model to finally obtain important parameters such as affinity values and binding and dissociation constants (see table 5). E in the numerical values denotes scientific notation, e.g. 2.716E-09 denotes 2.716X 10-9The affinity value of the toxoplasma gondii virulence factor nano antibody No.22 clone strain is as high as 2.716E-09.
TABLE 5
Sample numbering Binding constant Dissociation constant Affinity of
22 1.214E+04 3.045E-0.5 2.716E-09
Example 7
The enzyme-linked immunosorbent assay is carried out by using the specific toxoplasma gondii virulence factor nano antibody obtained by screening, and the steps are as follows:
(1) in order to detect whether the screened specific toxoplasma gondii virulence factor nano antibody can identify toxoplasma gondii antigen, adding toxoplasma gondii whole insect antigen (2 mu g/mL) into a 96-hole enzyme label plate, 100 mu L/hole, and standing 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 virulence factor nano antibody with dilution of 1:100, and acting at 37 ℃ 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, the virulence factor nanobody can specifically recognize toxoplasma gondii whole worm antigen, but has no reaction with the control group schistosoma japonicum, sparganium mansoni, plasmodium falciparum, trypanosoma evansi and cryptosporidium parvum whole worm antigen.
(4) The toxoplasma toxicity factor nano antibody can be applied to further develop a reagent for detecting toxoplasma antigen.
Sequence listing
<110> Hangzhou college of medicine
<120> nano antibody aiming at toxoplasma gondii virulence factor ROP18 and coding sequence and application thereof
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<400> 4
catgtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggacagtc tctgagactc 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> 5
<211> 375
<212> DNA
<213> dromedarius camel (Camelus dromedarius)
<400> 5
catgtgcagc 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 ggggacccag 360
gtcaccgtct cctca 375
<210> 6
<211> 375
<212> DNA
<213> dromedarius camel (Camelus dromedarius)
<400> 6
catgtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgtag cctctcgata cacctatacc 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 cgtcgtacgg ttataagtgc atgtataact ctcgacacaa ggggaccctg 360
gtcaccgtct cctca 375
<210> 7
<211> 375
<212> DNA
<213> dromedarius camel (Camelus dromedarius)
<400> 7
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 ggggacccag 360
gtcaccgtct cctca 375
<210> 8
<211> 375
<212> DNA
<213> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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> dromedarius camel (Camelus dromedarius)
<400> 22
Ile Asp Ser Asp Gly Gly Ala
1 5
<210> 23
<211> 38
<212> PRT
<213> dromedarius camel (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> dromedarius camel (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> dromedarius camel (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 VHH chain of a nanobody against toxoplasma gondii ROP18 comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is shown as SEQ ID No.23, the amino acid sequence of CDR2 is shown as SEQ ID No.24, and the amino acid sequence of CDR3 is shown as SEQ ID No. 25.
2. The VHH chain according to claim 1, characterized in that the amino acid sequence is according to SEQ ID No. 18.
3. A nanobody against toxoplasma gondii factor ROP18, 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. 9.
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.
CN202111294817.0A 2021-11-03 2021-11-03 Nanometer antibody for toxoplasma virulence factor ROP18 and coding sequence and application thereof Active CN114133451B (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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