CN1164331C - Human hepatitis B nucleic acid vaccine - Google Patents

Abstract

The present invention relates to a human hepatitis B nucleic acid vaccine which belongs to the technical field of medicinal biology. The human hepatitis B nucleic acid vaccine is eukaryotic biologic expression plasmid pVAX-PS-CpG jointly composed of a transcription expression unit and a human body specific immunity activation sequence (CpG sequence: 20062103), wherein the transcription expression unit is composed of human hepatitis B adr subtype surface antigen genes preS2-S. Animal experiment results indicate that the present invention possibly performs favorable action of immunization and protection on patients of human hepatitis B and performs the therapeutic action. The nucleic acid vaccine has the advantages of simple production and purification processes, stable physicochemical property, and convenient storage and transportation.

Description

A kind of human hepatitis B nucleic acid vaccine

The present invention relates to the medical biotechnology field, is a kind of human hepatitis B nucleic acid vaccine.

Hepatitis B is a kind of infectious disease that is caused by hepatitis B virus (HBV), is the whole world important health problem that particularly developing country faced.Therapeutic strategy to hepatitis B mainly contains three aspects at present: Chinese medicine and western medicine associating liver protecting therapy, chemical synthetic drug (as LAMIVODINE etc.) suppresses hepatitis virus and duplicates, and cytokine (as IFN-α etc.) protects the liver and suppresses virus replication synergy treatment hepatitis.But limitation is in various degree all arranged, cause therapeutic effect not satisfactory.The control that appears as a series of infectious disease such as hepatitis of dna vaccination provides new hope.Nucleic acid vaccine has following outstanding advantage: (1) can bring out body fluid and the comprehensive immunne response of cell, and existing preventive effect has therapeutical effect again; (2) immunological adjuvant CpG sequence can be integrated in the nucleic acid vaccine, improve the immunne response level of body and need not to add in addition adjuvant; (3) the immunoprotection time is long; (4) simple, with low cost, good stability of production technology and storing are convenient.

The mechanism of action of nucleic acid vaccine is: eukaryon expression plasmid is gone in clone's hepatitis B virus surface antigen (HBsAg) gene recombinaton, be injected in human muscular tissue, make it at myocyte's invading the exterior da virus surface antigen, through the angtigen presentation process, can activate intravital immunity system and cell immune system.Activated immunity system can produce special antibody, eliminates the virus that is free in the blood, the invasion of prevention hepatitis B virus; And activated cell immune system can produce cellulotoxic lymphocyte (CTL), attacks by the hepatocyte of viral infection, eliminates the virus that is hidden in the cell, and therefore, hepatitis B nucleic acid vaccine not only has preventive effect but also therapeutical effect is arranged.

Experimentation in recent years shows, CpG sequence in the DNA of bacteria plays important effect in bringing out immune response, can produce the extensive influence to body immune system by the direct and indirect action to B cell, M φ, DC, NK cell, T cell and hemopoietic forebody cell.CpG immune activation sequence has broad application prospects as the adjuvant that a kind ofly effectively excites, enhancing human body immunity is replied.

Though existing lot of documents report carries out the study on prevention of hepatitis B with hbsag gene and CpG sequence construct nucleic acid vaccine, but used gene multi-source is in the common ayw hypotype hepatitis B virus of foreign country, simultaneously the CpG sequence only also mostly is the sequence of can special stimulation mouse immune replying, and people's immune system is not but had activation.Do not see bibliographical information as yet with the hepatitis B virus surface antigen PreS2-S gene of the modal adr hypotype of Chinese and the hepatitis B nucleic acid vaccine of human body specific immune activation sequence structure.

The purpose of this invention is to provide a kind of hepatitis B nucleic acid vaccine that makes up with the hepatitis B virus surface antigen PreS2-S gene and the human body specific immune activation sequence CpG of the modal adr hypotype of Chinese.

The present invention is pVAX (available from Invitrogen company, its sequence and physical map are seen the catalogue of the said firm) in order to the basic framework that makes up carrier for expression of eukaryon; In building process and the preparation process, the used host bacterium of plasmid amplification is DH10B (available from a GIBCO company); The exogenous gene that inserts is: the hepatitis B virus surface antigen PreS2-S gene of the modal adr hypotype of Chinese; The CpG sequence of inserting is the immune activation sequence at human body; Its structure division is as follows:

(1) the hepatitis B virus surface antigen PreS2-S gene of adr hypotype

A) sequence signature

Length: 846bp

Type: nucleic acid

Chain number: two strands

Geometry: linearity

B) molecule type: DNA

C) source: adr hypotype hepatitis B virus

D) sequence description: SEQ ID NO:PreS2-S

1 atgcagtgga?actccaccac?atttcaccaa?gtcctgctag?atcccagagt?gaggggccta

61 tattttcctc?ctggtggctc?cagttccgga?acagtaaacc?ctgttgcgac?tactgcctca

121?cccatatcgt?caatctcctc?gaggactggg?gaccctgcac?cgaacatgga?gagcacaaca

181?tcaggattcc?taggacccct?gctcgtgtta?caggcggggt?ttttcttgtt?gacaagaatc

241?ctcacaatac?cacagagtct?agactcgtgg?tggacttctc?tcaattttct?agggggagca

301?cccacgtgtc?ctggccaaaa?ttcgcagtcc?ccaacctcca?atcactcacc?aacctcttgt

361?cctccaattt?gtcctggcta?tcgctggatg?tgtctgcggc?gttttatcat?attcctcttc

421?atcctgctgc?tatgcctcat?cttcttgttg?gttcttctgg?actaccaagg?tatgttgccc

481?gtttgtcctc?tacttccagg?aacatcaacc?accagcacgg?gaccatgcaa?gacctgcacg

541?attcctgctc?aaggaacctc?tatgtttccc?tcttgttgct?gtacaaaacc?ttcggacgga

601?aactgcactt?gtattcccat?cccatcatcc?tgggctttcg?caagattcct?atgggagggg

661?gcctcagtcc?gtttctcctg?gctcagttta?ctagtgccat?ttgttcagtg?gttcgtaggg

721?ctttccccca?ctgtttggct?ttcagttata?tggatgatgt?ggtattgggg?gccaagtctg

781?tacaacatct?tgagtccctt?tttacctcta?ttaccaattt?tcttttgtct?ttgggtatac

841?atttga

(2) at the immune activation sequence C pG of human body

A) sequence signature

Length: 83bp

Type: nucleic acid

Chain number: two strands

Geometry: linearity

B) molecule type: DNA

C) source: synthetic

D) sequence description: SEQ ID NO:CpG (20062103)

ggc?cgc?tcg?tcg?ttt?tgt?cgt?ttt?gtc?gtt?ttt?cgt?cgt?ttt?gtc?gtt?ttg?tcg?ttt?ttc

gtc?gtt?ttg?acg?ttt?tga?cgt?tt

Adr hypotype hepatitis B virus surface antigen gene clone scheme adopts the method for PCR to carry out, the roughly flow process of this method is: according to the two ends sequential design PCR primer of this gene, be template with the virus genom DNA that extracts in the adr hypotype hepatitis B patient serum then, the corresponding genetic fragment of pcr amplification gets final product the extension amplification outcome order-checking again.CpG sequence clone scheme is taked the artificial synthetic oligonucleotide, and the annealing back connects, transforms the back order-checking.Concrete grammar and reaction system thereof are referring to " molecular cloning " (Science Press published in 1992) related Sections.

Description of drawings

Fig. 1 contains the plasmid map of PreS2-S antigen gene and CpG sequence

The structure flow chart of Fig. 2 nucleic acid vaccine pVAX-PS-CpG

Fig. 3 pVAX-PS-CpG stimulation human peripheral blood lymphocytic emiocytosis cytokine relatively

The time graph of Fig. 4 mice serum HBsAb level

Serum antigen level in the mice body of Fig. 5 hepatitis B nucleic acid vaccine inoculation back

Fig. 6 pVAX-PS-CpG brings out the specific CTL reaction that body produces

The induce antibody potency ratio is in hepatitis B transgenic mice and general mice body for Fig. 7 pVAX-PS-CpG

The structure of this nucleic acid vaccine is to carry take eukaryotic expression---pVAX is the recombinant plasmid dna molecule of skeleton, general structure See Fig. 1. The about 3.9kb of total length (kilobase to) is except containing plasmid replication initiation site pMB1, kalamycin resistance gene Outside the elements such as Kanamycin, a complete eukaryotic transcription translation unit and one group of CpG sequence have also been comprised. Eukaryotic transcription Translation unit contains eukaryotic transcription translates necessary element: eukaryotic promoter CMV (being cytomegalovirus promoter), corresponding Encoding gene and 3 ' end polyadenylic acid BGHpA; The CpG sequence is made up of two kinds of immune activation sequences: 2006 and 2103; Wherein 2006 have 2 copies, and its sequence is tcg tcg ttt tgt cgt ttt gtc gtt; 2103 have 1 copy, Its sequence is tcg tcg ttt tga cgt ttt gac gtt. On the whole, this vaccine be one can be in eukaryotic But express adr hypotype HBsAg and the lymphocytic recombinant plasmid dna of human activin.

The building process of this nucleic acid vaccine is as shown in Figure 2:

1, make up the pVAX-CpG carrier and synthesize the 83mer oligonucleotides (including the special CpG repetitive sequence of people) of two complementations with chemical synthesis, its sequence is:

A chain: 5 ' ggc cgctcg tcg ttt tgt cgt ttt gtc gtt tt t cgt cgt ttt gtc gtt ttg tcg ttt t tc gtc gtt ttg acg ttt tga cgt tt 3’

B connects: 5 ' cta gaa acg tca aaa cgt caa aac gac gaa aaa cga caa aac gac aaa acg acg aaa aac gac aaa acg aca aaa cga cga gc 3 '

83mer oligonucleotides heat denatured 10min in 75 ℃ of water-baths with two complementations slowly is annealed to room temperature. Annealing After double-stranded short-movie section 5 ' end contain the NotI site, 3 ' end contains the XbaI site. With this fragment and through the two enzymes of NotI/XbaI The carrier large fragment pVAX1 that cuts rear recovery connects CaCl₂Method transformed into escherichia coli DH10B, specific embodiments is: earlier Host Strains DH10B 0.5ml is inoculated in 50ml LB culture medium and (contains 1% tryptone, 0.5% yeast extract and 1% sodium chloride, PH7.0) in, 37 ℃ of shaken cultivation 90 minutes are to OD₆₀₀Value is 0.4 o'clock, and 4 ℃ of 5000rpm are centrifugal, collects the place Main bacterium; Ice the CaCl of the 100mmol/L of precooling with 40ml₂Solution is resuspended, ice bath 30 minutes; Turning to 5000rpm again 4 ℃ of speed are lower centrifugal, collect Host Strains; Ice the CaCl of the 100mmol/L of precooling with 1-2ml₂Solution is resuspended, makes impression The attitude cell is for subsequent use. After getting 200 μ l competent cells and 10 μ l being connected the product mixing, ice bath 30 minutes, 42 ℃ of heat treatments 90 Second, ice bath 2 minutes adds the LB (the same) of 800 μ l, cultivates 1 hour in 37 ℃, gets 200 μ l converted products and is coated with Be distributed on the LB agar plate that contains kanamycins, cultivated 12-24 hour for 37 ℃, screening obtains recombinant plasmid pVAX-CpG The bacterial clone at place. After this bacterial strain amplification, the alkaline lysis extracting and purifying with classical can obtain recombinant plasmid pVAX-CpG.

2, make up nucleic vaccine plasmid pVAX-PS-CpG

From adr hypotype great three positive B-type hepatitis human serum, extract virus genom DNA, get 100 μ l serum, add 5 μ l albumen Enzyme K solution, it is by 0.25% SDS, 5mmol/L EDTA, 10mmol/L Tris-HCl (pH8.0) preparation, Proteinase K Content be 20mg/ml; The rearmounted 50 ℃ of reaction 30min of mixing; With phenol and the chloroform extracting of equal proportion, get molten after the extracting Liquid 4 μ l are template, with primer 1:cgg gat cca tgc agt gga act cca cca and primer 2: cgg aat tct caa atg tat Acc caa aga amplification Hepatitis B virus-DNA DNA, the pcr amplification product of 860bp is cut with BamHI and EcoRI enzyme, Expose cohesive end. With BamH I/EcoRI double digestion carrier pVAX-CpG, electrophoresis reclaims the carrier large fragment simultaneously. Will with Use above-mentioned CaCl behind upper two segment ligations₂Method transformed into escherichia coli DH10B, screening obtains positive recombinant, namely obtains to contain heavily The engineering bacteria of group plasmid pVAX-PS-CpG. (concrete reaction system and experimental program are referring to aforementioned " molecular cloning " related content)

3, preparation nucleic acid vaccine pVAX-PS-CpG

Nucleic acid vaccine of the present invention prepares with above-mentioned engineered strain. Cultivate amplification and refining can by conventional Bacteria Culture, Plasmid amplification and purification process carry out (seeing aforementioned " molecular cloning " related content for details).

Training method is generally Liquid Culture. Available air shaking table shaken cultivation during a small amount of amplification; Needing when industrialness is produced to be used for Cultivate with the capable high density suspension ventilation of bacterial fermentation tank. Cultivation source in the culture medium is not had special requirement, generally use LB liquid Culture medium such as the need High Density Cultivation, then can select 2 * YT (to contain 1.6% tryptone, 1% yeast extract and 0.5% chlorination Sodium, PH7.0) or the SOC fluid nutrient medium (contain 2% tryptone, 0.5% yeast extract, 0.05% sodium chloride, 20mmol/L Glucose, 2.5mmol/L KCl, 10mmol/L MgCl₂, PH7.0). Also can add in case of necessity moving, plant, the mineral wet goods does Be defoamer. Cultivation temperature is generally 37 ℃, are decided by on incubation time and the opportunity of collecting bacterium culture medium, training method with And the setting of throughput condition.

Separation and purification DNA from the engineering bacteria after the amplification can adopt some conventional methods. Available detergent SDS for example The cracking thalline utilizes DNA and chromosomal DNA to become the difference of renaturation feature, by locating the denature and renature of DNA Reason is separated DNA and chromosomal DNA; Recycling DNA and impurity are in solubility, ions binding power, absorption Further purifying is carried out in the difference of the aspects such as affinity, and removes endotoxin. Specifically, be present in matter in the renaturation solution Grain DNA is through after the desalting processing, and available anion exchange resin (such as DEAE Sepharose) exchanges, and uses then TE (containing 10mmol/L Tris and 1mmol/L EDTA) buffer solution elution can make highly purified DNA and (specifically see Embodiment). Through packing, freeze-drying, can obtain the nucleic acid vaccine sterling of the present invention of translucent sheet. This product physicochemical property very Stable, can under normal temperature, preserve and transportation, before the use, get final product with the water for injection dissolving.

The vivoexpression of this nucleic acid vaccine and detection are that constructed nucleic acid vaccine is passed through electroporation transfection P815 cell, After the strain of G418 screening acquisition survivaling cell, in one week of Continuous Cultivation, detect culture supernatant and cell with double antibodies sandwich ELISA method The expression of HBsAg in the broken liquid, the result shows all expression, illustrates that nucleic acid vaccine of the present invention really can be thin mammal Express among the born of the same parents.

CpG sequence in this nucleic acid vaccine is the people's periphery in the external use fresh separated to the activation of human lymphocyte Blood lymphocyte and this nucleic acid vaccine Mixed culture be after 24-48 hour, quantitatively detect in the cells and supernatant IL-12 and The level of IFN-gamma, thus reflection (is seen below by the ability that human body specific immune stimulus sequence activates human lymphocyte Experiment 1). The result is as shown in Figure 3: but be incorporated into CpG sequence effective stimulus human lymphocyte secretion IL-12 in this nucleic acid vaccine And IFN-gamma, these cell factors are enabling signals of active cell immune response, indication CpG sequence can be used as immunity Adjuvant effectively strengthens the immune response level of body.

The expression in vivo of this nucleic acid vaccine and detection, be the nucleic acid vaccine sterling immune mouse that will obtain after, with the inspection of ELISA method Survey antigen, antibody horizontal (seeing experiment 2) in the serum, result such as Fig. 4, Fig. 5. The result shows that this nucleic acid vaccine immunity is moving Behind the thing, not only can express the surface antigen of hepatitis B, and the effectively interior HI reaction of inductor; Simultaneously, From the rising of antibody horizontal antigen levels is descended, antibody capable efficient neutralization antigen is described, have immanoprotection action.

This nucleic acid vaccine brings out the detection that body produces the cellullar immunologic response level, is that the nucleic acid vaccine sterling immunity that obtains is little Behind the mouse, the SPL action effect cell of separating mouse is with the P815 cell (target cell) of expressing adr hypotype HBsAg Mixed culture, the degree of being attacked cracking by target cell reflects cellullar immunologic response level (seeing experiment 3). Simultaneously, for testing Demonstrate,prove superiority of the present invention, we with its make up with my chamber, based on other vaccine of identical immune protective antigen, and Commercially available subunit vaccine compares in the cellullar immunologic response level. Being used for vaccine relatively is respectively: do not contain CpG The Yeast expression subunit epidemic disease that the hepatitis B nucleic acid vaccine of sequence (pVAXS2-S), Beijing institute of Biological Products and Dalian pharmaceutical factory produce The CHO that seedling (YHC, HYC) and North China Pharmaceutical Factory produce expresses subunit vaccine (CHOC), the results are shown in Figure 6: the nucleic acid epidemic disease The ctl response that seedling excites is higher than subunit vaccine far away, and the ctl response that pVAX-PS-CpG of the present invention excites also is higher than and does not contain The hepatitis B nucleic acid vaccine pVAXS2-S of CpG sequence shows that the result for the treatment of of this nucleic acid vaccine is better than other control group vaccines.

This nucleic acid vaccine is broken the hepatitis B transgenic mice to the detection of HBsAg immune tolerance, is to utilize hepatitis B transgenic mice pair The immune tolerance simulation Chronic Hepatitis B of HBsAg is to the hypoergia of hepatitis B, with the nucleic acid vaccine sterling immunity that obtains The hepatitis B transgenic mice is broken the ability of immune tolerance by this nucleic acid vaccine of reflection of tiring of mice serum anti-HBsAg antibody, Thereby embody the therapeutic action (see experiment 4) of this vaccine, the result as shown in Figure 7: the turnover of second behind HB vaccination base Because just detecting specific antibody in the mice serum. The rising that is in line of this antibody horizontal of coming weeks, and reach in the 7th week Peak value. The antibody level of serum of this and non-transgenic mouse changes basically identical. Point out hepatitis B nucleic acid vaccine of the present invention to break The immune tolerance that the HbsAb transgenic mice forms.

Advantage of the present invention and good effect: the present invention can bring out the immune response of body effectively, illustrates that it is to B-mode Hepatitis viruse carrier, CAH and chronic persistant hepatitis patient can have certain therapeutic action; And produce Technology is simple, and is with low cost, and physicochemical property is stablized, is convenient to store and transportation.

Use engineering bacteria of the present invention to prepare the embodiment of hepatitis B nucleic acid vaccine finished product:

From-80 ℃ of refrigerators, take out frozen engineered strain---DH10B (pVAX-PS-CpG), after room temperature is melted, dip in inoculating loop and to take a morsel the streak inoculation of bacterium liquid on 1.5% LB agar plate (containing kanamycin 50ug/ml), after 37 ℃ of overnight incubation, get monoclonal be inoculated in 50ml LB fluid medium (contain 1% tryptone, 0.5% yeast extract and 1% sodium chloride, kanamycin 50ug/ml, PH7.0) in, 37 ℃ of shaken cultivation 40 hours are as seed liquor.In 1: 20 ratio seed liquor is inoculated in (on the basis of aforementioned composition, other contains kanamycin 50ug/ml) in 1 liter of SOC fluid medium, cultivated 25 hours in 37 ℃ of violent joltings; Adding chloromycetin to final concentration is 170ug/ml, continues to cultivate 16 hours in 37 ℃ of violent joltings.With bacterium liquid in 4 ℃ with 6000rpm centrifugal 10 minutes; Remove supernatant, thalline is resuspended in the solution I (containing 50mmol/L glucose, 25mmol/L Tris.Cl, 10mmol/L EDTA PH8.0) of the alkaline lysis of 30ml and adds lysozyme 40mg, add 60ml solution II (0.2mol/LNaOH and 1%SDS), slowly put upside down mixing 5 times, room temperature was placed 5 minutes.The solution III (containing potassium ion 3mol/L, acetate 5mol/L) that adds the pre-cooling of 45ml ice is slowly put upside down mixing for several times, ice bath 10 minutes; In 4 ℃ with 10000rpm centrifugal 10 minutes, get supernatant, cross 500ml G25 post desalination after, last 20ml DEAE Sepharose anion-exchange column, treat in conjunction with adsorb finish after, press salt ion (Cl ^-) (0～1mol/L) progressive linear gradient elution method is slowly cleaned anion-exchange column with the PBS (PH8.0) and the sodium chloride solution of 10 column volumes to concentration, collects the eluent of 260nm place absworption peak, obtains the crude product of nucleic acid vaccine from low to high.Be further purified then, add the dehydrated alcohol precipitation of 2 times of volumes, 12000rpm4 ℃ centrifugal 10 minutes, remove supernatant, behind 70% ethanol rinsing airing, be dissolved in PBS (PH8.0) solution, promptly get the pure product of nucleic acid vaccine of the present invention; With ultraviolet spectrophotometry quantitatively after, make finished product by every part of 2mg/ml packing, lyophilizing again.

The scheme that the present invention treats hepatitis B is: after water for injection 1ml dissolving, intramuscular injection get final product, dosage be the 20ug/kg body weight (also can with local anaesthetics lignocaine 10mg joint injection, in order to the vaccine absorption).Supplementary immunization once can obtain to react than strong immune response after two weeks.As then effect is better with the subunit vaccine therapeutic alliance.

Test 1 CpG sequence of stimuli human lymphocyte secretion IL-12 and the experiment of IF-gamma horizontal detection

The separation of human peripheral blood lymphocyte is adjusted cell concentration to 1 * 10 ⁷/ ml adds 200ul (about 2 * 10 in every hole in 96 porocyte culture plates ⁶/ hole), nucleic acid vaccine pVAX-PS-CpG of the present invention is added in the hand-hole with 30ug/ml, with the positive contrast of DNA of bacteria, the negative contrast of calf thymus DNA.37 ℃ of 5% CO2 cultivated 48 hours; Get the 100ul cells and supernatant and detect IFN-gamma, IL-12.Result such as Fig. 3: but show that CpG sequence in this nucleic acid vaccine is as the enabling signal of immunological adjuvant effective stimulus human lymphocyte secretion active cell immunne response: and IL-12 and IFN-gamma, indication is integrated in the effective immunne response level of enhancing body of CpG sequence in this nucleic acid vaccine.

Intravital antigen-antibody horizontal detection is tested after testing 2 immunity inoculations

50 of the female BALB/c mouse (average weight 20g) in 6～8 ages in week are divided into three groups at random, first group of 20 immune nucleic acid vaccine (every inoculation 100 μ g), second group of 20 immune subunit vaccine (every inoculation 2 μ g), the 3rd group of 10 negative contrasts.

Utilize level (test kit is available from Hua Tai bio-engineering corporation) that the double antibodies sandwich method detects HBsAg in serum and HBsAb with 10 times of dilutions of standard substance HBsAb and sample to be checked in bag on same 96 hole elisa plate by, develop the color after, optical density (OD450) the value reference standard of sample and the standard curve of OD450 are calculated antibody in the serum, antigenic actual tiring.

With reference to the detection kit explanation, it is positive to set OD450 reading numerical values S/N＞2.1, analyzes the percentage rate of the positive mice of each time point and each group sum.The result shows, immunity 2～6 weeks of back, detect the positive mice of HbsAb gradually, and after immunity the 8th week, humoral immunoresponse(HI) all takes place in all immunized mices.See figure-4

Shown in figure-5; the rising gradually of tiring with HBsAb after the level of HBsAg reaches peak value in first week after the immunity and descending gradually; the humoral immunoresponse(HI) reaction that the prompting nucleic acid vaccine brings out can be effectively in and specific antigen in the body, indication will be brought into play certain immanoprotection action.

Test 3 nucleic acid vaccines and bring out the detection that body produces the cellullar immunologic response level

This experiment detects the CTL cytoactive with lactic acid dehydrogenase (LDH) method for releasing.50 of the female BALB/c mouse (average weight 20g) in 6～8 ages in week, be divided into five groups at random, the CHO that produces of yeast expression subunit vaccine (YHC, HYC) of producing with this nucleic acid vaccine pVAX-PS-CpG, the hepatitis B nucleic acid vaccine pVAXS2-S that does not contain the CpG sequence, Beijing institute of Biological Products and Dalian pharmaceutical factory and North China Pharmaceutical Factory expressed behind subunit vaccine (CHOC) immune balb/c mice 12 days respectively, separate mouse spleen lymphocyte, after counting, add ConA (5ug/ μ l) and cultivated 5 days for 37 ℃.Respectively each group lymphocyte (effector lymphocyte) is added to 96 orifice plates, cell concentration 1 * 10 ⁷/ ml, every hole 100 μ l are imitated target than adding the p815 target cell of cultivating in advance (MHC is restricted consistent with BALB/C mice, changes hepatitis B preS/S antigen gene and stably express over to) with difference, imitate target cell and hatch 37 ℃ of 5% CO jointly ₂4 hours, get supernatant, press the test kit explanation and add LDH effect substrate, cessation reaction behind the 30min, 490nm reads at the place OD value.Calculate the CTL cell killing activity, computing formula is as follows:

Killing activity (%)=[(experimental port D value-natural release aperture D value)/(maximum release aperture D value-natural release aperture D value)] * 100%

The results are shown in Figure 6, show through immunity inoculation after 12 days, can in the mice body, bring out the special ctl response of tangible HBsAg, illustrate hepatitis virus parasitic host cell will be subjected to the targeting sexual assault of CTL and be eliminated.Point out this vaccine certain therapeutical effect can be arranged to hepatitis b virus carrier, chronic active hepatitis and chronic persistent hepatitis patient.

Test 4 nucleic acid vaccines and break of the detection of hepatitis B transgenic mice the HBsAg immunologic tolerance

Hepatitis B nucleic acid vaccine is inoculated HBsAg transgenic mice (every inoculation 100 μ g), and vein is got blood once weekly, utilizes the double antibodies sandwich method to detect the level of HBsAb in the serum, and the while with common BALB/C mice in contrast.

The results are shown in Figure 7, show in second all transgenic mice serum of inoculation behind the Hepatitis B virus vaccine just can detect specific antibody.The rising that is in line of this antibody horizontal of coming weeks, and reach peak value in the 7th week.The serum antibody level of this and non-transgenic mice changes basically identical.Point out this hepatitis B nucleic acid vaccine can break the immunologic tolerance that the HBsAb transgenic mice forms.

Can prove that according to above experiment nucleic acid vaccine of the present invention can bring out the immune response of body effectively, explanation can have certain therapeutical effect to hepatitis b virus carrier, chronic active hepatitis and chronic persistent hepatitis patient.

Claims (3)

1, a kind of human hepatitis B nucleic acid vaccine, with the carrier for expression of eukaryon is basic framework, the surface antigen gene and the immune activation sequence C pG that contain hepatitis B is characterized in that hepatitis b surface antigen gene is adr hypotype hbsag gene preS2-S, and its nucleotides sequence is classified as:

atgcagtgga?actccaccac?atttcaccaa?gtcctgctag?atcccagagt?gaggggccta

tattttcctc?ctggtggctc?cagttccgga?acagtaaacc?ctgttgcgac?tactgcctca

cccatatcgt?caatctcctc?gaggactggg?gaccctgcac?cgaacatgga?gagcacaaca

tcaggattcc?taggacccct?gctcgtgtta?caggcggggt?ttttcttgtt?gacaagaatc

ctcacaatac?cacagagtct?agactcgtgg?tggacttctc?tcaattttct?agggggagca

cccacgtgtc?ctggccaaaa?ttcgcagtcc?ccaacctcca?atcactcacc?aacctcttgt

cctccaattt?gtcctggcta?tcgctggatg?tgtctgcggc?gttttatcat?attcctcttc

atcctgctgc?tatgcctcat?cttcttgttg?gttcttctgg?actaccaagg?tatgttgccc

gtttgtcctc?tacttccagg?aacatcaacc?accagcacgg?gaccatgcaa?gacctgcacg

attcctgctc?aaggaacctc?tatgtttccc?tcttgttgct?gtacaaaacc?ttcggacgga

aactgcactt?gtattcccat?cccatcatcc?tgggctttcg?caagattcct?atgggagggg

gcctcagtcc?gtttctcctg?gctcagttta?ctagtgccat?ttgttcagtg?gttcgtaggg

ctttccccca?ctgtttggct?ttcagttata?tggatgatgt?ggtattgggg?gccaagtctg

tacaacatct?tgagtccctt?tttacctcta?ttaccaattt?tcttttgtct?ttgggtatac

atttga

Described immune activation sequence is the special immune activation sequence C pG20062103 of human body, and its nucleotides sequence is classified as:

ggc?cgc?tcg?tcg?ttt?tgt?cgt?ttt?gtc?gtt?ttt?cgt?cgt?ttt?gtc?gtt?ttg?tcgttt?ttc?gtc?gtt?ttg?acg?ttt?tga?cgt?tt。

2, by the described human hepatitis B nucleic acid vaccine of claim 1, it is characterized in that described carrier for expression of eukaryon basic framework is carrier for expression of eukaryon pVAX1.

3, claim 1 or 2 described human hepatitis B nucleic acid vaccines are in order to the purposes of preparation prevention or treatment hepatitis B medicament.

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