TFPR1 protein, a coding gene thereof and application thereof in immune regulation
A protein and encoding technology, applied in the biological field, can solve problems such as adverse reactions
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Embodiment 1
[0048] Example 1. Obtainment, expression and purification of protein TFPR1 and its encoding gene
[0049] 1. Obtaining the protein TFPR1
[0050] After bioinformatics analysis and prediction, a Triflin functional domain containing 145 amino acid residues was designed, and sequence 1 in the synthetic sequence table was synthesized. The gene shown in it was named TFPR1, and BamHI and Hind were added to the 5' and 3' ends respectively III, entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize the whole gene, and obtain the cloned plasmid pUC57-TFPR1 (obtained by inserting the TFPR1 shown in sequence 1 in the sequence table between the EcoRV sites of the pUC57 vector).
[0051] The protein encoded by the gene TFPR1 shown in the sequence 1 in the sequence listing is named TFPR1, and the amino acid sequence of the protein is the 13th-157th amino acid from the N-terminal of the sequence 2 in the sequence listing.
[0052] 2. Construction of recombinant vector pET-TFPR1
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Embodiment 2
[0072] Example 2. Application of TFPR1 as an immunomodulator
[0073] 1. Binding and activation test of TFPR1 and mouse splenocytes
[0074] The spleen of BALB / c mice was isolated under sterile conditions, and the mononuclear cells were isolated, and the binding of protein to spleen mononuclear cells was analyzed by flow cytometry. The specific methods are as follows:
[0075] Adjust the isolated mouse splenocytes to 10×10 6 / ml, add 50 μL to each tube, and add the renatured recombinant protein TFPR1 prepared in 5 of Example 1 with a final concentration of 10 μg / ml, mix well, incubate at room temperature for 30 min, and then wash with phosphate buffered saline (PBS) ( 1500rpm, 6min), and then FITC-labeled anti-His antibody (Miltenyi Biotec) was added.
[0076] The results showed that TFPR1 mainly binds to B cells with antigen-presenting effect in mouse splenocytes ( Figure 4 B) and macrophages ( Figure 4 D), and combined with a part of NK cells ( Figure 4 C), without b...
Embodiment 3
[0084] Example 3. Application of recombinant protein TFPR1 as vaccine adjuvant
[0085] 1. Animal immunization and grouping
[0086] Female BALB / C mice, SPF grade, 5-6 weeks old, were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences, randomly assigned to different experimental groups, and kept in SPF grade animal rooms. Using endotoxin-free ovalbumin OVA, yeast-expressed hepatitis B surface antigen (HBsAg) and HIV-1 polypeptide P5 (PKVSFEPIPIHYCAPAGFAILKCNNKTFN) as model antigens, the adjuvant activity of TFPR1 was observed. The route of inoculation is intramuscular, and the injection volume of each mouse is 200 μL. In all the following studies using aluminum adjuvant as an adjuvant control, the volume ratio of aluminum adjuvant and immunogen is 1:9 according to the optimized dose recommended in the product instructions. .
[0087] (1) Observing the adjuvant activity of TFPR1 with OVA as the model antigen: BALB / c mice aged 5 weeks wer...
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