Schistosoma japonicum thioredoxin glutathione reductase Sj TGR single-domain antibody and preparation method thereof
A single-region antibody and schistosomiasis technology, which is applied in the direction of anti-enzyme immunoglobulin, antibody, peptide library, etc., can solve the problem that anti-SjTGR single-region antibody has not been reported.
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Embodiment 1
[0066] Example 1: Recombinant Sj TGR protein immunization of Xinjiang Bactrian camels and preparation of serum antibodies
[0067] Before immunization, blood was collected from camel jugular vein, serum was separated, and stored at low temperature for later use. For the first immunization, the recombinant purified Sj TGR protein (500 μg) complete Freund's adjuvant antigen was injected subcutaneously into the neck skin of Bactrian camels in Xinjiang, followed by booster immunization with the same dose of incomplete adjuvant antigen, and the immunization interval was 2 Week, a total of 4 booster immunizations. Two weeks after the first immunization, 50 mL of blood was collected from the jugular vein of the camel, and the serum was separated. Coat the ELISA plate with carbonic acid buffer solution (10 μg / mL) of recombinant Sj TGR protein, overnight at 4°C, block with 5% skimmed milk powder PBS the next day, 37°C for 2h, and then mix with doubly diluted immune camel serum Then r...
Embodiment 2
[0075] Embodiment 2: Preparation of gene fragments encoding camel anti-Sj TGR nanobody
[0076] Anticoagulated blood from camels immunized with Sj TGR was centrifuged at 1000 rpm for 10 min to separate plasma and blood cells. Use percoll (GE Health product) to prepare camel white blood cell separation fluid with a specific gravity of 1.07, add anticoagulant blood cells to the upper layer of the white blood cell separation liquid, centrifuge at 4 °C for 30 min at a speed of 3000 g, and collect the white cell layer at the interface in a fresh In a sterile centrifuge tube, add sterile PBS to suspend and wash the cells, centrifuge at 1000 rpm for 10 min, remove the supernatant, then add PBS, wash the cells three times repeatedly, collect the cell pellet, and count.
[0077] Trizol was used to extract the total RNA of Sj TGR immunized camel peripheral blood leukocytes, and the method was as follows:
[0078] 1) Take about 1×10 7 One Sj TGR was used to immunize the peripheral ...
Embodiment 3
[0152] Example 3: Anti Sj Construction of phage display library of TGR single region antibody
[0153] The linearized phagemid pCANTab5E DNA and the DNA fragment encoding the nanobody were prepared by Sfi1 and Not1 double digestion method. The enzyme digestion reaction system is as follows:
[0154] pCANTab5E DNA fragment 30 μL
[0155] 10×Buffer4 5 μL
[0156] 100×BSA 0.5 μL
[0157] Not1-HF 1 μL
[0158] wxya 2 O 2.5 μL
[0159] 49 μL total
[0160] After mixing and centrifuging, place in a 37°C water bath to react for 4 h. Add 1 μL of Sfi1, mix well and centrifuge, then place in a water bath at 50 °C for 3 h.
[0161] Recovery of digested products: The digested products were subjected to 1% agarose gel electrophoresis, and after electrophoresis at 100 V for 60 min, the status of the digested products was observed. The gel block containing the target fragment was excised from the agaro...
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