CN105031613B - A kind of human beta-defensin 1 is preparing the application in treating or preventing hepatitis B virus infective medicament - Google Patents

Abstract

The invention discloses a kind of beta-defensin 1 to prepare the application in treating or preventing hepatitis B virus infective medicament.Influence of the beta-defensin 1 to hepatitis B replication is studied by way of the siRNA and HBV1.3 times of constitution grain of the overexpression plasmid of cotransfection beta-defensin 1 or targeting beta-defensin 1 into liver cell; it have detected the surface antigen and e antigens of viral secretory; and intracellular virus nucleocapsid correlation DNA level, display beta-defensin 1 suppress the duplication of hepatitis type B virus in liver cell.Beta-defensin 1 may influence HBV activity or influence HBV infection or metainfective reproduction process.Therefore, by being used alone or being used in combination with other Anti-HBV drugs, beta-defensin 1 can play a significant role in preventing or treating infection course of hepatitis B virus.Human beta-defensin 1 is a kind of antimicrobial polypeptide of the constructive expression in human body, does not have toxic side effect to human body under physiological concentration.

Description

A kind of people's beta-alexin -1 is preparing treatment or prevention hepatitis B virus infective medicament In application

Technical field

The present invention relates to antiviral drugs field, is related to a kind of the new of people's beta-alexin -1 (β-defensin-1, hBD1) Purposes, it is more particularly to a kind of people's beta-alexin -1 and is preparing treatment or prevention hepatitis type B virus (Hepatitis B Virus, HBV) application in infection medicine.

Background technology

Hepatitis type B virus (Hepatitis B virus, HBV) infection is to cause one of principal element of liver cancer, seriously Threaten the life and health of the mankind.China is hepatitis b virus infected district occurred frequently, there are about 1.2 hundred million Chronic Hepatitis Bs, often Year there are about 300,000 people and die from hepatocellular carcinoma caused by chronic hepatitis B.HBV belongs to Hepadnaviridae, and genome is partially double stranded Cyclic DNA.The HBV a diameter of 28nm of core granule, is 20 face bodies, and viral capsid is made up of core protein HBc.The table of nucleocapsid There is a coating in face, surface antigen preS1, preS2 and HBsAg containing three kinds of sizes on coating.The virion of infectious is again Referred to as Dane particles, a diameter of 42nm.HBV infection can cause a series of liver diseases, from oxyhepatitis (including fulminant liver MSOF) arrive chronic hepatitis, hepatic sclerosis and hepatocellular carcinoma.Hbv replication does not directly result in cytopathy, most HBV in itself Carrier is asymptomatic, and also very little, the antiviral immunity of host are to cause the main original of the clinical symptoms such as hepatic injury to hepatic lesion Cause.Symptom after HBV infection human body is related to the age of infected person and immunity.Hepatitis 99% is above is without disease in neonate Shape, in 1-5 year children, only 10% is Symptomatic./ 3rd adult acute's hepatitis B patient has clinical symptoms, Wherein about 1% is explosive hepatitis, and fatal rate reaches 70%.Explosive hepatitis is along with strong antiviral immunity, and virus is very It is eliminated soon.Virus can be spontaneously removed after 95% Adult infections, and 90% child and 30% children of 1-5 year are then Chronic infection can be caused.The diagnosis of HBV infection and relevant disease is according to a series of clinical biochemistry, tissue and serological Index is carried out.The main virulent several antigens of index of detection virus infection, and corresponding specific antibody and HBV DNA；Detection patient's hepatic disease situation mainly has blood alanine and glutamic-pyruvic transaminase (Alanine Transaminase, ALT), and hepatic tissue detection inflammation and fibrosis.

Clinically hepatitis b virus infected treatment mainly has two class medicines at present：Interferon and nucleoside analog.Face The interferon of bed application includes IFN α 2a, IFN α 2b and IFN α 1b, and these are traditional interferon, in addition to PEG-IFN α 2a with PEG-IFNα2b.Nucleoside analog Lamivudine (Lamivudine, LAM) and Sebivo (telbivudine, LDT)；Deoxidation Guanosine analogue Entecavir (Entecavir, ETV)；Nucleoside phosphate ester, Aldoforwe ester (Adefovir, ADV) and fumaric acid Tenofovir disoproxil (tenofovir disoproxil fumarate, TDF).Wherein ETV, TDF and PEG-IFN are the most frequently used.IFN The history of 30 years has been had more than for treating chronic HBV infection.Clinically IFN α was for patient positive HBeAg, at 6 months Interior, the patient HBeAg that inject three times, has 25% weekly turns out cloudy.The IFN α treatment results of 12 months show 10-17% patient HBsAg turns out cloudy.For the negative patients of HbeAb, examined after the IFN α treatment of 12 months in 60% patients serum less than HBV DNA.The IFN modified through PEG is PEG-IFN, and its antiviral-mechanism is identical with IFN α, but significantly improves the drug metabolism of IFN α Dynamics, frequency injection are weekly, and IFN α concentration can maintain higher level in serum.One research table of 2003 Bright, PEG-IFN therapeutic effect is better than IFN.But then, PEG-IFN also has its side effect and limitation, if the initial stage for the treatment of It is ineffective, then it should discontinue medication, consider other treatment schemes.Nucleoside analog is pressed down by suppressing HBV RNA reverse transcription HBV processed duplication.Compared with IFN, nucleoside analog has some advantages, such as Small side effects, conveniently etc..But also there are some to lack Point, such as high relapse rate, low HBsAg negative conversion rates, drug resistance are easily produced during single drug.

Alexin (defensin) is a kind of antimicrobial polypeptide, including α-, β-, θ-alexin, be that host resists an invasion The part of the innate immune reaction of pathogen, can resist includes gram-positive bacterium, gramnegative bacterium, fungi, There are the various microorganisms including coating and nonencapsulated virus.People's beta-alexin -1 (Human β-defensin-1, hBD1) is more Constructive expression in the epithelial cell of kind organ, there are some researches show the missing of the hBD1 constructive expression in Crohn disease may It is relevant with PPAR γ mutation, and expression quantity increase in microorganism infection and inflammatory reaction, it is considered to be in epithelial cell A kind of most important antimicrobial polypeptide.HBD1 expression is also regulated and controled by epigenetic, and HDAC1 adjusts hBD1 expression, suppression The expression of system and siRNA silences HDAC1 up-regulations hBD1.HBD1 is at testis epithelial cell, intestinal epithelial cell, cornea, star angle High expression in the epithelial cells such as cell plastid.HBD1 high expression, concentration in milk in galactophore epithelial cell can reach 1- 10 μ g/ml, hBD1 is prompted to be played an important role in mammary gland antibacterium and neonate resist pathogenic microorganism.HBD1 is in liver cell Also there is higher expression, the expression quantity for having studies have shown that hBD1 declines in HCC, but temporarily not on hBD1's and HBV The research of correlation.Research to the antivirus action of alexin family member is concentrated mainly on α-alexin to HIV, HSV Deng effect.HNP1-4 AntiHIV1 RT activity effect mainly includes, and directly inactivates virion；Prevention disease is combined with gp120 and CD4 Poison invasion；Raise chemotactic factor (CF) MIP-1 α and MIP-1 β；Suppress PKC activity etc..On -1 pair of viral effect of beta-alexin Research there was only its HIV-resistant activity, its specific mechanism is unclear.

The content of the invention

The purpose of the present invention is to be the provision of a kind of people's beta-alexin -1 to prepare treatment or prevention hepatitis type B virus Application in infection medicine, it is safe and efficient and small toxic side effect more so as to provide one kind for the treatment of clinically hepatitis B Peptide.By being used alone or being used in combination with other Anti-HBV drugs, people's beta-alexin -1 can prevent or treat it is B-mode Played a significant role during hepatites virus infections.

In order to realize above-mentioned purpose, the technical solution adopted by the present invention is：

A kind of people's beta-alexin -1 is preparing the application in treating or preventing hepatitis B virus infective medicament, its step It is：

A, the evaluation of the anti-hepatitis B virus activity of people's beta-alexin -1：

B, by people hBD1 overexpression plasmid (this laboratory structure) or siRNA (synthesis of Guangzhou Rui Bo companies) and energy HBV plasmid pHBV1.3 (D types, construction method see below) cotransfections are expressed to hepatic cell line HepG2 (coming from ATCC) or Huh7 In (coming from ATCC), the expression quantity of HBV secretion antigens HBeAg and HBsAg in cells and supernatant (China of Shanghai section) is then detected DNA related to intracellular hbv replication intermediate nucleocapsid level.

This experimental result is as shown in Figure 1.It is overexpressed and interference experiment all proves that hBD1 can be lowered in cells and supernatant HBV secretion antigens HBeAg DNAs related to intracellular hbv replication intermediate nucleocapsid to HBs expression quantity level, explanation HBD1 suppresses hbv replication, has the activity of anti-hepatitis B virus.

Described hBD1 overexpression plasmid, its building process are shown in embodiment 1.

Described plasmid pHBV1.3, its building process are the DNA of extraction HepG2.2.15 cells (coming from ATCC), amplification HBV DNA and insertion vector pBluescript II (being purchased from Invitrogen).

Described anti-hepatitis B medicine is using people's beta-alexin -1 as active constituents of medicine, can be made using routine techniques Piece agent, capsule, granule, oral liquid, sustained release preparation, controlled release preparation, nanometer formulation (active component content 5% (mass ratio, It is same as below), remaining is corresponding auxiliary material (90% starch and 5%PVP)) or injection (specification 1mg/mL, is dissolved in physiology salt Water) any formulation pharmaceutically received.

The present invention compared with prior art, has advantages below and effect：

1. people's beta-alexin -1 is a kind of antimicrobial polypeptide of the constructive expression in human body, to human body under physiological concentration There is no toxic side effect.

2. the Antiviral mechanism of alexin is by directly acting on peplos, or influences viruses adsorption, or is changed Become intracellular signal path, so as to antiviral, because of its antiviral mechanism variation, be not easy to make virus produce drug resistance.

Brief description of the drawings

Figure 1A is a kind of schematic diagram for proving to be overexpressed e antigens and the secretion of s antigens that hBD1 suppresses HBV.Wherein e antigens press down Restrict 50%, s antigens and suppress about 30%.

Figure 1B is a kind of schematic diagram for proving to be overexpressed the amount for the nucleocapsid correlation DNA that hBD1 lowers HBV.Suppress about 40%.

Fig. 1 C are that the diagram that a kind of proof si-hBD1 has preferable interference effect is anticipated.

Interference falls 80%.

Fig. 1 D are a kind of e antigens for proving to disturb hBD1 to promote HBV and the schematic diagram of s antigens secretion.

E antigens and s antigens raise more than 2 times.

Fig. 1 E are a kind of schematic diagram of the amount for the nucleocapsid correlation DNA for proving to disturb hBD1 to raise HBV.

About 1.8 times of up-regulation.

Fig. 2 is the graph of a relation for transfecting different pCAGGS-hBD1 plasmids amounts and cell survival rate.

Embodiment

Content for a better understanding of the present invention, present invention is made furtherly with reference to specific implementation method It is bright, but the protection content of the present invention is not limited to following examples.

Embodiment 1：By the Anti-HBV effect for being overexpressed experimental evaluation people beta-alexin -1.

A kind of people's beta-alexin -1 is preparing the application in treating or preventing hepatitis B virus infective medicament, its step It is：

1. experiment material：

1.1 cells, plasmid：

HepG2 cells (coming from ATCC) and pHBV1.3 plasmids.Using the total mRNA extracted from Huh7 cells as template, first Total cDNA is expanded by reverse transcription PCR, then using the cDNA as template, amplifies complete hBD1 genetic fragments with special primer, draws Thing sequence is as follows：

hBD1CDS Forward:5’-CGCGAATTCGATGAGAACTTCCTACCTTC-3’

hBD1CDS Reverse:5’-TATCTCGAGCTTGCAGCACTTGGCCTTC-3’

By specific restriction restriction endonuclease (EcoR I and Xho I) digestion, connect hBD1 gene clonings to carrier On pCAGGS (being purchased from addgene), pCAGGS-hBD1 overexpression plasmid, and sequencing identification are obtained.

1.2 reagent：

DMEM culture mediums are purchased from GIBCO companies；Restriction enzyme and T4 ligases are purchased from NEB companies.

1.3 laboratory apparatus：

Roche LightCycler 480

2. experimental method：

Human liver cell system HepG2 is inoculated into 24 orifice plates, after about 12h, after cell density grows to about 70%, turned Dye.With Lipofectamine-2000 by pCAGGS/pCAGGS-hBD1 and pHBV1.3 cotransfections, renew within 4 hours after transfection fresh Nutrient solution.After transfection after 48 hours, the expression of the e antigens and s antigens in cells and supernatant is detected with ELISA kit Amount；And DNA related to detection HBV nucleocapsids is extracted, method is as follows：A, supernatant is abandoned, 100 microlitres of cell pyrolysis liquid is added per hole (50mM Tris, 0.5%NP-40,1mM EDTA and 100mM NaCl), crack 1 hour on 4 DEG C of shaking tables, be transferred to 1.5 milliliters In EP pipes.B, often pipe adds 1 microlitre of 1M magnesium chloride, and 1 microlitre of DNaseI, and 37 DEG C are incubated 2 hours.C, often pipe adds 3.5 microlitres 0.5M EDTA and 22.5 microlitre of 35%PEG, 4 DEG C are incubated 1 hour；4 DEG C, 12000rpm is centrifuged 1 minute.D, supernatant is abandoned, is often managed Add 200 microlitres of re-suspension liquids (10mM Tris, 100mM Nacl, 1mMEDTA and 1%SDS), and 5 microlitres of Proteinase K (25mg/ ML), 55 DEG C of overnight incubations.E, often pipe adds 100 microlitres of Tris saturated phenols and 100 microlitres of chloroforms, and vibration mixes, room temperature (20- 25 DEG C are same as below) 12000rpm centrifuge 10 minutes.F, supernatant is taken, adds isometric isopropanol precipitating, room temperature, 12000rpm Centrifugation 10 minutes, supernatant is removed, receive precipitation.G, the ethanol that 1 milliliter of volume ratio is 75% is added, 12000rpm is centrifuged 5 minutes, is repeated Once, residual ethanol is blotted, is dried.H, it is dissolved in 20 microlitres of dH2O.I, fluorescence quantitative PCR detection HBV, with gradient dilution PHBV1.3 plasmids do standard curve, calculate the HBV DNA copy numbers in sample.The primer and probe sequence are HBV DNAForward:5’-ACCCAAGGCACAGCTTGGAGG-3’；HBV DNAReverse:5’- AGATGATTAGGCAGAGGTGAAAAA-3’；HBV probe sequences：5’-TGGCTAGTTTACTAGTGCAATTTTG-3’

3. experimental result：

As shown in Figure 1A, 1B, expression quantity and intracellular hbv replication intermediate that hBD1 lowers e antigens and s antigens are overexpressed Nucleocapsid correlation DNA level, illustrate that hBD1 suppresses hbv replication, hBD1 has Anti-HBV effect.

Embodiment 2：Pass through the Anti-HBV effect of interference experiment appraiser beta-alexin -1.

A kind of people's beta-alexin -1 is preparing the application in treating or preventing hepatitis B virus infective medicament, its step It is：

1. experiment material：

RNA interfering set meal is purchased from Guangzhou Rui Bo companies.

2. experimental method is same as Example 1.

3. experimental result：

The interference effect of RNA interfering is have detected first, and as shown in Figure 1 C, it is about 80% to suppress efficiency.As shown in Fig. 1 D, 1E Disturb hBD1 up-regulations HBeAg DNAs related to intracellular hbv replication intermediate nucleocapsid to HBsAg expression quantity level, explanation HBD1 suppresses hbv replication, and hBD1 has Anti-HBV effect.

Other steps are same as Example 1.

The experimental result of above example 1 and embodiment 2 illustrates that hBD1 has the function that the duplication for preferably suppressing HBV, Again because hBD1 is the less secretory protein of molecular weight of constructive expression in human body, and the machine with a variety of suppressing virus replications System, is not likely to produce drug resistance, the antiviral drugs for also not having alexin or derivatives thereof to develop so far, has innovation well Property.

Influences of the embodiment 3MTS detection transfections pCAGGS-hBD1 to cell survival rate

The pCAGGS-hBD1 plasmids of transfection corresponding amount are opposed into HepG2 cells, and with pCAGGS zero loads as shown in Figure 2 According to, detect cell survival rate with MTS (being purchased from Promega) within 48 hours after transfection, test result indicates that, transfect pCAGGS-hBD1 HepG2 cell survival rates are not influenceed.

Claims (2)

1. people's beta-alexin -1 is preparing the application in treating or preventing hepatitis b virus infected medicine.

2. application as claimed in claim 1, it is characterised in that：Described medicine is to be used as pharmaceutical activity using people's beta-alexin -1 Composition, tablet, capsule, granule, oral liquid, sustained release preparation, controlled release preparation, nanometer formulation or injection is made.