Antigenic determinants and their applications
A Mycoplasma pneumoniae, encoding technology, applied in the direction of antibodies, specific peptides, antibacterial drugs, etc., can solve the problems of unsatisfactory specificity and sensitivity
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Embodiment 1
[0042] Example 1 Expression and purification of target protein
[0043] The gene sequence encoding the P1 protein of Mycoplasma pneumoniae (GI number: 15213522) was selected from Genebank, and the hydrophilicity and antigenicity of the encoded protein were analyzed by DNASTAR.Lasergene v7.1 software, and the high hydrophilicity and good antigenicity were selected Amino acids 1160-1498 were used as the target protein and named as P1'. The DNA sequence corresponding to P1' is the 3601-4617 nucleotide sequence of the p1 gene. According to the mycoplasma codon table, when designing PCR primers, the "TGA" codon (termination codon in E. coli) is designed as " TGG" codon, which encodes tryptophan in E. coli. Entrust BGI to synthesize the DNA sequence and introduce enzyme cutting sites EcoR I and xho I, mixed it with the vector pET-30a(+) and ligated overnight at 16°C to construct the recombinant vector pET-30a(+)-p1', and transformed the recombinant vector into Escherichia col...
Embodiment 2
[0046] Example 2 Preparation and purification of P1' polyclonal antibody
[0047] 200 μg of recombinant protein P1' was mixed with Freund's complete adjuvant and fully emulsified, and New Zealand rabbits (2-month-old weighing about 2 kg) were injected subcutaneously at multiple points on the back. At the same time, a PBS control group was set up, and 3 rabbits were immunized in each group. Booster immunization was carried out in two weeks, and immunized 3 times in total. Blood was collected from the heart 14 days after the last immunization, and the serum was saturated with (NH 4 ) 2 SO 4 Preliminary purification was carried out using the Millipore ultrafiltration centrifuge tube to concentrate and remove inorganic salt ions. Use cyanogen bromide-activated agarose to perform affinity chromatography on the preliminarily purified rabbit serum to obtain polyclonal antibodies with affinity to the target protein. The steps are briefly described as follows: cyanogen bromide-activ...
Embodiment 3
[0049] Example 3 Panning of phage display random 12-peptide library
[0050] Biopanning was performed on the phage display random 12 peptide library, and the steps are briefly described as follows: In the first round of panning, 100 μg of purified P1’ antibody was used to coat the ELISA plate, and the blocking solution (0.1M NaHCO 3 , 5 mg / mL BSA) at 4°C for 3 h, washed 10 times with 0.1% TBST, and put in about 4×10 10 pfu of the original phage library, gently shaken to bind for 1 h, washed 8 times with 0.1% TBST, eluted with 100 μL of elution buffer (0.2M glycine-HCl, pH=2.2) for 10 min, and collected the eluate. In the second and third rounds, an empty ELISA plate and negative serum were used for reverse adsorption, respectively, to exclude non-specific phages that were adsorbed to the ELISA plate or had affinity with the negative serum. The fourth round of screening was similar to the first round, but the coating concentration of polyclonal antibody was appropriately reduc...
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