Glycosylation variants of human chorionic gonadotropin hcg β subunit and hCG containing it
A technology of glycosylation and β subunits, applied in the field of hCG biological activity, can solve the problems of reducing hCG activity in vivo, shortening half-life, etc.
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Embodiment 1
[0079] Example 1 Class analysis of different glycosylation variants contained in hCG
[0080] Sample processing: hCG protein is a commercially available product produced by Merk Serono (Switzerland) (imported drug registration number: S20130091). Concentrate the hCG protein to 1 mg / ml and divide it into two groups. Group 1 (non-reduced) is directly injected for the following (RP-HPLC method, SDS-PAGE method, CE-SDS method) detection; Group 2 (reduced) is added with 5 % β-mercaptoethanol, heated in a water bath at 70°C for 10 minutes, and then injected for the following detection.
[0081] Subunit preparation: large-volume injection of reduced and non-reduced samples, and post-column collection of the peaks of each component of the sample, and then the collected fractions were centrifuged and concentrated under reduced pressure for 3 hours, and finally used a molecular weight cut-off of 3KD The ultrafiltration tube is concentrated, and the replacement solvent is water.
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Embodiment 2
[0128] Example 2 Preparation method of hCG β1 subunit
[0129] The hCG β1 subunit can be prepared by RP-HPLC and SDS-PAGE. The source of the hCG protein sample is the same as in Example 1.
[0130] RP-HPLC preparation hCG β1 subunit, the method is as follows:
[0131] The hCG sample was concentrated to 1-2 mg / mL, added 5% β-mercaptoethanol, heated in a water bath at 70°C for 10 minutes and cooled to room temperature, and injected into RP-HPLC. The RP-HPLC chromatographic conditions can adopt the conditions of Example 1. details as follows:
[0132] Chromatographic column: C18 bonded phase, 5μm, 4.6×250mm;
[0133] Mobile phase: A. Water + 0.1% trifluoroacetic acid + 0.01% heptafluorobutyric acid; B. Acetonitrile + 0.1% trifluoroacetic acid + 0.01% heptafluorobutyric acid;
[0134] Column temperature: 25°C;
[0135] Flow rate: 0.8ml / min;
[0136] Injection volume: 100μL / needle;
[0137] The elution rate is shown in Table 4 below.
[0138] Table 4.HPLC elution gradie...
Embodiment 3
[0143] Example 3 Determination of β1 content (or β1:β2 ratio) by CE-SDS method
[0144] Through the analysis of hCG molecules in Example 1, the (reduction) CE-SDS method can be used to measure the content of hCG β1 or the ratio of β1:β2. This method can be carried out by using the CE-SDS method in Example 1. details as follows:
[0145] Sample preparation: hCG samples were concentrated to 1-2 mg / mL, added with 5% β-mercaptoethanol, heated in a water bath at 70°C for 10 minutes and cooled to room temperature.
[0146] Instrument initial settings: set the capillary temperature to 20°C, and the sample tray temperature to 10°C. The PDA wavelength is 220nm and the bandwidth is 10nm.
[0147]Capillary balance: Before the instrument is tested every day, the balance of the capillary and gel solution is required. The settings are as follows in turn, use alkali washing solution to rinse at 20psi for 10min, pickling solution at 20psi for 5min, ultrapure water at 20psi for 2min, SD...
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