Uses of elderberry lectin and haptoglobin-related protein
A technology that binds to haptoglobin and related proteins, which is applied to the general structure of gas analyzers for detailed detection, instruments, and analysis materials. It can solve the problems of long cultivation period, difficult clinical promotion, harsh cultivation conditions, etc., and achieve good application value. Effect
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Embodiment 1
[0037] Example 1 High-throughput lectin chip screening and verification of differential glycosylation modification
[0038] 1. Sample quantification and biotin labeling
[0039] After the sample was thawed, centrifuged at 13 200 r / min for 15 min at 4°C, and the clarified part was taken from the middle, and the protein concentration was determined by BCA method. Then take 2μL of each sample and use the labeling kit of Full Moon Company to label. The steps are as follows: add 62μL of labeling buffer to every 2μL of serum sample, add 6μL of biotin-labeled reagent after mixing, and react for 2h at room temperature; add 30μL of stop solution and react for 30min at room temperature, then perform chip experiment or freeze at -80°C for use.
[0040] 2. High-throughput lectin chip detection
[0041] The chip is equilibrated at room temperature for 20 minutes and placed in a drying oven at 25°C for 1 hour. It was blocked with PBST solution containing 2% BSA for 1 h at room temperature (50r / mi...
Embodiment 2
[0049] Example 2 Mass spectrometric identification and western blotting verification of SNA affinity enrichment glycoprotein
[0050] 1. Lectin affinity enrichment of glycoprotein and elution
[0051] Incubate 1 mg of mycoplasma pneumonia patient serum sample with 250 μg biotin-labeled elderberry agglutinin at 4°C overnight. The next day, incubate with streptavidin-immobilized agarose beads for 1 hour at room temperature, discard the supernatant, and wash away unbound samples. Then eluted with 0.5mol / L lactose solution at room temperature for 15min. After the eluted part was concentrated by ultrafiltration, it was subjected to denaturing polyacrylamide gel electrophoresis and silver staining, and the 42kDa band was taken for mass spectrometry identification.
[0052] 2. Mass identification
[0053] After the strips are decolorized, protein reductive alkylation, and enzymatic hydrolysis, the enzymatic polypeptide is extracted. The dried polypeptide sample was added to the loading buf...
Embodiment 3
[0061] Example 3 Detection and analysis of large sample volume of binding globin related protein
[0062] 1. Western blot detection of binding globin related proteins
[0063] The serum of children with mycoplasma pneumonia (n=22), the serum of children with bacterial pneumonia (n=21), the serum of children with viral pneumonia (n=22), and the serum of healthy children (n=21) were prepared into 50-fold dilutions The electrophoresis samples from 10 healthy children were mixed in equal proportions to prepare 50-fold diluted electrophoresis samples (as a reference). All samples were loaded with an equal volume of 5μL (equivalent to 0.1μL of original serum). Other operations were the same as in the example 2.
[0064] 2. Data analysis
[0065] The exposure image was read with AlphaView SA software to read the signal value of a specific band. The detection value of each sample was divided by the detection value of the reference product, and the relative value obtained was used for statis...
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