Human chlamydia pneumoniae quantum dot immunochromatographic detection card and its preparation method and application
A technology of chlamydia pneumoniae and immunochromatography, which is applied in the field of medical detection, can solve the problems that chlamydia pneumoniae cannot be directly identified, it is not suitable for clinical application, and the cell culture time is long.
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[0075] 1. Preparation of conjugated pads
[0076] (1) Preparation and purification of recombinant M98-His fusion protein:
[0077] Bioinformatics analysis of the 98KD membrane protein of human Chlamydia pneumoniae was carried out to obtain the most abundant epitope peptide in the extracellular domain of the 98KD membrane protein of human Chlamydia pneumoniae, and to find its corresponding gene sequence; the 5' end of the two gene sequences Restriction sites were introduced into the 3' and 3' ends respectively, and the whole gene sequence was chemically synthesized respectively, and marked as m98 at the same time. See the sequence listing for its gene sequence. The gene sequence was cloned into the expression vector pET-28a(+) according to the conventional method, and then the recombinant M98-His fusion protein was expressed. The fusion protein exists in the genetically engineered bacteria in the form of inclusion body expression. Purify the recombinant M98-His and fusion pr...
Embodiment 1
[0116] Embodiment 1 (preparation embodiment)
[0117] Conjugate pad preparation
[0118] (1) Preparation and purification of recombinant M98-His fusion protein
[0119] 1. Cloning of related genes
[0120] Bioinformatics analysis was performed on the 98KD membrane protein of Chlamydia pneumoniae (its accession number in the NCBI protein database is CAA04672), and the peptide with the most abundant antigenic epitope in its extracellular conserved domain was obtained, and its corresponding DNA coding sequence was found. At the same time, the whole gene sequence was chemically synthesized after introducing the restriction site NdeI at the 5' end, the termination signal TAA and the restriction site XhoI at the 3' end (the whole sequence synthesis was completed by GenScript Biotechnology Co., Ltd. The synthetic gene fragment was connected to the vector pUC57), which was denoted as m98. The full sequence of its gene is shown in the sequence listing. Specifically, the protein seq...
Embodiment 2
[0145] Embodiment 2 (preparation embodiment)
[0146] Preparation of sample pads
[0147] Prepare sample pad treatment solutions with different formulations, observe the release effect of quantum dot-labeled antibodies, and optimize through multiple orthogonal experiments to obtain the optimal sample pad treatment solution formulation (that is, described in the present invention). Take a piece of glass cellulose membrane, soak it in the sample pad treatment solution for at least 3 hours, then place it in a biological safety cabinet at 37°C, ventilate and dry it, and cut it into a size of 4cm*2.5cm / strip to prepare the sample pad , Store in airtight and dry place at 25°C. Tests have proved that the use of the sample pad greatly improves the release rate of the quantum dot-labeled antibody on the binding pad and achieves a better application effect.
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