ApuA protein antigen polypeptide and application thereof
A technology of protein antigen and antigen polypeptide, which is applied in the direction of bacterial antigen components, anti-enzyme immunoglobulin, anti-bacterial drugs, etc., can solve the problems of non-specificity of antibodies, low protein yield, low protein purity, etc., and achieve good immunogenicity, good specific effect
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Embodiment 1
[0031] Embodiment 1 Design and synthesis of Streptococcus suis ApuA protein-specific antigen polypeptide
[0032]1. Obtain the amino acid sequence (accession number: CAZ52647.1) and related information of the ApuA protein of Streptococcus suis type 2 in the GenBank database. The length of the protein is 2094 amino acids (amino acid, aa).
[0033] 2. Analyze the secondary structure, hydrophilicity and hydrophobicity, and antigenicity of the amino acid sequence of ApuA, and obtain 3 polypeptide fragments with high comprehensive evaluation ( figure 1 ): Pep1 (TGKSYQAIEKDGKW, SEQ ID NO. 1), Pep2 (VYDKDNGYYYETKLD, SEQ ID NO. 2) and Pep3 (IQQKDYSFKDLKNQ, SEQ ID NO. 3). Pep1 has good specificity, and no homology of this fragment in Streptococcus suis or other bacteria was found; while Pep2 and Pep3 failed to avoid homologous proteins or protein superfamily in Streptococcus suis or other bacteria, and the specificity was poor . After the above analysis, Pep1 (871-884aa) was selected...
Embodiment 2
[0035] Embodiment 2 animal immunization
[0036] 1. The experimental animals are New Zealand white rabbits with a weight of about 2.0kg. Before immunization, negative blood was collected from the ear vein, left at room temperature for 2 hours, centrifuged at 5000rpm for 10 minutes, and negative serum was prepared and stored at -80°C. Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma.
[0037] 2. See Table 1 for the immunization program. Take 400 μg or 200 μg of Pep1-KLH immunogen (3 mg / mL), dilute it to 200-500 uL with normal saline, and then add an equal volume of Freund’s adjuvant (freund’s complete adjuvant for initial immunization, and incomplete Freund’s adjuvant for booster immunization) agent), the antigen and adjuvant were mixed with a shaker to form a water-in-oil emulsion.
[0038] Table 1 specific immunization program
[0039] Number of immunizations number of days antigen dose primary immunization Day 1 (2...
Embodiment 3
[0042] Example 3 ELISA titer detection
[0043] 1. Coating: Dilute the Pep1-BSA antigen with coating solution (sodium carbonate-sodium bicarbonate buffer, pH 9.6) to a final concentration of 2 μg / mL, add to the microplate, 100 μL per well, overnight at 4 °C.
[0044] 2. Blocking: Wash 3 times with washing solution PBS-T (PBS buffer containing 0.05% Tween), add PBS buffer containing 1% skimmed milk powder to block, 200 μL per well, and incubate at 37° C. for 2 hours.
[0045] 3. Washing: Discard the blocking solution, wash 3 times with washing solution, and pat dry.
[0046] 4. Primary antibody incubation: use the blocking solution to dilute the rabbit polyantisera 2 times from 200 times to 102400 times, the blank control is the blocking solution, and the negative control is the rabbit negative serum diluted 200 times with the blocking solution; add to the enzyme label Incubate at 37°C for 1 hour at 100 μL per well.
[0047] 5. Washing: Discard the primary antibody, wash 3 ti...
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