CN1315535C - Adjuvant for improving hepatitis B vaccine immune effect and preparing method thereof - Google Patents

Abstract

The present invention relates to an adjuvant used for strengthening the immunological effect of hepatitis b nucleic acid vaccines and a preparation method of the adjuvant. The adjuvant contains fusion gene fragment carriers of thymin alpha 1 (TA1) and interferon alpha 8 (IFN alpha 8), wherein the carriers are obtained by that the fusion gene fragments of thymin alpha 1 and interferon alpha 8 are connected to various non-replicative form mammalian cell high-level expression carriers, and the high-level expression carriers and the fusion gene fragments are amplified and purified. When the adjuvant and S2S nucleic acid vaccines are simultaneously inoculated to a BALB/c mouse, humoral immune response of S2S nucleic acid vaccines can be strengthened; when the adjuvant is singly inoculated to a hepatitis b virus transgenic C57BL/6 mouse, duplication and expression of HBV DNA of the mouse can be obviously inhibited.

Description

A kind of adjuvant and preparation method that is used to strengthen the hepatitis b vaccine immune effect

Technical field:

The present invention relates to biological technical field, be specifically related to a kind of adjuvant and preparation method that is used to strengthen the hepatitis B nucleic acid vaccine immunological effect.

Background technology:

Hepatitis B (Viral hepatitis B) endangers the most serious infectious disease of China's people's health at present.According to investigations, China chronic hbv-infection person reaches more than 1.2 hundred million, and wherein the chronic viral hepatitis B patient reaches more than 2,000 ten thousand.Some cases can develop into hepatitis gravis, liver cirrhosis even hepatocarcinoma in carrier and the chronic hepatitis patient, dies from hepatitis and diseases related not following 300,000 examples of patient thereof every year.It is estimated that pay up to 300-500 hundred million yuan because of hepatitis and the diseases related economy that causes thereof every year.Hepatitis B virus infection does not still have the specific treatment method so far.All there are many-sided defectives such as curative effect is not fully up to expectations, side effect is many in the treating chronic hepatitis B medicine of using clinically at present, far can not satisfy the needs of hepatitis B virus infection control, and this has brought great difficulty for the control of hepatitis B.Therefore, develop anti-hepatic-B virus medicine efficiently, have urgent clinical needs.

Nucleic acid vaccine and therapeutic nucleic acids vaccine are the emerging biometric technologies that grows up after the nineties in 20th century in the world, have vast potential for future development aspect prevention and the treatment chronic hbv-infection.

The hepatitis B therapeutic nucleic acid vaccine is compared with conventional medicament has following superiority: (1) conventional medicament only can reduce HBV dna replication dna level in reversibility ground, and part is removed HBeAg, and invalid substantially to removing HBsAg; Theoretically, the therapeutic nucleic acids vaccine is expected to develop into the radical cure medicine that HBV infects, and not only can remove HBV gene expression product (HBsAg, HBeAg), and can reduce significantly or removes HBV DNA, makes treatment more complete, thorough.(2) Davis, Hui etc. find by the transgenic mice Research of Animal Model for Study, can not cause the rising of liver function index in the therapeutic nucleic acids vaccine application process, do not cause the hepatic pathology infringement, and these characteristics also obviously are superior to existing antiviral drugs.(3) hepatitis B therapeutic nucleic acid vaccine expression vector is a plasmid vector, the safety concerns of anosis toxicity carrier, simple, the easy row of method of vaccination, but duplicate injection, the destination gene expression persistent period is not long, but is enough to mediate good immunne response, and genes of interest does not need accuracy controlling.(4) because treatment target is HBV the infected, and target gene fragment do not contain complete virion, on the body chromosomal DNA random integration rate low, use safer.

The subject matter that runs in the hepatitis B nucleic acid vaccine research and development at present is that immune effect can't meet clinical needs, and associated mechanisms is carrying out many-sided improvement and perfect both at home and abroad, and relevant research is main concentrates in the following areas:

1. the selection of target gene fragment

Multinomial studies show that, HBV S gene is active essential by inducing the HBsAg specific CTL, and preS ₁Or preS ₂But the immunne response of the adding enhancing body of gene.Davis etc. induce in the active research of CTL at the HBV gene vaccine and find, gene vaccine can very induce the HBV specific CTL to reply effectively, thereby provide strong theoretical foundation for development of new hepatitis B therapeutic nucleic acid vaccine.Davis etc. adopt in research subsequently and comprise HBV DNA S and preS ₂The plasmid pCMV-S of gene ₂S immunity HBsAg transgenic mice is observed the part mice as far back as dna vaccination injection 2 weeks of back, and serum HBsAg promptly disappears, continued to for 12 weeks without booster injection more than, and other mice, the HBsAg level obviously descends and maintains reduced levels.Simultaneously, anti-HBs produces in the serum.All mices there is no liver and tangible necrosis and inflammation pathological change occur.Hui etc. have made up and have comprised HBV S gene and preS ₁The recombiant plasmid pCMV-SS of 21-47 position peptide coding gene ₁, be used for immune HBsAg transgenic mice, found that pCMV-SS ₁Administration causes removing or the level of HBsAg in the circulation of 80% transgenic mice obviously descend (drop to medication before 1/1000 level), apparently higher than pCMV-S (40%), and unloaded plasmid is invalid, and non-NULL charge material grain administration group is all observed higher anti-HBs level, and pCMV-SS ₁The plasmid group is also observed anti-preS ₁Generation.This also removes chronic hbv-infection for nucleic acid vaccine strong experimental basis is provided.Adopt HBe/cAg antigen encoding gene fragment to make up Study on DNA Vaccine Against and also be seen in report, but studies show that of Matti etc. comprises HBe/cAg expression of gene plasmid and there is no the effect of removing HBV chronic infection gorilla body inner virus.Hepatitis B nucleic acid vaccine genetic fragment and the compound mode that has adopted both at home and abroad mainly contains at present: S gene, S+preS ₁21-47 position peptide coding gene, preS ₂S, C, SC, preS ₁PreS ₂Genetic fragments such as S, relevant patent is as follows:

Table 1 hepatitis B nucleic acid vaccine genetic fragment is selected relevant patent

The patent No.	Date of application	The inventor	Genetic fragment	Carrier
					1 US05024938 2 US06025341 3 WO00016802 4 CN1108306 5 CN175585A 6 CN1209340A 7 CN1316518A 8 CN1324661A	1991.07.18 2000.02.15 2000.03.20 1995.09.13 1998.03.13 1999.03.03 2000.06.19 2001.05.23	Nozaki, et al. Jack R, et al. Shan LU, Li Guang ground, the et al Li Guang ground big magnificent Chen Guangming Sun Shu Chinese of having mercy on	(SC) 3 S 1/S 2/S+HCV core C(35nts deleted) SSl C(AA1-144)(AAl-44/69) S 1/S 2/S S 2S+GM-CSF/IL-2IFN-γ S 2S+CpG	PSHB 3 pcDNA3 PJW4303 V.V pcDNA3 pcDNA3.1 pVAX

2. the selection of expression vector and carrier construction method

Nucleic acid vaccine is carrier with high efficiency mammalian cellular expression plasmid often or adopts retroviral vector.Plasmid vector must be can be in escherichia coli high copy amplification, efficiently express in the mammalian cell, and reproducible does not contain simultaneously the sequence of integrating yet in host cell gene.The expression plasmid that is usually used in making up nucleic acid vaccine at present mainly comprises:

(1) the commercialization plasmid vector of pcDNA3:Invitrogen company exploitation, it is the cloning and expression genes of interest carrier of using always, contain CMV immediate early promoter, multiple clone site and bovine growth hormone polyadenylic acid signal sequence, adopt neomycin resistance gene to carry out resistance screening.

(2) the commercialization plasmid vector of pVAX1:Invitrogen company exploitation for the unique expression vector that is used for nucleic acid vaccine of FDA approval, contains CMV and T7 promoter, BGH polyadenylic acid signal tail and Kan resistant gene.

(2) the commercialization plasmid vector of pRC/CMV:Clontech company exploitation is widely used in research, contains the CMV immediate early promoter, and exogenous gene has higher expression efficiency, can produce higher humoral immunization and cellullar immunologic response after the injection.

(3) the commercialization plasmid vector of pCI:Promega company exploitation contains CMV immediate early promoter, multiple clone site, SV40 polyadenylic acid in late period signal sequence and can improve the mosaic type intron of destination gene expression level.

The selection of carrier and construction method are most important for the immunological effect that improves nucleic acid vaccine.

Its influence factor mainly comprises:

(1) optimization of target gene fragment and method of attachment

Nucleic acid vaccine itself is not with the protein-synthesizing system of genes of interest, and the host cell that places one's entire reliance upon is regulated and control.Exogenous gene is optimized for the codon of host cell preference, can improves the synthetic of destination protein, thus the enhance immunity effect.For the coexpression form that a plurality of fragments are formed, the connected mode between each fragment is also extremely important.Most scholars advocates to adopt Gly at present ₄Ser ₁2-4 repetitive sequence of encoding gene be as the catenation sequence between the different genes fragment, is beneficial to that protein is expressed better and biologic activity stable.In addition, ribosome binding site should be contained in genes of interest transcription and translation sintering, to improve genetic transcription and protein expression efficient.

(2) transcription of foreign genes promoter/enhancer sequence

One class is pathogenic viral promotors, has the transcription initiation ability of higher level in most mammalian cells, thinks that at present the hCMV promoter can play a role better than SV40 and MPSV promoter.Another kind of is mammalian promoter, be adapted at using in human body or the animal, as from mammiferous MHCI promoter/enhancer BL3-60prmtr (Harms JS, et al.Braz J Med Biol Res1999,32:155-62), people's muscle specific creatine kinase promoter (Baztl ett et al., 1996); Mus metallothionein gene promoter, exempt from-β cardiac muscle heavy chain and light chain 1/3 enhancer; β-actin promoter and cmv enhancer share etc. (Niwa et al., 1991).

(3) intron sequences

The intron sequences that inserts in the carrier helps antigenic efficiently expressing, may be because the cause that the RNA shear rate that RNA polyadenylation and/or consideration convey record connect improves.Brinster etc. studies show that intron can improve transcription of foreign genes efficient 10-100 times of transgenic mouse.The upstream that intron sequences is positioned at the antigen gene coding region could promote expression of exogenous gene preferably, and enhance immunity is replied.At present existing carrier itself has comprised the intron sequences that can strengthen the transcription of foreign genes translation, as containing the mosaic type intron sequences in the pCI carrier.

(4) polyadenylation (poly A) signal

The polyadenylation signal sequence is positioned the downstream of multiple clone site, can improve the stability and the translation efficiency of transcribe rna, promotes effective processing of genes of interest.In the nucleic acid vaccine carrier polyadenylation terminator sequence often derive from bovine growth hormone or late period the SV40 polyadenylation signal, as pcDNA3.lmychis (BGH, SV40), pCI (SV40), VR1012 (BGH), pVAXl (BGH) etc.

(5) other immune enhancement sequences

Reports such as Sato, the carrier that the carrier immunne response that contains the Amp resistant gene contains the Kan resistant gene is strong, and reason may be to contain two immunostimulatory sequences (ISS, 5 '-AACGTT-3 ') in the Amp resistant gene, and does not have ISS in the Kan resistant gene.

3. the route of administration of nucleic acid vaccine

The immunization route of nucleic acid vaccine has multiple (seeing Table 2), comprises that mainly intramuscular injection, gene rob 4 kinds of inoculations, mucosal vaccination, intravenous injection.Intramuscular injection still is to use the widest, that effect is more sure a kind of mode; The advantage of mucosal immunity is to induce preferably secretory IgA, sIgA antibody; The serum antibody level that gene gun inoculation induces is the highest, and cellullar immunologic response is slightly disliked deficiency; Intravenous injection easily diffuses to lymph node even bone marrow place, might the mitotically active stem cell of transfection and cause malignant change of cell, so be difficult to be applied to clinical.Therefore according to the type and the purposes of nucleic acid vaccine, adopt corresponding inoculation measure, just might obtain best immune protection effect.

After nucleic acid vaccine entered body, being taken in also by body cell, expressed exogenous gene was minority eventually.Developed multiple Novel DNA introduction method in recent years in succession.As gene gun technology (Yang Ns, et al., 1980), cationic-liposome (Gregoriadis et al., 1997), the Cochleate bag is by (Mannino RJ et al., 1997), degradable microgranule parcel (Jone et al., 1997), some antibacterial attenuated strain (Darji A et al., 1997) etc.External some drug-supplying system patented products of exploitation in recent years see Table 3.

The immunization route that table 2 nucleic acid vaccine is relevant

Injecting pathway	The report author
		Injection in the injection mammary gland in the intramuscular injection hypodermic injection intracutaneous injection lumbar injection intravenous injection intra arterial injection oral mucosa nasal membrane tunica mucosa bronchiorum genital tract mucous membrane cheek	Davis，et al.，1996 Yokeyama，et al.，1996；Liu et al.，1997 Gramzinski，et al.，1993；Condon et al.，1996 Fynan et al.，1993 Fynan et al.，1993；Zhang GF，et al.，1999 Nabel，et al.，1993 Jones DH，et al.，1997；Chen SC.，et al.，1998 Fynan EF，et al.，1993；Sasaki S，et al.，1998 Tsan MF，et al.，1995；Meyer KB，et al.，1995 Wang B，et al.，1997；Livingston JB，et al.，1998 Etchart et al.，1997；Hinkula et al.，1997 Furth et al.，1992

The drug-supplying system patented product of the external exploitation of table 3

Drug-supplying system	The exploitation/or patent have company
		1.PowderJect system 2.Lipoplex-Biovector system 3.Biojector 2000 jet injection delivery system 4.Geneswitch 5.Bacfotection	PowderJect vaccines Inc (Oxford，London and Madison，Wisconsin) BITVECTOR THERAPEUTICS SA(France) Bioject Medical Technologies Inc (Vical，U.S.A) Gene Medicine Inc(U.S.A) Institute of Human Virology(IVH，U.S.A)

4. the application of adjuvant in nucleic acid vaccine

Have found that the various kinds of cell factor has immunoadjuvant function, intensity that immunity-regulating is replied and direction.IL-2, IL-12 and β chemotactic factor TCA ₃Deng to strengthen TH ₁The type cellular immunization is main, to the humoral immunization facilitation a little less than.IL-4, GM-CSF can obviously strengthen humoral immunization, cause antibody to raise.Wherein IL-12 is at TH ₀To TH ₁Differentiation in play key effect, and IL-4 can obviously promote TH ₁To TH ₀Transform.

Also find multiple molecule in recent years, can improve the level of nucleic acid vaccine antibody response as report C3d such as Ross with immunoregulation effect; Endresz V etc. observes IFN α can make the medium degree of antibody horizontal raise.The oral nucleic acid vaccine coupling that studies show that thymosin expression plasmid and Pseudorabies virus glycoprotein of Shiau etc. can significantly strengthen the latter's cellullar immunologic response etc.

Summary of the invention:

Technical problem solved by the invention is to provide a kind of novel molecular adjuvant that improves the hepatitis B nucleic acid vaccine specific immune response.

Enhancing hepatitis B nucleic acid vaccine effect disclosed by the invention adjuvant for containing the preparation of thymosin (TAl) and interferon-ALPHA 8 (IFN α 8) fusion gene fragment carrier, this carrier is by thymosin and interferon-ALPHA 8 fusion gene fragments being connected in the various non-replicating mammal cell with high efficient expression vectors, obtaining after a large amount of amplification purification.

Thymosin of the present invention and interferon-ALPHA 8 fusion gene fragment combination are TAl+linker+IFN α 8, and this sequence total length is 633bp (base sequence is seen sequence 5, and aminoacid sequence is seen sequence 6); Wherein TA1 gene 87bp is a synthetic; Connecting portion linker is [(GGT) ₄TCT] n, n=2-4, its coding aa is (Gly ₄Ser ₁); Downstream IFN α sequence and IFN α 8 are in full accord, do not contain start codon, long 501bp, and preceding 3 of start codon is ACC, comes from China's Healthy adult's peripheral blood leucocyte.

Preparation formulation of the present invention comprises acceptable liquid preparation on the physiology, emulsion formulations, lyophilized formulations or colloid gold particle bag quilt etc.

Non-replicating mammal cell with high efficient expression vector of the present invention is pcDNA series, pRc/CMV, pCI or pVAX1 etc.

Another technical problem of solution of the present invention is the preparation method that discloses above-mentioned enhancing hepatitis b vaccine immune effect adjuvant.

The preparation method that contains the preparation (adjuvant that contains TA1-IFN α 8) of thymosin and interferon-ALPHA 8 fusion gene fragment carriers disclosed by the invention comprises the steps:

(1) amplification of IFN α gene

From normal person's peripheral blood leucocyte, obtain IFN α, design primer PF1 (sequence 1), PB1 (sequence 2), by RT-PCR, the reverse transcription and the IFN alpha gene fragment that increases are cut glue and are reclaimed standby;

(2) IFN α gene and pGEM ^The connection of-T carrier

Order-checking behind the IFN α gene insertion pGEM-T carrier is confirmed;

(3) TA1 and IFN α gene is connected

In the upstream of IFN α gene (removing start codon), whole 87 bases of synthetic TA1 (do not have stop codon) and the coupling part is [(GGT) ₄TCT] n, acquire fusion gene TA1-IFN α fragment;

(4) structure of TA1-IFN alpha expression plasmid

TA1-IFN α (being called for short TI) is connected in the non-replicating mammal cell with high efficient expression vector by double enzyme site (KpnI, NotI), obtains expression plasmid;

(5) large-scale production of TA1-IFN alpha expression plasmid

With recombiant plasmid and unloaded plasmid transformed competence colibacillus JM109, through a large amount of amplification purification, double digestion identify, ultraviolet spectrophotometer quantitatively after, the physiological saline solution dilution promptly get hepatitis B nucleic acid vaccine potentiation molecule adjuvant, packing postposition-80 ℃ refrigerator preservation.

A technical problem more to be solved by this invention is to disclose the application of above-mentioned TA1-IFN α 8 adjuvants in preparation hepatitis B virus immune medicine and enhancing hepatitis B nucleic acid vaccine immunological effect.

Adjuvant of the present invention is used the immunization therapy effect that has at hepatitis B virus separately; With existing hepatitis B nucleic acid vaccine use in conjunction, can strengthen the latter's immunoprophylaxis and therapeutic effect.

Prevention crowd of the present invention is not for being subjected to HBV after testing and infecting colony or the individuality of non-responsiveness after conventional HBV vaccine injection.Described treatment crowd comprises the hepatocarcinoma HBV dna replication dna person of enlivening after HBV carrier, various chronic viral hepatitis B patient, hbv-liver cirrhosis and the hepatitis B, the HBV DNA person etc. that is the integrated state for HBV persistent infection person.

Described adjuvant can adopt certain dosage form such as lyophilizing, powder pin, colloid gold particle bag by etc., unite with hepatitis B nucleic acid vaccine, by corresponding route of administration (muscle, subcutaneous injection, lumbar injection, mouth, nasal mucosa medicine administration etc.) be applied to the organism (mankind, primate, large and small Mus etc.), be used for the prevention and the treatment of chronic hbv-infection.

Hepatitis B nucleic acid vaccine of the present invention means the preceding S of HBV ₁, preceding S ₂, S, preceding C, C, gene such as P, X or its various combinations expression plasmid.

With molecule adjuvant TA1-IFN alpha expression plasmid of the present invention to nucleic acid vaccine pVAX1-S ₂The influence of immunological effect is tested behind the S inoculation mice:

One. experimental technique

1. laboratory animal and grouping

Healthy female BALB/c (H-2 ^d) mice (Shanghai Xi Puer-Bi Kai company), age in 6-8 week, body weight 18-20g, the cleaning level, standardized environment is raised.

Random packet is as follows: experimental group is pVAX1-S 1. ₂S+pVAX1-TI

②pVAX1-S ₂S

Blank: normal saline

Negative control: pVAX1

2. vaccination ways:

The injection of bilateral tibialis anterior, total amount 100 μ g//times; 2,4 week each booster shot of back are 1 time.

Blank winding kind 100 μ l normal saline/only/time

3. the detection of immunne response

(1) antigen presentation of inoculation position

The part mice is put to death in hepatitis B nucleic acid vaccine inoculation back the 10th day, gets inoculation position muscular tissue, and neutral formalin is fixed, the routine paraffin wax embedding, and it is fast to make the single wax of organizing.

To make organization chip with batch tissue, and adopt conventional section, immunocytochemical technique detects the preS of inoculation position muscular tissue ₂Ag, HBsAg express.The anti-employing respectively of I resists preceding S ₂Monoclonal antibody, anti-HBs monoclonal antibody.The DAB colour developing.

(2) mice serum specific antibody

3 weeks, 5 weeks, 8 weeks, 12 weeks behind the primary vaccination, gather mice socket of the eye venous blood, detect pre-s2 antibody, anti--HBs.

Reagent adopts respectively: S before the hepatitis B ₂Antibody assay kit (Beijing Medical University hepatitis reagent center), hepatitis B surface antibody detection kit (magnificent).

Two. experimental result

(1) antigen presentation of inoculation position

The routine immunization cytochemical methods detects and shows: the S gene of experimental group is all efficiently expressed, and preceding S ₂Antigen presentation a little less than.The matched group testing result is all negative.

(2) serological specificity antibody

1. preceding S ₂Antibody

S ₂The antibody male rotary rate of S nucleic acid vaccine group reaches 66.7%, with the antibody male rotary rate of TA1-IFN alpha expression plasmid combined immunization group be 50%, the two differences is not obvious.S ₂The highest titre of pre-s2 antibody of S group only 1: 10; And the combined immunization group reaches more than 1: 50.S before the matched group serum ₂The antibody test total negative.

2. resist-HBs

S ₂The antibody male rotary rate of S group and combined immunization group is 50%; The highest titre of antibody is 1: 50.The anti-HBs of matched group serum detects total negative.

Three. conclusion

Behind the mouse inoculation hepatitis B nucleic acid vaccine 10 days, the antigenic expression of purpose appearred in inoculation position.

Combined immunization group and S ₂The S group all induces out preceding S ₂Antibody.Antibody male rotary rate difference is little, but the antibody top level is S ₂More than 5 times of S group.Show that TA1-IFN alpha expression plasmid can strengthen nucleic acid vaccine pVAX1-S ₂The preceding S of S ₂Antibody response, this is for the prevention of hepatitis B and treat significant.

Combined immunization group and S ₂Anti--the HBs of S group induces the ability no significant difference.

With TA1-IFN alpha expression plasmid of the present invention to HBV DNA transgenic C ₅₇BL/6 (H-2 ^b) therapeutical effect of mice tests:

One. experimental technique

1. laboratory animal: HBV DNA transgenic C ₅₇BL/6 (H-2 ^b) mice, male and female half and half, the 3-5 monthly age, body weight 25-30g, the SPF level, the SPF standard environment is raised down.

2. animal grouping

Experimental group: pVAX1-TI matched group: pVAX1

3. vaccination ways: identical with BALB/c mouse

4. the detection of immunological effect

(1) serum HBV dna level: adopt HBV DNA quantitative PCR detection kit, the by specification operation.

(2) antigen presentation in the hepatic tissue: inoculate preceding 3 days and inoculate 3 weeks of back, 5 weeks, 8 weeks, respectively with 1% pentobarbital sodium 50mg/Kg body weight intraperitoneal anesthesia C ₅₇The BL/6 mice, aseptic operation is cut open the belly, and cuts 0.5 * 0.5GM ²The hepatic tissue of size, neutral formalin is fixed.Close the abdominal cavity after the operation and continue to observe raising.Make organization chip with batch murine liver tissue, conventional section and immunocytochemical technique detect HBsAg, HBcAg.

Two. experimental result

1. the antigen presentation in the hepatic tissue

Before the experimental group inoculation, all there is the expression of HBsAg and HBcAg in murine liver tissue and mainly is distributed in the cytoplasm.The inoculation back is during the 5th～8 week, and antigen presentation is reducing tendency, part mice antigen presentation even complete obiteration.

Meanwhile, the above-mentioned antigen presentation in the control group mice hepatic tissue did not relatively have significant change in the 5th, 8 weeks before inoculation and after the inoculation.

2. serum HBV DNA is quantitative

Experimental mice serum HBV dna level seemingly is the trend that reduces one by one; The serum HBV dna level of matched group does not have significant change.

Three. conclusion

Behind the transgenic mice inoculation TA1-IFN alpha expression plasmid, the HBV dna level has the trend that reduces one by one, and HBsAg and HBcAg express and be reducing tendency even complete obiteration in the hepatic tissue.Thereby for HBV chronic infection person's treatment provides strong theoretical foundation.

Employed nucleic acid vaccine pVAX1-S in the present invention's experiment ₂S is the nucleic acid vaccine of the inventor's disclosed novel hepatitis B virus infection in another Chinese patent application.S/S district and C district before the employed encoding gene of most of hepatitis B nucleic acid vaccine derives from, the natural compound mode of gene in preceding S/S district has S, preS ₂/ S, preS ₁/ preS ₂Three kinds of/S, the main albumen of the hepatitis B virus envelope antigen of encoding respectively, middle albumen and large protein.The inventor has adopted preS ₂/ S mode, reason is: 1) preceding S ₂Has dependency with integrating in the infectivity of hepatitis B virus, immune evasion, the host cell nuclear etc.2) preceding S ₂, (major histocompatibility complex, preceding S is used in MHC) site domination to the antigenic immunne response of S by different major histocompatibility complexs ₂Antigen immune can overcome the antigenic unresponsiveness to S.Therefore adopt preS ₂The hepatitis B nucleic acid vaccine of S genetic fragment has better prevention and treatment effect theoretically.Preparation to this vaccine in the embodiment of the invention has detailed description.

Above-mentioned result of the test confirm preparation that the present invention contains thymosin and interferon-ALPHA 8 fusion gene fragment carriers be definite effect enhancing hepatitis B nucleic acid vaccine effect adjuvant.

Description of drawings:

Fig. 1 IFN α segment (PCR product) electrophoretogram

Wherein 1 is the PCR product of IFN α; M is GeneRuler ^TM100bp DNA Ladder

The structure sketch map of Fig. 2 recombiant plasmid pVAX1-TI

Fig. 3 pVAX1-TI collection of illustrative plates that checks order

The specific embodiment:

The structure (see figure 2) of embodiment 1 molecule adjuvant TA1-IFN alpha expression plasmid

1.IFN the amplification of α gene

Centrifugal peripheral blood cells of collecting health adult adds erythrocyte cracked liquid (Qiagen) mixing, places 10-15min on ice; 4 ℃ of centrifugal 10min of 400rpm.Add write cell lysis buffer in the precipitation and jolt mixing, move in the RNA adsorption column, behind cyclic washing, add the pure water (through the DEPC pretreatment) that 50ul removes the RNA enzyme, the centrifugal 1min eluting of 8000g.Contain the total RNA of leukocyte in the eluent, comprising the mRNA of IFN α.Adopt primer PF1, PB1, by RT-PCR, the reverse transcription and the IFN alpha gene fragment that increases are cut glue and are reclaimed standby.IFN α segment PCR product electrophoretogram is seen Fig. 1.

2.IFN α gene and pGEM ^The connection of-T Vector

Get IFN α 2 μ l, add an amount of pGEM ^-T Vector and T4 dna ligase mixing, room temperature connects 1h.Transformed competence colibacillus bacterium JM109, and coat LB/Ap resistant panel (surface is coated with IPTG/X-gal in advance), 37 ℃ of overnight incubation.The picking white colony changes sample to be cultivated, and the extraction recombiant plasmid carries out the NotI enzyme action to be identified, confirms to have the plasmid of genes of interest insertion to deliver order-checking through preliminary.

3.TA1 with being connected of IFN α gene

In the upstream of IFN α gene (removing start codon), whole 87 bases of synthetic TA1 (do not have stop codon) and the coupling part is [(GGT) ₄TCT] ₃, acquire fusion gene TA1-IFN α fragment.

4.TA1-IFN the structure of alpha expression plasmid

TA1-IFN α (being called for short TI) is connected in the pVAX1 carrier by double enzyme site (Kpn I, NotI), obtains expression plasmid pVAX1-TI.

Sequence analysis confirms TI total length 633bp, TA1 gene 87bp wherein, and connecting portion is [(GGT) ₄TCT] ₃Downstream IFN α sequence and IFN α 8 are in full accord, do not contain start codon, long 501bp.Preceding 3 of start codon is ACC, meets the requirement of Kozak sequence.The order-checking collection of illustrative plates is seen Fig. 3.

A large amount of preparation methoies of embodiment 2 molecule adjuvant TA1-IFN alpha expression plasmids

With recombiant plasmid plasmid pVAX1-TI and the unloaded plasmid transformed competence colibacillus JM109 that embodiment 1 obtains, coating LB flat board (Amp or Kan resistance) overnight incubation; The single bacterium colony of picking increases in a large number, uses Qiagen Mega Endofree purification system, and the by specification operation obtains highly purified plasmid: superhelix partly is no less than 2/3, and no RNA and protein are residual, no contaminated with endotoxins.Through double digestion identify, ultraviolet spectrophotometer quantitatively after, physiological saline solution is diluted to 1ug/ul, is hepatitis B nucleic acid vaccine or molecule adjuvant.Packing postposition-80 ℃ refrigerator is preserved.

Embodiment 3 nucleic acid vaccine pVAX1-S ₂The preparation of S

One, hepatitis B virus gene fragment preS ₂The amplification of S

1.HBV the preparation of dna profiling

(1) specimen source

Chronic hbv-infection person pooled serum, the research department provides by the Huaxi Hospital Attached to Sichuan Univ viral hepatitis.HBsAg (+), anti-HBc (+), HBV DNA carrying capacity is 10 ⁵～10 ⁹Between the copies/mL.

(2) preparation method

Get above-mentioned serum 200 μ l, add (face time spent add) TNES (10mmol/L Tris.Cl PH8.0,150mmol/L NaCl, 25mmol/L EDTAPH8.0,1%SDS (the W/V)) mixing contain 200 μ g/mL E.C. 3.4.21.64s, hatched 4 hours; Add the abundant mixing of equal-volume phenol again, centrifugal 10 minutes of 10000r/min; Get supernatant, add equal-volume chloroform/isoamyl alcohol mixing, centrifugal 10 minutes of 10000r/min; Abandon supernatant, add 200 μ l, 70% washing with alcohol, centrifugal 5 minutes of 12000r/min in the precipitation; Remove supernatant, precipitation is dried, and is dissolved among the 30ul TE.

2.preS ₂The amplification of S gene

Infect main type (genotype B, C) gene order design universal primer according to the Chinese patient HBV that has reported among the GenBank, entrust precious biological engineering (Dalian) company limited to synthesize (PAGE purification), with the pure water dissolved dilution is 10 μ mol/L concentration, and packing is stored in-20 ℃.

Get HBV template 5 μ l, dNTPmix200 μ mol/L, pyrobest polymerase0.5U/50 μ l and primer PF2 (sequence 3), PB2 (sequence 4) carries out pcr amplification.Loop parameter is set to: pre-94 ℃ of 5min of degeneration; 94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃ of 45sec circulate 35 times; 72 ℃ are extended 10min.Reaction is got PCR product electrophoresis on 1% agarose gel after finishing, and cuts glue and reclaims the purpose band (preS that molecular weight is about 870bp ₂S), press Qiagen glue and reclaim the operation of test kit description.Use 50 μ l pure water eluting standby at last.

Two, animal cell expression vector nucleic acid vaccine pVAX1-S ₂The structure of S

1. target gene fragment S ₂The double digestion of S and carrier pVAX1 and being connected

KpnI, NotI double digestion pVAX1 plasmid vector and DNA insert fragment, 37 ℃ of 4h.Carrier behind the separation and purification enzyme action and DNA insert fragment, get and insert fragment 4 μ l, carrier (1: 5～20 dilution) 1 μ l mixing, add isopyknic sol I (Takara), and 16 ℃ of insulation 1h or connection are spent the night.

2. connect product transformed competence colibacillus JM109 and recombinant screen

In the product of coupled reaction, add 5mol/L NaCl to final concentration 150mmol/L, again with competence bacteria JM109 60～80 μ l mixings of prepared fresh, ice bath 30min; 37 ℃ of 2min; Ice bath 2min immediately.Add SOC culture medium 500 μ l mixings, 37 ℃ of 250rpm jolt 1h.Get 200 μ l transformed bacteria liquid, coat on the LB/Amp resistant panel 37 ℃ of overnight incubation.

3. the screening of recombiant plasmid

From 15 bacterium colonies of LB/Amp resistant panel central authorities' picking, place 5ml LB (containing Amp100 μ g/mL) fluid medium respectively, 37 ℃ of 250rpm jolt 8～16h.Sample is extracted plasmid, and get 1 μ l and carry out agarose gel electrophoresis, be contrast with unloaded plasmid., continue to identify greater than unloaded plasmid as the recombiant plasmid molecular weight with KpnI and NotI double digestion.When the carrier behind the enzyme action conforms to desired value with insertion fragment molecular weight size, continue sequencing analysis.

Confirm through the polyclone order-checking, obtained nucleic acid vaccine mammalian cell expression vector pVAX1-S ₂The S plasmid.

Sequence analysis confirms, pVAX1-S ₂The S of S expression vector ₂The S fragment length is 846bp, is defined as genotype B, blood serum subtype adw2 according to the S gene order.Start codon ATG upstream is ACC, meets the requirement of mammalian cell expression vector Kozak sequence.

Three, nucleic acid vaccine pVAX1-S ₂A large amount of preparation methoies of S expression plasmid

With recombiant plasmid pVAX1-S ₂S and unloaded plasmid transformed competence colibacillus JM109, coating LB flat board (Amp or Kan resistance) overnight incubation; The single bacterium colony of picking increases in a large number, uses QiagenMega Endofree purification system, and the by specification operation obtains highly purified plasmid: superhelix partly is no less than 2/3, and no RNA and protein are residual, no contaminated with endotoxins.Through double digestion identify, ultraviolet spectrophotometer quantitatively after, physiological saline solution is diluted to 1ug/ul, is hepatitis B nucleic acid vaccine or molecule adjuvant.Packing postposition-80 ℃ refrigerator is preserved.

Embodiment 4 pVAX1-S ₂S cell transfecting and detection of expression

1.SP2/0 going down to posterity of cell cultivated and the minimum complete lethal quantitative determination of G418

The SP2/O cell derives from the BALB/c mouse myeloma cell line, and culture fluid is RPMI 1640 complete mediums.Face with preceding adding 20%FBS, L-glutaminate 2mmol/L, penicillin 200u/mL, streptomycin 200u/mL, HEPES 25mmol/L, 0.15%NaHCO ₃, regulate pH to 7.4.Condition of culture is 37 ℃, 5%CO ₂, 95% relative humidity.Every 3-5d goes down to posterity 1 time.

Through adopting the MTT analytic process, the minimum complete lethal amount of G418 that draws the SP2/O cell is 400 μ g/mL.

2. recombiant plasmid transfection SP2/O cell

Adopt calcium phosphate method, with recombiant plasmid pVAXl-S ₂S and empty carrier stable transfection SP2/0 cell, the cotransfection plasmid adopts pSV2-neo.

Transfectional cell is after containing about 2 weeks of G418 resistance culture medium continuous action, and survivaling cell adopts limiting dilution assay, isolates monoclonal cell and continues and cultivate and identify.

3.RT-PCR the mRNA of testing goal genetic transcription

Transfectional cell adds 1mL Trizol mixing and also leaves standstill 5min after washing with PBS.Add equal-volume chloroform mixing, room temperature is placed 2～3min; Get supernatant behind 4 ℃ of centrifugal 15min of 12000g, add equal-volume isopropyl alcohol mixing, room temperature leaves standstill 10min; 4 ℃ of centrifugal 10min of 12000g, precipitation adds DEPC treating water 20 μ l dissolving with drying after 75% washing with alcohol.This is total RNA of transfectional cell, adopts specific primer, by RT-PCR amplifying target genes fragment S ₂S, PCR product electrophoresis observation have or not the purpose band to occur.

The result: 3 kinds of different carriers recombiant plasmid transfection SP2/0 cells, the clonal cell line that obtains extracts total RNA, and RT-PCR amplifies respectively and S ₂The DNA band that the S molecular weight conforms to, unloaded plasmid transfection cell is then negative.

4.ELISA detecting HBsAg expresses

Through the cell strain that RT-PCR alleged occurrence genes of interest is transcribed, collecting cell also adds an amount of cell pyrolysis liquid, lashes repeatedly to no viscose silk appearance with the 20gauge syringe needle, places 10min on ice; 4 ℃ of centrifugal 10min of 12000g.Get supernatant, adopt the ELISA detection kit of HBsAg, detect the monoclonal cell strain and whether express the purpose antigen protein.

Result: point out 3 kinds of different carriers recombiant plasmid transfection SP2/0 cells all to obtain S ₂S detects the positive cell strain.

5.Western blot detects

Get ELISA testing result positive cells strain crack protein, carry out the SDS-PAGE electrophoresis, with the negative contrast of unloaded plasmid transfection cell, with the positive contrast of HBVer's serum (the HBsAg positive).After electrophoresis finished, to pvdf membrane, room temperature was sealed 1h with gel protein matter electrotransfer.Add anti-(the HBs monoclonal antibody 1: 200 of corresponding I; Preceding S ₂Monoclonal antibody 1: 100), 4 ℃ are spent the night.After washing film, add II anti-(sheep anti-mouse igg-AP, 1: 1000) again, room temperature 3h; Develop the color with NBT/BCIP at last.

Result: ELISA detects the positive strain of HBsAg, identifies the S of expression through Western blot ₂S albumen is about 33KD size strip, and molecular weight conforms to expection.

Sequence table

＜110〉Medicine Research Institute,Chengdu Di'ao Group

＜120〉a kind of adjuvant and preparation method that is used to strengthen the hepatitis b vaccine immune effect

＜130〉description, claims

<160>6

<170>PatentIn version 3.1

<210>1

<211>21

<212>DNA

＜213〉primer PF1

<400>1

tgtgatctgc ctcagactca c 21

<210>2

<211>20

<212>DNA

＜213〉primer PB1

<400>2

ttattcctta ctcttcaatc 20

<210>3

<211>36

<212>DNA

＜213〉primer PF2

<400>3

gcgggtacca tgcagtggaa ctccacaaca ttccac 36

<210>4

<211>38

<212>DNA

＜213〉primer PB2

<400>4

ggggcggccg cttaaatgta tacccaaaga caaaagaa 38

<210>5

<211>633

<212>DNA

＜213〉TA1-IFN α base sequence

<400>5

atgagcgacg ccgccgtgga caccagcagc gagatcacca ccaaggacct gaaggagaag 60

aaggaggtgg tggaggaggc cgagaacggt ggtggtggtt ctggtggtgg tggttctggt 120

ggtggtggtt cttgtgatct gcctcagact cacagcctgg gtaacaggag ggccttgata 180

ctcctggcac aaatgcgaag aatctctcct ttctcctgcc tgaaggacag acatgacttt 240

gaattccccc aggaggagtt tgatgataaa cagttccaga aggctcaagc catctctgtc 300

ctccatgaga tgatccagca gaccttcaac ctcttcagca caaaggactc atctgctgct 360

ttggatgaga cccttctaga tgaattctac atcgaacttg accagcagct gaatgacctg 420

gagtcctgtg tgatgcagga agtgggggtg atagagtctc ccctgatgta cgaggactcc 480

atcctggctg tgaggaaata cttccaaaga atcactctat atctgacaga gaagaaatac 540

agctcttgtg cctgggaggt tgtcagagca gaaatcatga gatccttctc tttatcaatc 600

aacttgcaaa aaagattgaa gagtaaggaa tga 633

<210>6

<211>210

<212>PRT

＜213〉TA1-IFN α encoding amino acid sequence

<400>6

Met Ser Asp Ala Ala Val Asp Thr Ser Ser Glu lle Thr Thr Lys Asp

1 5 10 15

Leu Lys Glu Lys Lys Glu Val Val Glu Glu Ala Glu Ash Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Asp Leu Pro

35 40 45

Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile Leu Leu Ala Gln

50 55 60

Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe

65 70 75 80

Glu Phe Pro Gln Glu Glu Phe Asp Asp Lys Gln Phe Gln Lys Ala Gln

85 90 95

Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr Phe Asn Leu Phe

100 105 110

Ser Thr Lys Asp Ser Ser Ala Ala Leu Asp Glu Thr Leu Leu Asp Glu

115 120 125

Phe Tyr Ile Glu Leu Asp Gln Gln Leu Asn Asp Leu Glu Ser Cys Val

130 135 140

Met Gln Glu Val Gly Val Ile Glu Ser Pro Leu Met Tyr Glu Asp Ser

145 150 155 160

Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Thr

165 170 175

Glu Lys Lys Tyr Ser Ser Cys Ala Trp Glu Val Val Arg Ala Glu Ile

180 185 190

Met Arg Ser Phe Ser Leu Ser Ile Asn Leu Gln Lys Arg Leu Lys Ser

195 200 205

Lys Glu stop

210

Claims (14)

1, a kind of adjuvant that is used to strengthen the hepatitis b vaccine immune effect is characterized in that this adjuvant is the preparation that contains thymosin and interferon-ALPHA 8 fusion gene fragment carriers.

2, adjuvant according to claim 1 is characterized in that wherein said carrier is by thymosin and interferon-ALPHA 8 fusion gene fragments being connected in the various non-replicating mammal cell with high efficient expression vectors, obtaining after a large amount of amplification purification.

3, adjuvant according to claim 1 and 2 is characterized in that wherein said thymosin and interferon-ALPHA 8 fusion gene fragment combination are TA1+linker+IFN α 8, and this sequence total length is 633bp, has following base sequence and aminoacid sequence:

1) TA1-IFN α 8 base sequences:

atgagcgacg ccgccgtgga caccagcagc gagatcacca ccaaggacct gaaggagaag 60

aaggaggtgg tggaggaggc cgagaacggt ggtggtggtt ctggtggtgg tggttctggt 120

ggtggtggtt cttgtgatct gcctcagact cacagcctgg gtaacaggag ggccttgata 180

ctcctggcac aaatgcgaag aatctctcct ttctcctgcc tgaaggacag acatgacttt 240

gaattccccc aggaggagtt tgatgataaa cagttccaga aggctcaagc catctctgtc 300

ctccatgaga tgatccagaa gaccttcaac ctcttcagca caaaggactc atctgctgct 360

ttggatgaga cccttctaga tgaattctac accgaacttg accagcagct gaatgacctg 420

gagtcctgtg tgatgcagga agtgggggtg atagagtctc ccctgatgta cgaggactcc 480

atcctggctg tgaggaaata cttccaaaga ataaecctat atctgacaga gaagaaatac 540

agctcttgtg cctgggaggt tgtcagagca gaaatcatga gatccttctc tttatcaatc 600

aacttgcaaa aaagattgaa gagtaaggaa tga 633

2) TA1-IFN α 8 encoding amino acid sequences

Met Ser Asp Ala Ala Val Asp Thr Set Ser Glu Ile Thr Thr Lys Asp

1 5 10 15

Leu Lys Glu Lys Lys Glu Val Val Glu Glu Ala Glu Asn Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Asp Leu Pro

35 40 45

Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile Leu Leu Ala Gln

50 55 60

Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe

65 70 75 80

Glu Phe Pro Gln Glu Glu Phe Asp Asp Lys Gln Phe Gln Lys Ala Gln

85 90 95

Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr Phe Ash Leu Phe

100 105 110

Ser Thr Lys Asp Ser Ser Ala Ala Leu Asp Glu Thr Leu Leu Asp Glu

115 120 125

Phe Tyr Ile Glu Leu Asp Gln Gln Leu Asn Asp Leu Glu Ser Cys Val

130 135 140

Met Gln Glu Val Gly Val Ile Glu Ser Pro Leu Met Tyr Glu Asp Ser

145 150 155 160

Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Thr

165 170 175

Glu Lys Lys Tyr Ser Ser Cys Ala Trp Glu Val Val Arg Ala Glu Ile

180 185 190

Met Arg Ser Phe Ser Leu Ser Ile Asn Leu Gln Lys Arg Leu Lys Ser

195 200 205

Lys Glu stop

210

4, adjuvant according to claim 3 is characterized in that TA1 gene 87bp is a synthetic in wherein said thymosin and the interferon-ALPHA 8 fusion gene fragment combination.

5, adjuvant according to claim 3 is characterized in that connecting portion linker is [(GGT) in wherein said thymosin and the interferon-ALPHA 8 fusion gene fragment combination ₄TCT] n, n=2-4, its coding aa is (Gly ₄Ser ₁).

6, adjuvant according to claim 3, it is characterized in that wherein said thymosin and interferon-ALPHA 8 fusion gene fragment combination middle and lower reaches IFN α sequences and IFN α 8 are in full accord, do not contain start codon, long 501bp, preceding 3 of start codon is ACC, comes from China's Healthy adult's peripheral blood leucocyte.

7, adjuvant according to claim 2 is characterized in that wherein said non-replicating mammal cell with high efficient expression vector is pcDNA series, pRc/CMV, pCI or pVAX1.

8, adjuvant according to claim 1 is characterized in that wherein said preparation formulation comprises physiologically acceptable liquid preparation, emulsion formulations or lyophilized formulations.

9, a kind of adjuvant preparation method that is used to strengthen the hepatitis b vaccine immune effect as claimed in claim 1 is characterized in that the preparation of this adjuvant comprises the steps:

(1) amplification of IFN α 8 genes

From normal person's peripheral blood leucocyte, obtain IFN α 8, design primer PF1, PB1, by RT-PCR, the reverse transcription and IFN α 8 genetic fragments that increase are cut glue and are reclaimed standby;

(2) IFN α 8 genes and pGEM ^The connection of-T carrier

Order-checking behind the IFN α 8 genes insertion pGEM-T carrier is confirmed;

(3) TA1 and IFN α 8 genes is connected

In the upstream of IFN α 8 genes, remove start codon, whole 87 bases of synthetic TA1 do not have the codon of termination, and the coupling part are [(GGT) ₄TCT] n, acquire fusion gene TA1-IFN α 8 fragments;

(4) structure of TA1-IFN α 8 expression plasmids

TA1-IFN α 8 by double enzyme site KpnI, NotI, is connected in the non-replicating mammal cell with high efficient expression vector, obtains expression plasmid;

(5) large-scale production of TA1-IFN α 8 expression plasmids

With recombiant plasmid and unloaded plasmid transformed competence colibacillus JM109, through a large amount of amplification purification, double digestion identify, ultraviolet spectrophotometer quantitatively after, the physiological saline solution dilution promptly get hepatitis B nucleic acid vaccine potentiation molecule adjuvant, packing postposition-80 ℃ refrigerator preservation.

10. according to the preparation method of the described adjuvant of claim 9, it is characterized in that wherein said primer PF1 has following sequence:

tgtgatctgc ctcagactca c

11,, it is characterized in that wherein said primer PB1 has following sequence according to the preparation method of the described adjuvant of claim 9:

ttattcctta ctcttcaatc

12, the application of a kind of adjuvant as claimed in claim 1 in preparation hepatitis B virus immune medicine is characterized in that described medicine is a hepatitis B nucleic acid vaccine.

13, application according to claim 12 is characterized in that wherein said hepatitis B nucleic acid vaccine means the preceding S of HBV ₁, preceding S ₂, S, preceding C, C, P, X gene or its various combinations expression plasmid.

14, application according to claim 12, it is characterized in that wherein said medicine is the medicine that is used for HBV persistent infection person, comprise hepatocarcinoma after various chronic viral hepatitis B patients, hbv-liver cirrhosis and the hepatitis B and the HBV dna replication dna person of enlivening, HBV DNA are integrated state person's medicine.