CN102649814A

CN102649814A - Earthworm protein with HBeAg degrading enzyme activity and application thereof

Info

Publication number: CN102649814A
Application number: CN2010105929213A
Authority: CN
Inventors: 赫荣乔; 周园
Original assignee: Institute of Biophysics of CAS
Current assignee: Institute of Biophysics of CAS
Priority date: 2011-02-24
Filing date: 2011-02-24
Publication date: 2012-08-29

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

Earthworm protein and application thereof with HBeAg degrading enzymatic activity

Technical field

The invention provides a kind of earthworm protein of the HBeAg of having degrading enzymatic activity, and the pharmaceutical composition that contains it.The present invention also provides the application of said earthworm protein in the diseases related medicine of preparation treatment HBeAg; The diseases related hepatitis B that comprises of said HBeAg; The liver cirrhosis that hepatitis B causes, and the hepatitis b virus carrier of e antigen positive and surface antigen positive etc.In addition, the present invention also provides the method for the thick enzyme extract that a kind of separation contains said earthworm protein and by the thick enzyme extract of earthworm protein and the application in the diseases related medicine of preparation treatment HBeAg thereof of said method preparation.

Background technology

Hepatitis B is called for short hepatitis B, is the transmissible disease that is caused by hepatitis B virus (HBV).Through blood and body fluid communication, has chronic carrier state.This disease is widely current in China.According to pertinent data, hepatitis detects the male patient and has reached 1.89 hundred million, and the number (carrier) nearly 400,000,000 of should not going to a doctor.It is the most serious transmissible disease of current harm people ' s health.Be more common in children and person between twenty and fifty.Hepatitis B clinical manifestation variation is prone to develop into chronic hepatitis and liver cirrhosis, and few patients can change primary hepatocarcinoma into, and these diseases are more serious to patient and social harm.

Pathogenic process: in general, after liver cell received the hepatitis B virus invasion, hepatitis B virus itself did not directly cause hepatocellular pathology.The nutriment that hepatitis B virus just utilizes liver cell to absorb is depended on for existence and in liver cell, is duplicated.The surface antigen of virus replication, e antigen and cAg all are released on the liver plasma membrane, and the immunity system of exciting human is recognized, and reacted.This antigen antibody reaction that on liver plasma membrane, takes place can cause hepatocellular damage and destruction, thereby produces a series of clinical symptom.

Treatment means: China's most of patients is to enclose the living phase or infecting HBV when young and cause immunological tolerance, often forming chronic infection, and these needs of patients actively and efficacious therapy otherwise possibly develop into liver cirrhosis even liver cancer, cause death.Duplicating of HBV be can not remove or forever suppress yet still there is a kind of therapeutic method of reliable so far, thereby the inflammation and the necrosis of liver stopped.The present therapeutic destination of chronic hepatitis B is: 1. suppress hbv replication (HBsAg, HBeAg disappear); 2. non-TRAP can not detect the HBV dna level; 3. the state of an illness is improved, and ALT is normal again; 4. liver histological improves; 5. reduce the incidence that liver loses compensatory, liver cirrhosis, hepatocellular carcinoma.The drug main that is used to treat hepatitis B at present will comprise immunoregulation druges such as antiviral such as Interferon, rabbit, nucleotide analog and Zadaxin.

1. traditional Interferon, rabbit and polyoxyethylene glycol Interferon, rabbit: Interferon, rabbit is still a line medicine of treatment hepatitis B at present; It has the clear and definite course of treatment; Good effect; HBsAg is disappeared and anti-HBs produces, advantage such as have no drug resistance, but since exist simultaneously spinoff more, can not be used in and lose shortcomings such as compensatory liver cirrhosis patient, injection for curing bothers and cost dearly and limited its use.In the U.S., a lot of doctors tends to select nucleosides (acid) analogue treatment chronic viral hepatitis B.

2. lamivudine: short-term all is superior to Interferon, rabbit with lamivudine to restraining effect, HBeAg negative conversion rate, HBeAg frequence of seroconversion and the Serum ALT normalization rate of HBV DNA; And easy administration; Can be used to lose compensatory liver cirrhosis and liver transplantation front and back patient, but the too late Interferon, rabbit of its lasting response rate, and have outstanding resistance problem; And along with the prolongation of Time of Administration, resistance also can increase gradually.

3. adefovir ester: be novel nuclear former times (acid) analogue; Oral adefovir ester is the precursor of Adefovir; Can effectively suppress hepatitis B virus duplication, improve histology, delay PD and become liver cirrhosis, liver function to lose compensatory and liver cancer, its resistance is lower than lamivudine, and the lamivudine resistance person is had good result of treatment; But taken certain renal toxicity for a long time, needed periodic monitoring serum creatinine and serium inorganic phosphorus.

4. Entecavir: be ring valeryl guanosine analogue, approval just at China SFDA end of last year is used for the treatment of chronic hepatitis B.

Other: other also have emtricitabine, Te Bifuding, Clevudine at the medicine that carries out II, III clinical trial phase.

6. immune modulating treatment: immune modulating treatment is one of important means of treatment chronic hepatitis B, and the immunoregulation druge that is used to treat hepatitis B at present mainly is a Zadaxin.Zadaxin stimulates the T cell, strengthens non-specific immune function, and its untoward reaction is little, and better tolerance is safe in utilization, for the antiviral therapy pointer is arranged, can select Zadaxin for use but can not tolerate or be reluctant to accept the patient that Interferon, rabbit and nucleotide analog treat.

Hepatitis B virus e antigen (HBeAg): HBeAg is a kind of non-particulate secretor type nucleocapsid protein, is the product of preC protein translation post-treatment, and the serum mark that infects as HBV is found in 1973.Discovering subsequently, this antigen are not the necessary albumen of virus replication, by HBV DNA C district coding, increase with hbv replication, clinically with one of its index of duplicating as judgement HBV reactivity.U.S. health research center found that chronic viral hepatitis B e antigen lasting masculin can greatly increase the danger of liver cirrhosis and liver cancer in 2007.They have researched and analysed the influence that the hepatitis B gene type is removed HBeAg.1158 the local patients in investigated Alaska State, the mean age, the hepatitis B gene type was A, B, C, D, F at 20.5 years old.Discover that genotype is that the patient HBeAg of the C possibility of turning out cloudy is more difficult than other genotype, and e antigen clean-up time is longer.Wherein 50% A, B, D type patient had e antigen to turn out cloudy less than 20 years, and the C type needs 47.8.But after these HBeAg turned out cloudy, the HBeAg of C, F type will soon reply.Therefore, HBeAg turns out cloudy in the hepatitis B mark, and produces the important indicator that HBeAb is judgement state of an illness trend and curative effect of medication.

HBeAg has two confirmed antigenic determinants: first is positioned at its N and holds the 84th～99 amino acids residue; Be linear epitope; Second epi-position is positioned at 136～145 amino-acid residues, is non-linear epi-position, and it need interact with preceding C district partial amino-acid residue just can show antigenicity.The halfcystine formation disulfide linkage in the halfcystine in C district-7 and 61 in C district before being arranged in, formation HBeAg is different from the secondary structure of the uniqueness of HBcAg, and this disulfide linkage also suppresses HBeAg formation protein particulate simultaneously.

Though also there is dispute in the biological function of HBeAg in the HBV life cycle, virus replication does not need it, and HBeAg also has important immunoregulation effect in hepatitis and liver cancer patient.In hepatitis b virus marker (HBV-M) detected, before DNA detection began, HBeAg was the standard affinity tag of reactivity hbv replication and infection.The detection of e system is particularly important, it to hbv replication whether judgement, whether have infectivity and clinical antiviral therapy effect and all have important value like aspect how.

Earthworm is commonly called as earthworm, and it is of a great variety, aboundresources.Earthworm is as drug use, in the history in existing more than 1200 year of China.Primary method is to utilize whole bright earthworm or dried earthworms; Along with development, found afterwards that the pharmaceutical use of earthworm was relevant with the number of chemical composition that wherein contains.Be separated at present the earthworm composition of multiple different purity; Comprising plasmin, plasminogen activator, CDR and caldesmon, Pseudocholinesterase, katalase, superoxide-dismutase SOD, short myeloid cell propagation component, antimicrobial proteins, vasoconstriction albumen, dissolved blood protein, immunoglobulin-like adhesion thing and anti-tumor protein, antibacterial peptide etc., obtained bigger progress.

In the earthworm body, finding in recent years has a histone, its aminoacid sequence to be respectively SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4; SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8; SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12; SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16.Present this histone of experiment proof all is glycosylated Tryase, but other functions of this histone it be unclear that.

Summary of the invention

For this reason; The inventor has carried out lot of test; And all of a sudden find first; The albumen of SEQ ID NO.1-16 is in that hatch in the process with HBeAg can be rapidly with its degraded, and proves also that through test cell line, animal experiment they all have HBeAg degrading enzyme (also being called " HBeAgase " in a following embodiment and the accompanying drawing) activity with external in vivo.

Therefore, aspect first, the purpose of this invention is to provide a kind of earthworm protein, it has a kind of sequence that is selected from following (a)-(b): (a) aminoacid sequence of SEQ ID NO.1-16 shown in each; (b) with (a) described in the aminoacid sequence of any aminoacid sequence with the homology more than 80%, wherein above-mentioned aminoacid sequence all has by H ⁴³, D ⁹¹, S ¹⁸⁸The catalysis triplet of forming, and said earthworm protein has the HBeAg degrading enzymatic activity.

Aspect second, the invention provides a kind of pharmaceutical composition, comprise earthworm protein of the present invention as activeconstituents, and one or more medicinal carriers, vehicle, thinner or auxiliary material.

Aspect the 3rd, the present invention relates to the application in the medicine of preparation treatment or prevention HBeAg relative disease of earthworm protein of the present invention or pharmaceutical composition.In a preferred embodiment, the said HBeAg relative disease liver cirrhosis or the tumour that are selected from the hepatitis b virus carrier of hepatitis B, e antigen and/or surface antigen positive, cause by hepatitis B.

Aspect the 4th, the invention provides the preparation method of the thick enzyme extract of a kind of earthworm protein, it may further comprise the steps:

1) earthworm was also at room temperature left standstill 10～48 hours with distilled water wash, immersion, to get rid of intravital silt;

2), supernatant is dialysed with earthworm homogenate, centrifugal;

3) will be through the supernatant of dialysis through the absorption of Trypsin inhibitor SBTI-affinity column, remove do not adsorb foreign protein after, with balance liquid thick enzyme extract of the said earthworm protein of wash-out from the said chromatography column.

Aspect the 5th, the invention provides a kind of thick enzyme extract of earthworm protein of method for preparing.

In a preferred embodiment, the thick enzyme extract of earthworm protein of the present invention comprises at least a earthworm protein with following amino acid sequences: (a) aminoacid sequence of SEQ ID NO.1-16 shown in each; (b) with (a) described in the aminoacid sequence of any aminoacid sequence with the homology more than 80%, wherein above-mentioned aminoacid sequence all has by H ⁴³, D ⁹¹, S ¹⁸⁸The catalysis triplet of forming, and said earthworm protein has the HBeAg degrading enzymatic activity.

In another preferred embodiment, the thick enzyme extract of earthworm protein of the present invention comprises the albumen with following aminoacid sequence: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.15.In a preferred embodiment; The abundance of SEQ ID NO.1 is 9.65%, and SEQ ID NO.2 is 3.65%, and the abundance of SEQ ID NO.11 and SEQ ID NO.12 is 5.1%; The abundance of SEQ ID NO.14 is 2.6%, and the abundance of SEQ ID NO.15 is 4.3%.

Aspect the 6th, the invention provides a kind of pharmaceutical composition, it comprises the thick enzyme extract of earthworm protein of the present invention as activeconstituents, and one or more medicinal carriers, vehicle, thinner or auxiliary material.

Aspect the 7th, the application of the pharmaceutical composition that the present invention relates to the thick enzyme extract of earthworm protein of the present invention or contain the thick enzyme extract of this earthworm protein in the medicine of preparation treatment or prevention HBeAg relative disease.In a preferred embodiment, the said HBeAg relative disease liver cirrhosis or the tumour that are selected from the hepatitis b virus carrier of hepatitis B, e antigen and/or surface antigen positive, cause by hepatitis B.

The present invention has following beneficial effect:

1. earthworm protein of the present invention have the degraded HBsAg enzymic activity, and this activity based in the aminoacid sequence by H ⁴³, D ⁹¹, S ¹⁸⁸The catalysis triplet of forming.

The HBsAg degrading enzymatic activity of earthworm protein of the present invention to the action site of HBeAg near 141 l-arginine and two amino acid of 142 L-glutamic acid.

3. the present invention also provides the preparation method of the thick enzyme extract of a kind of earthworm protein; Based on top 1) and 2) teachings, those skilled in the art can be as required just can prepare the thick enzyme extract of earthworm protein of the earthworm protein of the present invention that contains different sorts, various combination through the test of limited number of time.

4. earthworm protein of the present invention, thick enzyme composition and the pharmaceutical composition that contains them respectively are to the HBsAg relative disease; In the treatment such as hepatitis B; Especially the treatment to chronic hepatitis B has remarkable advantages; The HBsAg because it is degraded specifically, but not be directed against HBV DNA, and do not have the caused spinoff of non-specific targeting of the other drug of present clinical use.

Description of drawings

Fig. 1 is that earthworm protein SEQ ID NO.1 and HBeAg albumen are hatched back SDS-PAGE figure jointly, and wherein Figure 1A is that HBeAg and different concns SEQ ID NO.1 scheme (M: standard molecular weight at 37 ℃ of SDS-PAGE of hatching 1h; 1: contrast (HBeAg that does not add SEQ ID NO.1); 2-5:HBeAg and SEQ ID NO.1 are hatched jointly; 6: 6 μ M SEQ ID NO.1 only); Figure 1B is that HBeAg albumen is hatched different SDS-PAGE figure (M: standard molecular weight with SEQ ID NO.1 at 37 ℃; 1: contrast (the HBeAg albumen that does not add SEQ ID NO.1); 2:15min; 3:30min; 4:60min; 5:90min; 6:120min; 7: SEQ ID NO.1 only).

Fig. 2 is the western blotting figure after earthworm protein SEQ ID NO.1 and HBeAg albumen are hatched jointly, wherein 1: contrast (the HBeAg albumen that does not add SEQ ID NO.1); 2-5:HBeAg and SEQ ID NO.1 are hatched jointly; 6: 6 μ M SEQ ID NO.1 only.

Fig. 3 is the ELISA figure after SEQ ID NO.1 and HBeAg albumen are hatched jointly, wherein 1: contrast (the HBeAg albumen that does not add SEQ ID NO.1); 2-5:HBeAg and SEQ ID NO.1 are hatched jointly.

Fig. 4 is the synoptic diagram that diagram adopts the method flow of Trypsin inhibitor SBTI-thick enzyme extract of affinity chromatography series connection Para-Anisidine affinity chromatography separation and purification earthworm protein.

Fig. 5 is that 8 kinds of isozyme native-PAGE of the thick enzyme extract of earthworm protein of separation and purification of the present invention scheme and SDS-PAGE figure.Wherein the native-PAGE of Fig. 5 A:8 kind earthworm HBeAg degrading enzyme isozyme schemes (1:EfP-0-1; 2:EfP-0-2; 3:EfP-I-1; 4:EfP-I-2; 5:EfP-II-1; 6:EfP-II-2; 7:EfP-III-1; 8:EfP-III-2; 9: earthworm HBeAg degrading enzyme blending ingredients); SDS-PAGE figure (the 1:EfP-0-1 of Fig. 5 B:8 kind earthworm HBeAg degrading enzyme isozyme; 2:EfP-0-2; 3:EfP-I-1; 4:EfP-I-2; 5: standard molecular weight; 6:EfP-II-1; 7:EfP-II-2; 8:EfP-III-1; 9:EfP-III-2; ).

Fig. 6 is the figure of the thick enzyme extract of diagram earthworm protein of the present invention to the therapeutic action of HBV transgenic mice.The variation of Fig. 6 A diagram ALT; The variation of Fig. 6 B diagram AST; The variation of Fig. 6 C diagram HBsAg; Fig. 6 D is the pathological observation result.Wherein A is saline water group (10 times); B is the thick enzyme extract group of earthworm protein of the present invention (10 times); C is lamivudine group (10 times).

Fig. 7 is the figure of diagram SEQ ID NO.1 to the effect of HepG 2.2.15 cell levels.Fig. 7 A is that SEQ ID NO.1 is to HBsAg and the influence of HBeAg excretory; Fig. 7 B is that lamivudine is to HBsAg and the influence of HBeAg excretory;

Embodiment

Following this paper will describe the present invention through concrete embodiment.As do not specialize part; Can be according to the book of reference that those skilled in the art were familiar with: like protein TM (Wang Jiazheng, Fan Ming chief editor; Science Press), antibody technique experiment guide (Shen Guanxin, Gong Feili etc. translate Science Press), pharmacological experimental methodology (third edition, chief editor: Xu Shuyun; The People's Health Publisher), molecular biology experiment instructs (Liu Jinyuan; Press of Tsing-Hua University), listed method is implemented in molecular cloning experiment guide (third edition, Huang Peitang etc. translate, Science Press) etc. and the reference that this paper quoted.

Definition:

Described in the present invention " having the homology more than 80% " specifically finger derives from the proteic aminoacid sequence of earthworm equally; The sequence of itself and SEQ ID NO.1-18 of the present invention has more than 80%, and is preferred more than 85%, more preferably more than 90%; Also more preferably more than 95%; Even 99% above homology, and most preferably, the difference of certain locational amino-acid residue is merely conservative amino acid replacement between the sequence of itself and SEQ ID NO.1-16.

" disease " described in this paper should be from broadly understanding; Be that above-mentioned disease can be idiopathic or insecondary (for example hepatitis, hepatitis secondary infection, hepatitis Secondary cases ephrosis etc.); Or (for example hepatitis virus carrier, viral hepatitis etc.) recessive or dominance; Or complication and/or complication (for example, hepatitis B nephritis, hepatogenous diabetes, hepatitis merge haemolysis, gestation merges blood transfusion property hepatitis B etc.).

" HBeAg relative disease " described in this paper is meant generation, development, progress, the deterioration of disease and/or its complication; The prevention of disease and/or its complication, alleviate, alleviate, disappear; The diagnosis of disease and/or complication, prognosis, regimen are selected, therapeutic response monitoring, clinical existence, expression, variation and the removing etc. that lapse to etc. with HBeAg have close ties, or cause-effect relationship is arranged each other.For example, be used for turning out cloudy of hepatitis B virus carriers or reactivity hepatitis B patient HBeAg; Be used for the treatment of HBeAg male liver cirrhosis or liver cancer patient; Be in the immunodeficient patient's of hepatitis B virus infection high-risk (for example, transfuse blood in a large number or have frequent virus carrier to contact possibility) prophylactic application; Certain stage in the liver cancer treatment need be controlled the reactivity of HBV; The prophylactic application of other types hepatitis; Be used for diagnosis (for example coming the content of indirect quantitatively determined HBeAg) of HBeAg or the like through the consumption of earthworm protein of the present invention.Therefore, although mention hepatitis B in a lot of places among this paper, in fact the present invention is not limited to hepatitis B, if in the diagnosis of certain disease, prevention, treatment HBeAg have certain clinical meaning, all be applicable to the present invention.

" thick enzyme extract " of the present invention be meant with earthworm with distilled water wash, soak and at room temperature leave standstill 10～48 hours after, with earthworm homogenate, centrifugal, be thick enzyme extract with the protein solution of gained after the supernatant dialysis.

The present invention invents described " catalysis triplet " is meant that the zymophore of serine stretch protein enzyme family exists three polar residues---His, Asp and Ser, and these three polar residues form catalysis triplet (catalytic triad).

Earthworm protein with HBeAg degrading enzymatic activity:

In one embodiment of the invention, a kind of earthworm protein is provided, it has a kind of sequence that is selected from following (a)-(b): (a) aminoacid sequence of SEQ ID NO.1-16 shown in each; (b) with (a) described in the aminoacid sequence of any aminoacid sequence with the homology more than 80%; Preferred more than 85%; More preferably more than 90%, also more preferably more than 95%, even 99% above homology; And most preferably, the difference of certain locational amino-acid residue is conservative amino acid replacement between the sequence of itself and SEQ ID NO.1-16; Wherein above-mentioned aminoacid sequence all has by H ⁴³, D ⁹¹, S ¹⁸⁸The catalysis triplet of forming, and said earthworm protein has the HBeAg degrading enzymatic activity.

In the present invention, earthworm selects for use Lumbricidae to win the Eisenia foetida (Eisenia foetida) that earthworm belongs to, and earthworm protein mainly is distributed in the front end of the part of endless belt and stomach, particularly intestines.

The inventor finds that the general character of SEQ ID NO.1-16 aminoacid sequence is to have common domain: H ⁴³, D ⁹¹, S ¹⁸⁸The catalysis triplet that (numbering is corresponding to SEQ ID NO.1 aminoacid sequence on it) formed, wherein SEQ ID NO.2-SEQ ID NO.10 has the homology more than 85%.In addition, on function, their common trait is that its HBeAg degrading enzymatic activity is the C end beginning from HBeAg, at 141 l-arginine and 142 L-glutamic acid place degraded HBeAg (seeing embodiment 2 and table 1).

In another embodiment of the invention, a kind of pharmaceutical composition is provided, comprise earthworm protein of the present invention as activeconstituents, and one or more medicinal carriers, vehicle, thinner or auxiliary material.This pharmaceutical composition can be made for suitable formulation by those skilled in the art according to conventional pharmaceutical methods as required.

Comprise in the formulation process of pharmaceutical composition of the present invention in preparation; Pharmaceutical composition of the present invention usually prepares through technologies such as the mixing of routine, dissolving, granulation, the grinding of system ingot, emulsification, encapsulation, embedding or freeze-drying with carrier and/or vehicle; Drug dilution of the present invention or be encapsulated in wherein (see Remington ' s Pharmaceutical Sciences, the 15 edition, Hoover; J.E. edit Mack Publishing Co. (2003)).

Above-mentioned formulation can be taked any suitable administering mode, and this mode comprises approach such as part, per os, whole body, intranasal, injection, transdermal.

For topical, pharmaceutical composition of the present invention can be made into solution, gel, ointment, emulsifiable paste, suspension-s etc.

The whole body preparation comprises design through drug administration by injection, in for example subcutaneous, intravenously, intramuscular, the knurl, in the sheath or the formulation of peritoneal injection, and the formulation that is designed for transdermal, passes through mucous membrane oral cavity or pulmonary administration.

Injectable dosage formulations comprises sterile suspensions, solution or the emulsion of pharmaceutical composition of the present invention in water-based or oiliness media.Said formulation also can contain suspension agent, stablizer and/or dispersion agent etc.Injection type can provide by unit dosage, for example, in ampoule or in the multidose container, can contain sanitas.

Injectable dosage formulations also can be selected form of powder, uses suitable vehicle before use, includes but not limited to that aseptic no heat source water, damping fluid, glucose solution etc. prepare again.Injectable dosage formulations can be through direct intravenous injection, the quick dropleting medicine-feeding of vein, or carries out the infusion administration through adding infusion solution like 0.9% sodium chloride injection or other compatible infusion solutions.

For oral administration, pharmaceutical composition of the present invention can use the acceptable vehicle of pharmacy and/or additive to be prepared into for example lozenge, tablet or capsular formulation through the mode of routine.

The liquid dosage form that is used for oral administration can be taked the for example form of elixir, solution, syrup or suspension, or but powder makes water or other suitable vehicle prepare before use.This liquid dosage form can use the pharmacy acceptable additive to prepare through the mode of routine.

Oral Preparation can be formulated into and be the controlled release formulation, and its preparation technology and method are known in the art.Preferred tablet and capsule formulation.

Send for long-term medicine, pharmaceutical composition of the present invention can be made into sustained release preparation so that through implanting or the intramuscular injection administration.Pharmaceutical composition of the present invention can use suitable polymers or hydrophobic material (for example, in the acceptable oil of pharmacy, forming emulsion) or ion exchange resin to prepare.Optional is, can use to be made for the paster transdermal delivery system, can discharge pharmaceutical composition of the present invention lentamente and absorb through skin.Can use penetration enhancer to promote the transdermal penetration of pharmaceutical composition of the present invention.

The various formulations of pharmaceutical composition of the present invention will confirm that according to correlative factor route of administration, each patient's age, body weight and patient that concrete dosage, these factors comprise the disease of being treated, selection are to the response situation of treatment, the seriousness of patient's symptom etc. by the doctor.

In one embodiment of the invention, relate to the application in the medicine of preparation treatment or prevention HBeAg relative disease of earthworm protein of the present invention or pharmaceutical composition.

In a preferred embodiment, the said HBeAg relative disease liver cirrhosis or the tumour that are selected from the hepatitis b virus carrier of hepatitis B, e antigen and/or surface antigen positive, cause by hepatitis B.

The patient who mentions among the present invention can be any Mammals, and is preferred human.

This area professional and technical personnel it should be understood that scope of the present invention is not limited to content recited above and specific embodiments.According to content of the present invention; It is obvious that earthworm protein of the present invention can use separately for this area professional and technical personnel; Also can be multiple (for example; 2,3,4,5,6,7,8,9,10,11,12,13,14,15 or 16 kind, even more kinds of earthworm protein of the present invention) use together, be determined on a case-by-case basis.In addition, earthworm protein of the present invention can also be united use with other drug and treat-ment, can be confirmed according to correlative factor by those skilled in the art.

The thick enzyme extract of earthworm protein

In one embodiment of the invention, the preparation method of the thick enzyme extract of a kind of earthworm protein is provided, it may further comprise the steps:

1) Eisenia foetida (E.fetida) of choosing 2 monthly ages, 5-8cm length cleans in zero(ppm) water, then earthworm at room temperature is soaked in 12h in the zero(ppm) water, makes it tell the dirt in the most digestive tube.

2) with the homogenate in 0.05M Tris-HCl damping fluid of 1 kilogram of earthworm, in 4 ℃, the centrifugal 30min of 8000rpm.Get supernatant, the ammonium sulfate balance liquid to 85% is in 4 ℃ of dialysed overnight.

3) be carrier with Sepharose-4B (10ml), after carbonyl dimidazoles (800mg) activation, with the coupling of Trypsin inhibitor SBTI aglucon, the preparation affiliation carrier.The affiliation carrier dress post for preparing is used 0.1mol/L NaHCO ₃Damping fluid (pH 8.0) balance pillar.Supernatant adsorbs through last affinity column, and the enzyme of treating effluent is lived when consistent with upper prop liquid, and it is saturated to show that absorption has reached, uses 0.01mol/L NaHCO ₃Damping fluid (pH 8.0) balance liquid flush away does not adsorb foreign protein, and in order to the 6mol/L urea soln wash-out earthworm protein enzyme of balance liquid preparation, urea is removed in dialysis to water, gets earthworm protein enzyme total composition again, and freeze-drying is preserved.

4) with the DEAE-Cellulose-52 dress post of handling well, with 0.01mol/L pH8.0 phosphate buffered saline buffer balance; The crude enzyme liquid that affinity chromatography obtains is dialysed to above-mentioned balance liquid; Upper prop; Use earlier the balance liquid wash-out, more respectively with contain 0.05,0.1,0.15,0.20,0.25, the level pad stepwise elution of 0.3mol/L NaCl, collect the elutriant of each elution peak of correspondence.

5) the earthworm protein enzyme of SEQ ID NO.1 can be further purified according to the method that is provided among the Chinese invention patent ZL02116747.8, thereby obtains one-component.

Thus, aforesaid method according to the present invention obtains the thick enzyme extract of earthworm protein of the present invention.

In another preferred embodiment; The thick enzyme extract of earthworm protein of the present invention comprises the albumen with following aminoacid sequence: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.11; SEQ ID NO.12; SEQ ID NO.14, SEQ ID NO.15, wherein SEQ ID NO.1 abundance is the highest.

It should be noted that at this it is the initial coarse enzyme extract of wash-out from chromatography column that thick enzyme extract of earthworm protein of the present invention is not limited to, can be the mixture of otherwise combined after further purification is one-component.

The present invention also provides a kind of pharmaceutical composition, and it comprises the thick enzyme extract of earthworm protein of the present invention as activeconstituents, and one or more medicinal carriers, vehicle, thinner or auxiliary material.This pharmaceutical composition can be made for suitable formulation by those skilled in the art according to conventional pharmaceutical methods as required.

In another aspect of the present invention, the application of the pharmaceutical composition that relates to the thick enzyme extract of above-mentioned earthworm protein or contain the thick enzyme extract of this earthworm protein in the medicine of preparation treatment or prevention HBeAg relative disease.In a preferred embodiment, the said HBeAg relative disease liver cirrhosis or the tumour that are selected from the hepatitis b virus carrier of hepatitis B, e antigen and/or surface antigen positive, cause by hepatitis B.

Embodiment

Following embodiment only is used to explain the present invention, and the scope that is construed as limiting the invention never in any form.Therefore; For for simplicity; Following embodiment only explains with earthworm protein SEQ ID NO.1 of the present invention with according to one group of thick enzyme extract that process for extracting of the present invention extracts; Because the same catalysis triplet of other albumen disclosed by the invention, thereby has the effect (data not shown) of degraded HBeAg equally with catalysis HBeAg with SEQ ID NO.1.In like manner, the various thick enzyme extract that the present invention extracts is similar in nature, only might be on various proteic abundance difference to some extent, so basic variation takes place in the effect of its degraded HBeAg.Specify that all reagent all are commercially available among the following embodiment as not.

Embodiment 1 earthworm protein of the present invention and HBeAg albumen are hatched test jointly

Experimental technique and result

1. get HBeAg (the magnificent Sheng Ke in Beijing Bioisystech Co., Ltd) (66.7 μ M), add certain density earthworm protein of the present invention (SEQ ID NO.1 albumen) (0～6 μ M) and hatch in 37 ℃, respectively at 15; 30; 45,60,90; 120min sampling, simultaneously with blank HBeAg and earthworm protein of the present invention (SEQ ID No.1) (hatching 120min) at 37 ℃ as contrasting.

2. the above-mentioned sample of hatching is added 5 * SDS-PAGE sample-loading buffer, boiled 10 minutes, carry out 15%SDS-PAGE, at last with coomassie brilliant blue staining.

3.SDS-PAGE see Fig. 1.The result shows that blank HBeAg hatches the 120min protein band at 37 ℃ and do not have considerable change, HBeAg and earthworm protein of the present invention hatch in the product protein band degrade rapidly (seeing Figure 1A and Figure 1B).

Embodiment 2 earthworm proteins of the present invention are to the analysis of HBeAg proteolyze position

Experimental technique and result

1. 66.7 μ M HBeAg albumen and earthworm protein of the present invention (SEQ ID No.1) (6 μ M) are hatched 60min at 37 ℃, with enzymolysis product carry out 15%SDS-PAGE and go to pvdf membrane (Millipore, USA).

2. film is carried out coomassie brilliant blue staining, the main band that enzymolysis is produced carry out the order-checking of N end (instrument is Applied Biosystem Automated Protein Sequencer, Applied Biosystem Inc., USA).

3. proteic effect is from the beginning of the proteic C end of HBeAg to HBeAg to analyze earthworm protein of the present invention by sequencing result (seeing table 1); Right according to enzymatic fragment and former protein fragments molecular weight ratio, analyze earthworm protein of the present invention to the action site of HBeAg near 141 l-arginine and two amino acid of 142 L-glutamic acid.

Table 1.HBeAg albumen and the proteic N terminal sequence of enzymatic fragment

The protein fragments numbering	The N terminal sequence
		a	TMITN
b	TMITN

The western blotting that embodiment 3 earthworm proteins of the present invention and HBeAg albumen are hatched jointly

Experimental technique and result

1. get HBeAg (6.67 μ M); Adding certain density earthworm protein of the present invention (SEQ ID No.1) (0～0.6 μ M) hatches in 37 ℃; In 60min sampling, simultaneously with blank HBeAg and earthworm protein of the present invention (SEQ ID No.1) (hatching 60min) at 37 ℃ as contrasting.

2. the above-mentioned sample of hatching is added 5 * SDS-PAGE sample-loading buffer, boiled 10 minutes, carry out 15%SDS-PAGE.

3.70V constant voltage go to pvdf membrane (Millipore, USA), 1h.

4.5% skimmed milk-PBST (pH 7.4 for 0.01M phosphate buffered saline buffer, 0.05%Tween-20), room temperature sealing 1.5h.

5.1ml HBeAg antibody (Santa, USA), room temperature 1.5h.5% skimmed milk-PBST cleans 3 times, each 10min.

6.1ml the IgG two anti-(Bioisystech Co., Ltd of China fir Golden Bridge in Beijing) of horseradish peroxidase-labeled is hatched 1h for 37 ℃, the PBST washing.

7. film is soaked 3min at colour developing liquid (Bioisystech Co., Ltd of China fir Golden Bridge in Beijing).Exposure.Visible by Fig. 2, the HBeAg albumen after hatching with earthworm protein (SEQ ID No.1) can not be by its antibody recognition.

The ELISA that embodiment 4 earthworm proteins of the present invention and HBeAg albumen are hatched jointly

Experimental technique and result

1) gets HBeAg (6.67 μ M); Adding certain density earthworm protein of the present invention (SEQ ID No.1) (0～0.6 μ M) hatches in 37 ℃; In 60min sampling, simultaneously with blank HBeAg and earthworm protein of the present invention (SEQ ID No.1) (hatching 60min) at 37 ℃ as contrasting.

2) sample of getting after hatching adds hepatitis B virus e antigen diagnostic kit (Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.).

The result sees Fig. 3, and is visible by figure, is degraded after HBeAg and earthworm protein (SEQ ID No.1) are hatched.

The separation and purification of the thick enzyme extract of embodiment 5 earthworm proteins

Experimental technique and result

1. the Eisenia foetida (E.fetida) of choosing 2 monthly ages, 5-8cm length cleans in zero(ppm) water, then earthworm at room temperature is soaked in 12h in the zero(ppm) water, makes it tell the dirt in the most digestive tube.

2. with the homogenate in 0.05M Tris-HCl damping fluid of 1 kilogram of earthworm, in 4 ℃, the centrifugal 30min of 8000rpm.Get supernatant, the ammonium sulfate balance liquid to 85% is in 4 ℃ of dialysed overnight.

3. be carrier with Sepharose-4B (10ml), after carbonyl dimidazoles (800mg) activation, with the coupling of Trypsin inhibitor SBTI aglucon, the preparation affiliation carrier.The affiliation carrier dress post for preparing is used 0.1mol/L NaHCO ₃Damping fluid (pH 8.0) balance pillar.Supernatant adsorbs through last affinity column, and the enzyme of treating effluent is lived when consistent with upper prop liquid, and it is saturated to show that absorption has reached, uses 0.01mol/LNaHCO ₃Damping fluid (pH 8.0) balance liquid flush away does not adsorb foreign protein, and in order to the 6mol/L urea soln wash-out earthworm protein enzyme of balance liquid preparation, urea is removed in dialysis to water, gets earthworm protein enzyme total composition again, and freeze-drying is preserved.

4. with the DEAE-Cellulose-52 dress post of handling well, with 0.01mol/L pH8.0 phosphate buffered saline buffer balance; The crude enzyme liquid that affinity chromatography obtains is dialysed to above-mentioned balance liquid; Upper prop; Use earlier the balance liquid wash-out, more respectively with contain 0.05,0.1,0.15,0.20,0.25, the level pad stepwise elution of 0.3mol/L NaCl, collect the elutriant of each elution peak of correspondence.

5.SEQ the earthworm protein enzyme of ID NO.1 can be further purified according to the method that is provided among the Chinese invention patent ZL02116747.8, thereby obtains one-component.

(see figure 4).

The content and the activity of 68 kinds of thick enzyme extract isozymes of earthworm protein of embodiment

Experimental technique and result

1. assay is pressed the operation of the Gycoprotein Carbohydrate Estimation Kit of PIERCE company specification sheets.

2. the method for gray scale scanning behind the Fibrin-PAGE is adopted in the detection of relative reactivity.On the basis of conventional polyacrylamide gel electrophoresis, when the preparation separation gel, add the Fibrinogen of the 20mg/mL of 1.5mol/L pH8.9 Tris-HCl damping fluid preparation; Making its final concentration is 0.4mg/mL, adds an amount of zymoplasm, forms scleroproein with the catalysis fibre proteinogen; Add sodium lauryl sulphate (SDS), making its final concentration is 0.1%, with appearance on the earthworm homogenate of difference amount; Electrophoresis places 1%Triton-100 solution to soak 1h in glue after finishing, and then puts into one times of PBS (pH7.2) solution and soaks; In 37 ℃ of insulation certain hours, with Xylene Brilliant Cyanine G R-250 dyeing 10min, acetate decolouring (seeing table 3) with 7%.The corresponding relation of isolating each component and sequence of the present invention is seen table 4.

The combined compsn of table 3. earthworm HBeAg degrading enzyme

HB?eAgase	Sugar content (%)	Relative reactivity (%)	Apparent molecular weight (kD)
				EfP-0-1	6.13	6.2	22.5
EfP-0-2	4.30	12.8	22.4
				EfP-I-1	6.18	25.8	28.8
EfP-I-2	1.38	31.6	28.1

EfP-II-1	1.82	8.8	30.6
				EfP-II-2	7.40	2.1	29.1
EfP-III-1	6.55	12.5	34.8
				EfP-III-2	4.41	2.3	35.0
Blending ingredients	4.40	100	-

The corresponding relation of table 4. earthworm HBeAg degrading enzyme and each sequence of the present invention

HB?eAgase	SEQ?ID?NO.
		EfP-0	SEQ?ID?NO.14
EfP-I-1	SEQ?ID?NO.15
		EfP-I-2	SEQ?ID?NO.16
EfP-II-1	SEQ?ID?NO.11
		EfP-II-2	SEQ?ID?NO.12
EfP-III-1	SEQ?ID?NO.1
		EfP-III-2	SEQ?ID?NO.2

Embodiment 7 earthworm proteins of the present invention are to the therapeutic action of HBV transgenic mice

Experimental technique and result

Laboratory animal: C57BL/6J-HBV transgenic mice (from Department Of Medicine, Peking University laboratory animal portion).The gene fragment that changes over to is the S gene of encoded packets membranin HBsAg; (reference: Dong Yuhong, seat are magnificent, Tian Feng, Kang Aijun for preS gene and coding X antigenic X gene; Kurarinone is to the experiment of influence China and the clinical virology magazine of HBsAg transgenic mice serum T h1 and Th2 cytokine levels; 2004,18 (3) 277-280.Chisarl FV.Hepatitis B virus transgenic mice:insights into the virus and the disease.Hepatology; 1995,22:1316-1325.) mouse experimentizes in Department Of Medicine, Peking University laboratory animal portion meets the environment of SPF level animal rearing standard.

2. experiment is divided into groups: the screening of testing the action thing that advances; (operation steps of buying test kit according to commerce is measured and is passed judgment on to select the HBsAg positive; Hepanostika

HBsAg Ultra; Biom é rieux; Netherlands) 27 of laboratory animal divide into groups 9 every group according to the height (with the OD value representation) of secreting HBsAg in the HBV transgenic mice serum.

Negative control: saline water (0.9%NaCl)

Positive control: lamivudine (irritating stomach dosage is 100mg lamivudine/kg mouse body weight)

The thick enzyme extract of earthworm protein (lyophilized powder) that medicine: embodiment 5 obtains, irritating stomach dosage is 50mg extract/kg mouse body weight)

3. dosage regimen:

(1) irritates stomach: irritate stomach every day once, continue 21 days, observe to 30 days.

(2) get blood: before medication, blood was got in medication in 10 days, 20 days respectively, and preparation serum is measured glutamate pyruvate transaminase (ALT), and (IFCC of the northern control of life company recommends method to glutamate oxalo-acetate transaminase (AST) in the use, and instrument is the 7170A of a Hitachi type automatic clinical chemistry analyzer.Operation steps is operated according to the test kit specification sheets).After medication, got blood in 7,14,21,30 days; Preparation serum; Measuring HBsAg (uses test kit to be: Hepanostika HBsAg Ultra; Biom é rieux, Netherlands, detecting instrument are BIO-TEK Instruments EL311 type ELIASA.Operation steps is operated according to the test kit specification sheets).

Because the mouse Q volume of blood is limited, in order to guarantee the life quality of mouse, get the blood volume at every turn and generally be controlled at below the 0.2ml, and time enough at interval, so the blood time of getting of measuring ALT, AST with measure HBsAg get the blood asynchronism(-nization).

(3) get tissue: after experiment was got blood the last day and accomplished, disconnected neck was put to death mouse, collects hepatic tissue, and part for storage is fixing in 4% formaldehyde, the preparation paraffin section, and HE dyeing is carried out hepatic pathology and is changed and observe; Part for storage is subsequent use in liquid nitrogen.

4. detection index: (1) body weight; (2) glutamate pyruvate transaminase (ALT); (3) glutamate oxalo-acetate transaminase (AST); (4) HBsAg; (5) to the pathological observation of liver.

5. experimental result: along with the carrying out of experiment, three experimental group animal activity states do not have the difference of significance.Body weight does not have significant difference yet.Compare with the saline water group, the transgenic mice serum alt, AST rises and suppresses to some extent, and the result sees Fig. 5 A, 5B.Compare with the saline water group, the transgenic mice HBsAg in serum decreases, and the result sees Fig. 5 C.Pathological observation result to the transgenic mice liver sees Fig. 5 D.A: the saline water group, the liver lobule structure exists, and the liver cell oedema is outstanding with the liver lobule central area.Liver sinusoid and central vein are obviously expanded, some kitchen range shape hepatic necrosis, visible 12 the hepatic necrosis kitchen ranges of per 10 low-power fields.B: the liver lobule structure exists, and the liver cell oedema is outstanding with the liver lobule central area.Liver sinusoid and central vein are obviously expanded, some kitchen range shape hepatic necrosis and a small amount of monokaryon lymphocytic infiltration, visible 4 the hepatic necrosis kitchen ranges of per 10 low-power fields.C: the liver lobule structure exists, and the liver cell oedema is outstanding with the liver lobule central area.Liver sinusoid and central vein are obviously expanded, the sex change of special mess shape liver cell microvesicle shape fatty, visible 4 the hepatic necrosis kitchen ranges of per 10 low-power fields.6 transgenic mices of saline water group; 6 transgenic mices of earthworm protein group of the present invention show with the hepatic tissue pathology sections observation result of 6 transgenic mices of lamivudine group: compare with the saline water group; The hepatic necrosis pathology of earthworm protein laboratory animal group HBsAg transgenic mice alleviates, and curative effect and lamivudine group are similar.

The result shows, compares with the saline water negative control group, and the transgenic mice serum alt, AST rises and is suppressed, and the HBsAg secretion is suppressed, and these two indexs all are superior to the result of treatment of lamivudine.The serum FN of saline water group and lamivudine group transgenic mice increases with the prolongation of survival time, use earthworm protein of the present invention after content maintain the constant level basically.Pathological observation result to liver shows that the earthworm protein group has therapeutic action to hepatitis B, compares with the saline water group, and the hepatic necrosis pathology of HBV transgenic mice is alleviated, and curative effect and lamivudine group are similar.Therefore, the present invention proves that in HBV transgenic mice level earthworm protein of the present invention has certain restraining effect to the HBsAg secretion of hepatitis B transgenic mice, keeps the useful help of normal level to Serum ALT, AST.Hepatitis B transgenic mice pathology section examination finds that earthworm protein of the present invention has the certain protection effect to the hepatic disease of HBV transgenic mice.

Embodiment 8 earthworm proteins of the present invention are to the effect of HepG 2.2.15 cell levels

1. clone and culture condition: (Military Medical Science Institute gives HepG 2.2.15 clone; Derive from Mount Sinai medical center, New York at first); Be characterized in inserting in the genome HVB gene (reference: Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA, Proc.Natl.Acad.Sci.USA, 1987; 84,1005-1009).Substratum be the DMEM high glucose medium (Dulbecco ' s modified eagle medium, Gibco, Invitrogen Corporation; USA), add simultaneously 10% foetal calf serum (PAA, Austria); 380 μ g/ml microbiotic G-418 (Amresco; USA) and penicillium mould-Streptomycin sulphate solution (final concentration is 100units/ml penicillium mould and 100 μ g/ml Streptomycin sulphates, HyClone, USA).Culture condition is CO ₂Incubator, 37 ℃ of cultivations.

2. experiment is divided into groups:

Positive control: lamivudine treatment group;

Medication group: earthworm protein treatment group of the present invention (one-component SEQ ID NO.1).

3. therapeutic regimen

The HepG2.2.15 cell is inoculated (96 orifice plates, every hole 1 * 10 ⁴Individual cell).Behind the cell inoculation three days, add the earthworm protein of the present invention (substratum Chinese traditional medicine final concentration is 0-5 μ g/ml) and the lamivudine (substratum Chinese traditional medicine final concentration is 0-100 μ M) of different concns respectively.Cell inoculation is changed corresponding pastille substratum in the time of six days.The collecting cell substratum is used to measure HBsAg, HBeAg when the cell inoculation Ninth Heaven.

4.HBsAg; The measuring method of HBeAg: with the centrifugal 5min of cell culture medium 12000rpm of step 3 collection; Get in right amount, employing hepatitis B virus s antigen and e antigen diagnose reagent kit (ELISA, Huamei Bio-Engrg Co.) step are to specifications measured.

5. experimental result is seen Fig. 6, and the result shows, compares with blank cell, earthworm protein treatment group HBsAg of the present invention, and the secretion of HBeAg all is suppressed, HBsAg in the cell culture medium, HBeAg content descend to some extent (Fig. 7 A).

Experimental result shows, compares with blank cell, earthworm protein treatment group HBsAg of the present invention, and the secretion of HBeAg all is suppressed, HBsAg in the cell culture medium, HBeAg content descends to some extent.(Fig. 7 B) compares with the lamivudine positive controls, and earthworm protein of the present invention has stronger restraining effect to the secretion of HBeAg.

Reference

1.Stephen?E?L，Josephine?P?S，Lisa?R?B，et?al.Clearance?of?hepatitis?B?e?antigen?in?patients?with?chronic?hepatitis?B?and?genotypes?A，B，C，D?and?F.Gastroenterology，2007，133(5)：1452-1457

2.Milieh?D，Liang?T?J.Exploring?the?biological?basis?of?hepatitis?B?eantigen?in?hepatitis?B?virus?infection.Hepatology，2003，38(5)：1075-1086

3.Ou?J?H.Mlolecular?biology?of?hepatits?B?virus?e?antigen.Gastroenterol?Hepatol，1997，12：178-187

4.Parekh?S，Zoulim?F，Alan?S?H，et?al.Genome?replication，virion?secretion，and?e?antigen?expression?of?naturally?occurring?hepatitis?B?virus?core?promter?mutants.J?Virol，2003，77：6601-6612

5.Riedl?P，Stober?D，Oehninger?G，et?al.Priming?Thl?immunity?to?viral?core?particles?is?facilitated?by?trace?amount?of?RNA?bound?to?its?arginine-rich?domain.J?Immunol，2002，168：4951-4959

6.Luiz?Caetano?da?Silva；Maria?Luiza?da?Nova；Suzane?Kioko?Ono-Nita，et?al.Simultaneous?quantitation?of?serum?HBV?DNA?and?HBeAg?can?distinguish?between?slow?and?fast?viral?responses?to?antiviral?therapy?in?patients?with?chronic?hepatitis?B.Rev.Inst.Med.trop.S.Paulo(2009)，51(5)：261-268

Claims

1. earthworm protein has a kind of sequence that is selected from following (a)-(b):

(a) aminoacid sequence of SEQ ID NO.1-16 shown in each;

(b) with (a) described in the aminoacid sequence of any aminoacid sequence with the homology more than 80%,

Wherein above-mentioned aminoacid sequence all has by H ⁴³, D ⁹¹, S ¹⁸⁸The catalysis triplet of forming, and said earthworm protein has the HBeAg degrading enzymatic activity.

2. earthworm protein as claimed in claim 1 is characterized in that said HBeAg degrading enzymatic activity is the C end beginning from HBeAg, at 141 l-arginine and 142 L-glutamic acid place degraded HBeAg.

3. a pharmaceutical composition comprises at least a claim 1 or 2 described earthworm proteins as activeconstituents, and one or more medicinal carriers, vehicle, thinner or auxiliary material.

4. the preparation method of the thick enzyme extract of earthworm protein is characterized in that may further comprise the steps:

2), supernatant is dialysed with earthworm homogenate, centrifugal;

5. thick enzyme extract of earthworm protein by the preparation of the described method of claim 4.

6. the thick enzyme extract of earthworm protein as claimed in claim 5 is characterized in that comprising at least a or two above earthworm proteins according to claim 1 or claim 2.

7. like claim 5 or the thick enzyme extract of 6 described earthworm proteins; It is characterized in that comprising albumen: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.11 with following aminoacid sequence; SEQ ID NO.12; SEQ ID NO.14, SEQ ID NO.15, wherein SEQ ID NO.1 abundance is the highest.

8. pharmaceutical composition comprises among the claim 5-7 any thick enzyme extract of described earthworm protein as activeconstituents, and one or more medicinal carriers, vehicle, thinner or auxiliary material.

9. each described earthworm protein or pharmaceutical composition among the claim 1-3, or any thick enzyme extract of described earthworm protein or the application of pharmaceutical composition in the medicine of preparation treatment or prevention HBeAg relative disease among the claim 5-8.

10. application as claimed in claim 9 is characterized in that liver cirrhosis or tumour that said HBeAg relative disease is selected from the hepatitis b virus carrier of hepatitis B, e antigen and/or surface antigen positive, is caused by hepatitis B.

11. like each described earthworm protein or pharmaceutical composition among the claim 1-3, or like thick enzyme extract of the described earthworm protein of claim 5-8 or the application of pharmaceutical composition in the reagent of preparation diagnosis HBeAg.