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Earthworm protein with HBeAg degrading enzyme activity and application thereof

A technology for degrading enzymes and earthworms, applied in the field of earthworm proteins, can solve the problems of increased drug resistance, unclear other functions of the protein, and the sustained response rate is not as good as that of interferon.

Inactive Publication Date: 2012-08-29
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] 2. Lamivudine: short-term use of lamivudine is superior to interferon in its inhibitory effect on HBV DNA, HBeAg negative conversion rate, HBeAg seroconversion rate, and serum ALT normalization rate. Compensated liver cirrhosis and patients before and after liver transplantation, but its sustained response rate is lower than that of interferon, and there is a prominent problem of drug resistance, and with the prolongation of taking time, drug resistance will gradually increase
Current experiments prove that this group of proteins are all glycosylated serine proteases, but other functions of this group of proteins are still unclear

Method used

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  • Earthworm protein with HBeAg degrading enzyme activity and application thereof
  • Earthworm protein with HBeAg degrading enzyme activity and application thereof
  • Earthworm protein with HBeAg degrading enzyme activity and application thereof

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preparation example Construction

[0069] In one embodiment of the present invention, a kind of preparation method of earthworm protein crude enzyme extract is provided, it comprises the following steps:

[0070] 1) Select 2-month-old, 5-8cm long E. fetida and wash them in distilled water, then soak the earthworms in distilled water at room temperature for 12 hours to make them spit out the dirt in the digestive tract.

[0071] 2) Homogenize 1 kg of earthworms in 0.05M Tris-HCl buffer solution, and centrifuge at 4°C and 8000rpm for 30min. The supernatant was taken and dialyzed against 85% ammonium sulfate equilibrium solution overnight at 4°C.

[0072] 3) Sepharose-4B (10ml) was used as a carrier, activated by carbonyldiimidazole (800mg), and coupled with soybean trypsin inhibitor ligand to prepare an affinity carrier. The prepared affinity carrier was loaded into the column, and 0.1mol / L NaHCO 3 buffer (pH 8.0) to equilibrate the column. The supernatant is adsorbed by the upper affinity chromatography colum...

Embodiment 1

[0083] Embodiment 1 Earthworm protein of the present invention and HBeAg protein co-incubation test

[0084] Experimental methods and results

[0085] 1. Get HBeAg (Beijing Huamei Shengke Biotechnology Co., Ltd.) (66.7 μ M), add a certain concentration of earthworm protein (SEQ ID NO.1 protein) (0 ~ 6 μ M) of the present invention and incubate at 37 ° C, respectively at 15, Samples were taken at 30, 45, 60, 90, and 120 minutes, while blank HBeAg and earthworm protein (SEQ ID No.1) of the present invention (incubated at 37° C. for 120 minutes) were used as controls.

[0086] 2. Add the above incubated samples to 5×SDS-PAGE loading buffer, boil for 10 minutes, perform 15% SDS-PAGE, and finally stain with Coomassie brilliant blue.

[0087] 3. SDS-PAGE see figure 1 . Result shows that blank HBeAg hatches 120min protein band without obvious change at 37 ℃, and protein band degrades rapidly in the incubation product of HBeAg and earthworm protein of the present invention (see fi...

Embodiment 2

[0088] Embodiment 2 The analysis of the hydrolysis position of HBeAg proteolysis by earthworm protein of the present invention

[0089] Experimental methods and results

[0090]1. Incubate 66.7 μM HBeAg protein with earthworm protein (SEQ ID No.1) (6 μM) of the present invention at 37° C. for 60 min, and perform 15% SDS-PAGE on the enzymatic hydrolysis product and transfer to PVDF membrane (Millipore, USA).

[0091] 2. The membrane was stained with Coomassie Brilliant Blue, and the main band generated by enzymatic digestion was subjected to N-terminal sequencing (Applied Biosystem Automated Protein Sequencer, Applied Biosystem Inc., USA).

[0092] 3. analyze the effect of the earthworm protein of the present invention on the HBeAg protein by sequencing results (see Table 1) and start from the C-terminus of the HBeAg protein, and analyze the earthworm protein of the present invention on the HBeAg protein according to the molecular weight ratio of the enzymolysis fragment and th...

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Abstract

The invention provides earthworm protein with HBeAg degrading enzyme activity and a medicinal composition containing the earthworm protein. The invention further provides application of the earthworm protein to the preparation of medicines for treating HBeAg-related diseases. The HBeAg-related diseases comprise hepatitis B, cirrhosis caused by hepatitis B, hepatitis B virus vector with e antigen positive and surface antigen positive, and the like. In addition, the invention further provides a method for separating a crude enzyme extract containing the earthworm protein, an earthworm protein crude enzyme extract prepared by the method and application of the earthworm protein crude enzyme extract to the preparation of medicines for treating the HBeAg-related diseases.

Description

technical field [0001] The invention provides an earthworm protein with HBeAg degrading enzyme activity and a pharmaceutical composition containing it. The present invention also provides the application of the earthworm protein in the preparation of medicines for treating HBeAg-related diseases, and the HBeAg-related diseases include hepatitis B, liver cirrhosis caused by hepatitis B, and e antigen-positive and surface antigen-positive Hepatitis B virus carriers, etc. In addition, the present invention also provides a method for isolating the crude enzyme extract containing the earthworm protein, the crude earthworm protein enzyme extract prepared by the method and its application in the preparation of medicines for treating HBeAg-related diseases. Background technique [0002] Hepatitis B, referred to as hepatitis B, is an infectious disease caused by the hepatitis B virus (HBV). Transmitted through blood and body fluids, with a chronic carrier state. The disease is wid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N9/64A61K38/48C07K1/36A61P1/16A61P35/02C12Q1/37
Inventor 赫荣乔周园
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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