Mycoplasma pneumoniae antigen
A technology of Mycoplasma pneumoniae and antigen, applied in the field of molecular biology and biological detection, can solve the problems of easy quenching, long time consumption, low positive rate, etc., and achieve the effect of reducing the misdiagnosis rate
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Embodiment 1
[0027] Embodiment 1 New Mycoplasma pneumoniae P80 antigen preparation
[0028] The epitope-rich region sequence (nucleotide sequence such as SEQ ID NO.3) and corresponding antibody sequence (amino acid sequence such as SEQ ID NO.4) of Mycoplasma pneumoniae outer membrane adhesion protein is known, and its binding is predicted by Pymol biological software Site-related amino acid residues are subjected to site-directed mutation according to conventional methods in the art to obtain a mutant Mycoplasma pneumoniae antigen P80, the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2 . The mutant sequence was integrated into the pET28a plasmid through the routine operation of genetic engineering, transformed into DH5α, and then transformed into E. coli to construct a prokaryotic expression system to express the mutant antigen P80 exogenously. Purified by Ni column, the purity was greater than 90%. From figure 1 It can be seen that the s...
Embodiment 2
[0029] Example 2 New Mycoplasma pneumoniae antigen P80 reactivity test
[0030] In order to study the immunoreactivity of the novel Mycoplasma pneumoniae antigen P80, it was diluted to 100mg / L, and 20% bovine serum was added to the protein stock solution to simulate serum samples. The relative immunoreactivity of P80 was determined with FUJIREBIO reagent on the Siemens BNII system. The calculated actual effective concentrations of P80 and natural Mycoplasma pneumoniae antigen (nucleic acid sequence such as SEQ ID NO.3) are 91 mg / L and 40.7 mg / L, respectively. The measurement results are shown in Table 1, indicating that P80 has good immunoreactivity, and the measured concentration can reach 94.9% of the actual concentration after excluding the matrix effect, which is 34.6% higher than the natural antigen.
[0031] Table 1 Comparison of reactivity between recombinant antigen and natural antigen
[0032]
[0033] Actual concentration = relative molecular mass of natural / rec...
Embodiment 3
[0034] Embodiment 3 detects the preparation of Mycoplasma pneumoniae IgG, IgM quantum dot immunochromatography kit
[0035]The test strip adopts the principle of double-antibody sandwich method. Add 50 μL of the quantum dot stock solution into the EP tube containing the activation buffer, and place it in a vortex mixer to mix and activate for 0.5 h. Add 450 μL of coupling buffer and 75 μg of mouse anti-human IgM and mouse anti-human IgG antibodies, and place in a vortex mixer to shake and mix for 0.5 h. After centrifuging to remove the supernatant, block with 10% ethanolamine solution and 5% Casein solution, and mix with a vertical mixer for 45 minutes. Resuspend and remove the supernatant to prepare water-soluble quantum dot conjugates coated with mouse anti-human IgM and mouse anti-human IgG antibodies, add buffer and store in the dark at 2-8°C.
[0036] Take a marker pad with a width of 8 mm, use a gold sprayer to dilute the quantum dot-labeled mouse anti-human IgM and T...
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