Background[1-3]
Human lipoprotein phospholipase A2 (LP-PL-A2) ELISA kit uses a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) kit. To the coated microwells pre-coated with human lipoprotein phospholipase A2 (Lp-PL-A2) capture antigen, add specimens, standards, and HRP-labeled detection antibodies in sequence, incubate and wash thoroughly. The substrate TMB is used to develop color. TMB is converted into blue under the catalysis of peroxidase, and converted into the final yellow under the action of acid. The depth of color is positively correlated with human lipoprotein phospholipase A2 (Lp-PL-A2) in the sample. Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm, and calculate the concentration of human lipid Tosohin phospholipase A2 (LP-PL-A2) sample.
Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a novel inflammatory marker associated with low-density lipoprotein (LDL) cholesterol, atherosclerotic disease, and cardiovascular disease. Atherosclerosis and atherogenesis, a potential culprit associated with myocardial infarction, stroke, and other occlusive vessel-related diseases, are considered inflammatory diseases. Research has found that there may be three pathways that trigger atherosclerotic inflammation: first, oxidized LDL is cytotoxic and can cause arterial cell damage and recruit specific inflammatory T cells to migrate to the arterial wall; second, oxidized LDL It may directly activate inflammation by interacting with macrophage Toll-like receptors; third, by releasing bioactive metabolites, Lp-PLA2 hydrolyzes oxidized phospholipids in LDL to produce two inflammatory molecules-oxidized non-esterified fatty acids and Lysophosphatidylcholine (lysoPC).
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Prokaryotic expression, antibody preparation and epitope analysis of LP-PLA2
Lipoprotein–associated phospholipase A2 (LP-PLA2) belongs to type VIIA phospholipase A2, which can predict the risk of atherosclerosis and stroke related to coronary events. Obtain human lipoprotein-related phospholipase A2 protein, prepare corresponding antibodies, and verify the antigenicity of epitope peptides predicted by bioinformatics, laying the foundation for the development of LP-PLA2 in vitro immunodiagnostic reagents. Methods Total RNA was extracted from differentiated THP-1 cells using Trizol method, and the target fragment LP-PLA2 was amplified by RT-PCR; prokaryotic expression plasmid was constructed using molecular cloning technology; transformed into Escherichia coli BL21 (DE3), and induced expression conditions were optimized , perform multi-step purification and remove tags to obtain the target protein, immunize New Zealand white rabbits, and prepare polyclonal antibodies against LP-PLA2; use a variety of bioinformatics original dispersion methods imported from Germany to predict the LP-PLA2 epitope. And the purified LP-PLA2 polyclonal antibody was used to identify its antigenicity.
Result:
1. Recombinant expression plasmids such as PET-28a(+)-LP-PLA2, PGEX-4T-2-LP-PLA2, pCold TF-LP-PLA2, etc. were successfully constructed.
2. After IPTG-induced expression, SDS-PAGE electrophoresis showed that LP-PLA2 was not expressed in the PET-28a(+)-LP-PLA2 expression vector; it was expressed in the PGEX-4T-2-LP-PLA2 expression vector. A small amount of expression was obtained, and it mainly existed in the form of inclusion bodies; in the pCold TF-LP-PLA2 expression vector, a fusion protein with higher expression was obtained, mainly in the form of supernatant.
3. Purify the pCold TF-LP-PLA2 induced expression supernatant through nickel column affinity chromatography to obtain the TF-LP-PLA2 recombinant protein. After digesting the recombinant protein with HRV-3C prolease, use blue After gel separation and purification, a protein with a purity of about 90% and a molecular weight of about 45kD was obtained, which was consistent with expectations, and the protein could be recognized by commercially available LP-PLA2 polyclonal antibodies, proving that the target LP-PLA2 with higher purity was obtained. protein.
4. A polyclonal antibody against LP-PLA2 was prepared, with an antibody titer of >1:5.12×106. Western blot results showed that the antibody had high specificity.
