A kind of trypanosome evangelica nanobody against vsg and its coding sequence and application
A nanobody, sequence technology, applied in the field of biotechnology or biomedicine
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Embodiment 1
[0025] This embodiment constructs the Trypanosoma evanii VSG specific nanobody library, the steps are as follows:
[0026] (1) Firstly, trypanosome evangelia VSG antigen was purified, then 1 mg of VSG antigen was mixed with Freund’s adjuvant in equal volume, and an alpaca (Alpa-Vet, www.alpa-vet.be) was immunized once a week for a total of Immunized 7 times to stimulate B cells to express antigen-specific nanobodies;
[0027] (2) After the 7 times of immunization, extract 100ml alpaca peripheral blood lymphocytes and extract total RNA;
[0028] (3) Synthesize cDNA and amplify VHH by nested PCR;
[0029] (4) Digest 20ug of phage display vector and 10ul of VHH with restriction enzymes pstI and NotI and connect the two fragments;
[0030] (5) Transform the ligation product into electroporation-competent cells TG1, construct the VSG nanobody library and measure the storage capacity, the storage capacity is 5.1×10 9 .
Embodiment 2
[0032] In this embodiment, VSG-specific nanobodies are screened, and the steps are as follows:
[0033] (1) Dissolve in 100mM NaHCO 3 20ug VSG at pH 8.2 was coated on the NUNC microtiter plate and placed overnight at 4°C;
[0034] (2) Add 100ul 3% milk the next day, and block at room temperature for 2 hours;
[0035] (3) After 2 hours, add 100ul 10 11 tfu contains the helper phage of the VSG nanobody library, and acts at room temperature for 1 hour;
[0036] (4) Wash 10 times with 0.05% PBS+Tween-20 for the first round of panning / 20-25 times for the second round / 20 times for the third round to remove non-specifically bound phages;
[0037] (5) Use 100mM TEA (triethylamine) to dissociate the phage that specifically binds to VSG, and infect Escherichia coli TG1 in logarithmic growth phase, culture at 37°C for 1 hour, produce and purify the phage for the next round of screening, the same The screening process was repeated for 3-4 rounds, and the enrichment was obtained step b...
Embodiment 3
[0039] In this example, phage enzyme-linked immunosorbent assay (ELISA) was used to screen specific single positive clones, and the steps were as follows:
[0040] (1) From the cell culture dish containing phage after the above 3-4 rounds of screening, select 96 single colonies and inoculate them in TB medium containing 100ug / ml ampicillin, grow to the logarithmic phase, and add the final concentration 1 mM IPTG, culture overnight at 28°C.
[0041] (2) Use the infiltration method to obtain the crudely extracted antibody, transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1 hour;
[0042] (3) Wash off unbound antibodies with PBST, add mouse anti-His antibody (mouse anti-HIS antibody, R&D system), and place at room temperature for 1 hour;
[0043] (4) Wash off the unbound antibody with PBST, and add anti-mouse alkaline phosphataseconjugate (goat anti-mouse AP labeled antibody, sigma).
[0044] (5) Wash off the unbound antibody with PBS...
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