CN108277230A

CN108277230A - A kind of fusion dna and its vaccine of preparation

Info

Publication number: CN108277230A
Application number: CN201810124108.XA
Authority: CN
Inventors: 范雄林
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2018-02-07
Filing date: 2018-02-07
Publication date: 2018-07-13

Abstract

The present invention relates to a kind of fusion dna and its vaccines of preparation, belong to biomedicine field.The present invention fusion dna, encoding gene be Kozak sequences, tPA sequences, Rv0577, Rv2875, Rv3044 and Rv2073c, gene according to Kozak tPA Rv0577 Rv2875 Rv3044 Rv2073c sequential series.The fusion dna of the present invention can be used for preparing tuberculosis DNA vaccination and recombinant adenoviral vector vaccine, wherein a concentration of 0.2~0.8mg/ml of fusion dna in DNA vaccination.The DNA vaccination of the present invention can be used for auxiliary treatment pulmonary tuberculosis, shorten the chemotherapeutic drug therapy period, rAdKtCMFO vaccines need not use adjuvant, and need to only be inoculated with the primary body that can induce and generate stronger immune response, it can be used for latent infection and prevention lungy of being grown up, it is safe.

Description

A kind of fusion dna and its vaccine of preparation

Technical field

The invention belongs to biomedicine field, it is related to a kind of fusion dna KtCMFO and its vaccine of preparation.

Background technology

BCG vaccine (Mycobacterium bovis BCG) is the currently the only prevention tuberculosis in clinical application The vaccine of (tuberculosis, TB) can effectively prevent infant's tuberculosis.Although since 1974, BCG is immune just It is included in expansion Immunization programme, but tuberculosis is still the communicable disease for causing global death toll most at present, especially, at Human tuberculosis are the main reason for leading to TB endemic and death.Generation lungy of being grown up is mainly endogenous by latent infection Caused by property reactivation；Minor path is fallen ill by heteroinfection.Moreover, mycobacterium tuberculosis and human immunodeficiency Malicious (HIV) coinfection rate is continuously increased, on substance of medicines-resistant branched tubercle bacillus and extensive Drug-Resistant Mycobacterium tuberculosis infection rate continue It rises, significant difficulty is brought to prevention and treatment lungy, tuberculosis has been aggravated and public health is seriously threatened.Face at present Tuberculosis is treated on bed at least to be needed to take 6 months chemotherapeutics, and since treatment cycle is long, patient dependence is poor, easy tos produce Drug-fast bacteria.As attenuated live vaccine, BCG not recommended immunoprophylaxis defect crowd, especially HIV Positive Populations；And BCG is not It can prevent and/or treat latent tuberculosis infection and at human tuberculosis, and crowd repeats BCG is immunized cannot improve anti-infective protection Property.Especially so far from the 1970s, two kinds of new drugs (shellfish reaches quinoline and De Lamanni) are only developed for lungy Treatment, new drug development are difficult to keep up with the demand of clinical treatment.Therefore, there is an urgent need to develop more safer than BCG, more effective epidemic disease Seedling, for the above-mentioned prevention and/or treatment lungy for being difficult to prevent.

For vaccine other than it can prevent tuberculosis, vaccine activates cellular immunity in patient body by immunity inoculation patient Power is other than chemicals, for treating important means and method lungy.But so far, clinically there has been no one Kind vaccine can effectively treat tuberculosis, also can effectively prevent tuberculosis without a kind of vaccine.The construction strategy of novel Vaccinum Calmette-Guerini Including attenuated live vaccine, vector-viral vaccine, subunit protein vaccine and DNA vaccination etc..Wherein, vector-viral vaccine does not need Adjuvant, immunogenicity is strong, can pass through that respiratory mucosa is immune or the approach such as intramuscular injection are immune, induction and protein vaccine or its His different immunization type of type vaccine and play effective anti-infectious function, be the important development side of tuberculosis new generation vaccine To shortcoming is that the preservation of virus needs special equipment and condition；Protein subunit vaccine is mainly used for lungy pre- Anti-, it is that its purifying prepares more complex process to have good safety, shortcoming, needs effective cellular immunity adjuvant that could produce Raw anti-infectious protectiveness.Relative to live attenuated vaccine virus carrier bacterin and protein vaccine, DNA vaccination definite ingredients, safety Property it is high；DNA vaccination preparation method is simple, it is easier to which mass production is stablized, and easily stored and transport, cost of manufacture are significantly low In other types vaccine；And eukaryon expression plasmid pVAX1 is that can be used for preparing DNA vaccination and be applicable in by U.S. FDA certification In clinical application.Based on above-mentioned advantage, DNA vaccination is in the development field of tuberculotherapy vaccine by attention.So far, More than 60 kinds DNA vaccinations are constructed, still, these DNA vaccination generally existing immunogenicities are weak, resisting tuberculosis infection effect is paid no attention to The technical problems such as think.The difficulty of development, still main concentrate select suitable antigen of mycobacterium tuberculosis and construction strategy, it is necessary to It is widely tested, is likely to obtain effective vaccine.Single vaccine classes cannot solve the inhomogeneity clinically occurred The difficult prevention type tuberculosis of type, it is necessary to be directed to these different tuberculosis types, make full use of the advantage of different type vaccine, open Hairpin carries out accurate immune protection, reaches control and even eliminate tuberculosis to different type difficulty prevention property effective vaccine lungy The purpose of disease.

Application No. is 201510523087.5 patent applications to disclose a kind of fusion protein CMFO and its application, passes through choosing It selects antigen gene and fusion sequence produces fusion protein CMFO, coordinate corresponding vaccine adjuvant DMT as tuberculosis subunit Protein vaccine CMFO/DMT, it is safe for preventing working well for latent tuberculosis infection.Although it has significant pre- Anti- effect, still, protein vaccine may cause potential Koch's pathological reaction and cannot be used for clinic and control for treating tuberculosis It treats；And fused antigen CMFO is prepared by prokaryotic expression system, the preparation process complexity of protein will ensure that biology is lived Property cause production cost high, and need expensive adjuvant, it is still necessary to which research and development low cost and effective vaccine are for tying The treatment and prevention of core disease.In addition, application No. is 201510140710.9 patent applications to disclose therapeutic tuberculosis branch bar Bacterium DNA vaccination and the preparation method and application thereof, although the structure of the DNA vaccination is instructed with the secreting signal peptide of GM-CSF The secreting, expressing of Ag85A-ESAT6 fusion proteins, similar with other disclosed more than 60 kind DNA vaccinations, antigen only selects tuberculosis bar The Ag85A and ESAT6 that bacterium secretes under active growth state, these antigens only target the bacterium of internal fast breeding state, right Latent infection is invalid, and the less immunogenic of the vaccine of its structure, and the anti-infection effect in zoopery is still not as good as BCG.

Invention content

In view of the problems of the existing technology and defect, the present invention provides a kind of fusion dna KtCMFO and its preparations Vaccine, its object is to be carrier by comparing protein subunit vaccine, DNA vaccination and recombined adhenovirus based on same antigen Vaccine for preventing and treating tuberculosis, filter out more efficiently vaccine, thus solve BCG and cannot be used for immune deficiency people Group cannot prevent latent infection and treatment clinical problem lungy, and the technology of preparing of solution subunit protein vaccine is answered It is miscellaneous to limit its scale for clinical and existing Tubercle DNA disease vaccine immunogenicity to be weak, anti-infection effect is undesirable asks Topic.

To achieve the above object, of the invention first is designed to provide a kind of fusion dna, and the fusion dna is compiled Code gene is Kozak sequences, tPA sequences, Rv0577, Rv2875, Rv3044 and Rv2073c, and gene is according to Kozak-tPA- The sequential series of Rv0577-Rv2875-Rv3044-Rv2073c.

Preferably, the sequence of the fusion dna is：

(1) nucleic acid sequence shown in SEQ ID No.1；Or

(2) nucleic acid sequence homology limited with sequence SEQ ID No.1 encodes identical function albumen 80% to 100% The nucleic acid sequence of matter.

Second object of the present invention is to provide the application of fusion dna described above, is applied to prepare tuberculosis DNA Two aspect of vaccine and recombinant adenoviral vector vaccine.

The one side that the present invention applies provides a kind of DNA vaccination for tuberculosis prophylaxis and immunization therapy, containing upper State the fusion dna.

Further, DNA vaccination described in above-mentioned technical proposal is the pVAXI plasmids containing the fusion dna.

Preferably, the concentration of fusion dna described in above-mentioned technical proposal in 0.2mg/ml between 0.8mg/ml.

Preferably, DNA vaccination described in above-mentioned technical proposal contain 2mg/ml to 3mg/ml dimethyl dioctadecyl ammoniums, For 0.2mg/ml to 0.3mg/ml monophosphoryl lipid As and the trehalose of 0.4mg/ml to 0.6mg/ml, the vaccine is liposome Form.

Preferably, trehalose described in above-mentioned technical proposal is 6,6 '-two mycolic acids.

The another aspect that the present invention applies provides a kind of recombinant adenovirus for latent infection and tuberculosis prophylaxis of being grown up Poisonous carrier vaccine contains fusion dna described above.

Preferably, the concentration of the recombined adhenovirus described in above-mentioned technical proposal is 1 × 10⁷To 1 × 10⁹Between.

Compared with prior art, the present invention having the advantages that：

(1) Kozak can increase the transcriptional level 20 of fusion tPA-Rv0577-Rv2875-Rv3044-Rv2073c Times, be conducive to the high level expression of CMFO, improve the immunogenicity of DNA or adenovirus vaccine；Fusion can be improved in tPA The secreting, expressing of Rv0577-Rv2875-Rv3044-Rv2073c, to improve immunogenicity；Rv0577 is tubercle bacillus in work The antigen that characteristic is expressed under jump vegetative state, Rv2875, Rv3044 and Rv2073c are that tubercle bacillus resides in low-oxygen environment such as The antigen of characteristic expression, these single antigen-immunized animals inductions in granuloma or in latent tuberculosis infection person's body Anti-infective protectiveness not as good as BCG；When as fusion protein Rv0577-Rv2875-Rv3044-Rv2073c (i.e. CMFO), Its immunogenicity is better than BCG, and obtain with the protectiveness of the comparable anti-primary infections of BCG, even realize that BCG does not have it is anti-latent Volt infects and treats ability lungy.

(2) Kozak sequences of the invention, tPA sequences, antigen of mycobacterium tuberculosis Rv0577, Rv2875, Rv3044 and Rv2073c encoding genes are merged in a certain order, the fusion dna joint DMT adjuvants and recombined adhenovirus epidemic disease of preparation Seedling can effectively stimulate the Th1 type immune responses of mouse generation antigen-specific.DNA vaccination, that is, pKtCMFO/DMT vaccines, with The effect for the Killing Mycobacterium Tuberculosis primary infection that recombinant adenovirus vaccine, that is, rAd KtCMFO vaccines generate is suitable with BCG. For DNA vaccination due to its definite ingredients, safety is good, is readily produced, and is one of the main Types of Development of Novel Vaccinum Calmette-Guerini, this hair The DNA vaccination pKtCMFO/DMT of bright offer, prepares that purifying process is simpler, is not necessarily to the label of external source, relative to protein vaccine and There is BCG good safety, inoculation risk to substantially reduce.Importantly, pKtCMFO/DMT vaccines can be used for assisting Pulmonary tuberculosis is treated, the chemotherapeutic drug therapy period is shortened；RAd KtCMFO vaccines need not use adjuvant, and need to only be inoculated with primary It induces body to generate stronger immune response, obtains the protectiveness with the comparable anti-primary infections of BCG, and can be used for BCG cannot The immunoprophylaxis of the latent infection crowd of immunoprophylaxis.

Description of the drawings

Fig. 1 is the forming types figure of recombinant plasmid pVAX1-Kozak-tPA-CMFO (pKtCMFO) in embodiment 2；

Fig. 2 is that mice group injects pCFMO/DMT, pKCFMO/DMT, ptCFMO/DMT, pKtCMFO/ in embodiment 21 After DMT vaccine immunities 9 weeks, Th1 type immune response testing result figures：Fig. 2A is that the IgG that CMFO is special in each group mice serum is anti- Body testing result；Fig. 2 B are IgG1 antibody test results；Fig. 2 C are IgG2a antibody test results；Fig. 2 D are IgG2a/IgG1 ratios It is worth testing result；Fig. 2 E are the horizontal testing result of IFN-γ that CMFO is special in mouse boosting cell supernatant；Wherein-represent p< 0.05；

Fig. 3 is that mouse immune is after 9 weeks in embodiment 22, Th1 type of the pKtCMFO/DMT groups mouse to different subfraction antigens Immune response testing result：Fig. 3 A are the IgG antibody testing result of different subfraction antigen-specifics in each group mice serum；Fig. 3 B For IgG1 antibody test results；Fig. 3 C are IgG2a antibody test results；Fig. 3 D are IgG2a/IgG1 ratio figures；Fig. 3 E are mouse The IFN-γ of different subfraction antigen-specifics is horizontal in splenocyte supernatant；Wherein-represent p<0.05；

Fig. 4 is that mice group injects pKt0577/DMT, pKt2875/DMT, pKt3044/DMT in embodiment 23, After pKt2073c/DMT, pKtCFMO/DMT vaccine immunity 9 weeks, Th1 type immune response testing results：Fig. 4 A are each group mouse blood The IgG antibody testing result of antigen-specific in clear；Fig. 4 B are IgG1 antibody test results；Fig. 4 C are IgG2a antibody test results； Fig. 4 D are IgG2a/IgG1 ratio testing result figures；Fig. 4 E are the IFN-γ water of antigen-specific in each group mouse boosting cell supernatant It is flat；Wherein-represent p<0.05.

Fig. 5 be in embodiment 23 H37Rv collunariums infection respectively be immunized PBS, pKt0577/DMT, pKt2875/DMT, The C57BL/6 mouse of pKt3044/DMT, pKt2073c/DMT, pKtCMFO/DMT after 9 weeks, each group mouse lung lotus bacterium amount knot Fruit is schemed；Wherein-represent p<0.05；

Fig. 6 be in embodiment 24 H37Rv collunariums infection respectively be immunized PBS, DDA, DMT, pKtCMFO, pKtCMFO/DMT, C57BL/6 mouse of the BCG after 9 weeks, each group mouse lung lotus bacterium amount result figure；Wherein-represent p<0.05；

Fig. 7 be in embodiment 25 H37Rv infect C57BL/6 mouse, receive respectively pcD685A, CMFO/DMT, PKtCMFO/DMT vaccine therapies are after 4 months, with the mouse lung lotus bacterium amount testing result figure for not receiving treatment；Wherein-represent p <0.05；

Fig. 8 is the forming types figure of recombined adhenovirus rAdKtCMFO in embodiment 26；

Fig. 9 is that mice group injects pKtCFMO/DMT, CMFO/DMT, rAdKtCMFO in embodiment 27, and BCG is 9 weeks immune Afterwards, Th1 types immune response testing result：Fig. 9 A are the IgG antibody testing result of antigen-specific in each group mice serum；Fig. 9 B are IgG1 antibody test results；Fig. 9 C are IgG2a antibody test results；Fig. 9 D are IgG2a/IgG1 ratio testing result figures；Fig. 9 E For the horizontal testing result of IFN-γ of antigen-specific in each group mouse boosting cell supernatant；Wherein-represent p<0.05；

Figure 10 be in embodiment 27 H37Rv collunariums infection respectively be immunized PBS, pKtCFMO/DMT, CFMO/DMT, C57BL/6 mouse after rAdKtCMFO, BCG9 weeks, each group mouse lung lotus bacterium amount testing result；Wherein-represent p<0.05.

Specific implementation mode

Technical scheme of the present invention is described in detail below by specific embodiment and attached drawing.Following reality It is preferred embodiments of the present invention to apply example only, is not the restriction that other forms are done to the present invention, any skill for being familiar with this profession The equivalent embodiment that art personnel are changed to change on an equal basis possibly also with the technology contents of the disclosure above.It is every without departing from this hair Bright plan content, any simple modification made according to the technical essence of the invention to following embodiment or equivalent variations, fall Within the scope of the present invention.

The design and synthesis of 1 fusion CMFO of embodiment and single antigen gene sequences and primer

The coded sequence of Rv0577, Rv2875, Rv3044 and the Rv2073c of M.tb H37Rv are searched by Genbank. Whether signal peptide is carried with Signalp software predictions and UniProtKB database retrievals search sequence, then with DNAMAN softwares pair It carries out restriction enzyme site analysis.The translation effect that Kozak sequences (GCCACC) are used for enhancing target gene is added before target gene Rate.In order to make final expression product be secreted into extracellularly, before the gene order of splicing, tPA signal peptide sequences are added. The splicing of 4 target gene after tPA signal peptides need to follow following principle：If 1) there is the signal peptide that remove of signal peptide, including Initiation codon originally, while removing terminator codon (after the last one gene order that splicing finishes plus TAA)；2) nothing The gene order of signal peptide then retains initiator sequences.If initiation codon is GTG's, because its original expression product is GTG need to be corrected to ATG by Met rather than Val.The fusion and single antigen gene splicing sequence are respectively：Kozak sequences- TPA sequences-Rv0577-Rv2875-Rv3044-Rv2073c；Kozak sequence-tPA sequences-Rv0577；Kozak sequence-tPA sequences Row-Rv2875；Kozak sequence-tPA sequences-Rv3044；Kozak sequence-tPA sequences-Rv2073c.Sequence is by the handsome life in Shanghai Object company synthesizes and is implemented in pDC316 vector plasmids, is respectively designated as pDC316-KtCMFO, pDC316-Kt0577, PDC316-Kt2875, pDC316-Kt3044, pDC316-Kt2073c are compared errorless through sequencing.

The structure of 2 recombinant plasmid pVAX1-Koza-tPA-CMFO (pKtCMFO) of embodiment

1, target gene obtains：

(1) design of primers：According to the multiple cloning sites design on the complete sequence and eukaryotic expression vector pVAX1 of fusion Primer, primer sequence are synthesized by Shanghai Ying Jun biotech firms, and primer is diluted to 100pmol/ μ l, -20 DEG C of guarantors according to explanation It deposits spare.Primer sequence is shown in SEQ ID No.2 and SEQ ID No.3.

(2) PCR reaction systems：

(3) PCR reaction conditions：

98℃30s；98℃10s,68℃20s and 72℃90s,30cycles；72℃10min；4℃forever

2, the structure of recombinant plasmid：

The PCR product and carrier pVAX1 that are recycled by DNA gel electrophoresis are subjected to double digestion respectively, digestion system is：

The target gene KtCMFO segments that digestion is recycled are attached with carrier pVAX1 according to following condition and are reacted：

3, the identification of conversion and recombinant plasmid

PKtCMFO is through low temperature CaCl₂Method and heat shock are transferred to E.coli DH5 α, and positive gram is obtained after resistance screening It is grand.Recombinant plasmid pKtCMFO is through digestion and sequencing identification, correctly.The structure of pKtCMFO is as shown in Figure 1.

Embodiment 3 recombinant plasmid pVAX1-CMFO (pCMFO), pVAX1-Koza-CMFO (pKCMFO), pVAX1-tPA- CMFO (ptCMFO), pVAX1-Koza-tPA-0577 (pKt0577), pVAX1-Koza-tPA-2875 (pKt2875), pVAX1- The structure of Koza-tPA-3044 (pKt3044) and pVAX1-Koza-tPA-2073c (pKt2073c)

1, target gene obtains：

(1) design of primers：According to the multiple cloning sites design on the complete sequence and eukaryotic expression vector pVAX1 of fusion Primer, primer sequence are synthesized by Shanghai Ying Jun biotech firms, and primer is diluted to 100pmol/ μ l, -20 DEG C of guarantors according to explanation It deposits spare.Primer sequence is shown in No.4~10 SEQ ID.

(2) PCR reaction systems：

The template for expanding CMFO segments is pDC316-KtCMFO, and upstream and downstream primer sequence is respectively P1：SEQ IDNo.4； P2：SEQ ID No.3；

The template for expanding KCMFO segments is pDC316-KtCMFO, and upstream and downstream primer sequence is respectively P1：SEQ IDNo.5； P2：SEQ ID No.3；

The template for expanding tCMFO segments is pDC316-KtCMFO, and upstream and downstream primer sequence is respectively P1：SEQ IDNo.6； P2：SEQ ID No.3；

The template for expanding Kt0577 segments is pDC316-Kt0577, and upstream and downstream primer sequence is respectively P1：SEQ IDNo.1；P2：SEQ ID No.7；

The template for expanding Kt2875 segments is pDC316-Kt2875, and upstream and downstream primer sequence is respectively P1：SEQ IDNo.1；P2：SEQ ID No.8；

The template for expanding Kt3044 segments is pDC316-Kt3044, and upstream and downstream primer sequence is respectively P1：SEQ IDNo.1；P2：SEQ ID No.9；

The template for expanding Kt2073c segments is pDC316-Kt2073c, and upstream and downstream primer sequence is respectively P1：SEQ IDNo.1；P2：SEQ ID No.10；

(3) PCR reaction conditions：

98℃30s；98℃10s,68℃20s and 72℃50s,30cycles；72℃10min；4℃forever

2, the structure of recombinant plasmid：

The target gene fragment that digestion is recycled is attached with carrier pVAX1 according to following condition and is reacted：

3, the identification of conversion and recombinant plasmid

Recombinant plasmid is through low temperature CaCl₂Method and heat shock are transferred to E.coli DH5 α, are obtained after resistance screening positive Clone.Recombinant plasmid is through digestion and sequencing identification, correctly.Its nucleic acid sequencing result see sequence table SEQ IDNo.11~ 17。

Embodiment 4

A kind of tuberculosis DNA vaccination, the fusion dna pKtCMFO, a concentration of 0.5mg/ml prepared containing embodiment 2.

Embodiment 5

A kind of DDA vaccine adjuvants contain 2.5mg/ml dimethyl dioctadecyl ammoniums (DDA), are liposomal form.

Preparation method is as follows

DDA 2.5mg accurately are weighed, are dissolved in chloroform and in methyl alcohol mixed liquor (9:1, v/v), then it is slowly introducing nitrogen Organic solvent is dried up, until forming one layer of white film.Lipid membrane is dried overnight at room temperature, remaining is had to remove Solvent.It is dissolved with 1mlPBS, 60 DEG C of heating water bath 1h (vortex makes it fully dissolve per 10min) are prepared into institute after cooling DDA vaccine adjuvants are stated, are liposomal form.

Embodiment 6

A kind of DMT vaccine adjuvants contain 2.5mg/ml dimethyl dioctadecyl ammoniums (DDA), 0.25mg/ml monophosphoryl lipids The trehalose of matter A and 0.5mg/ml are liposomal form.The trehalose is 6,6 '-two artificial synthesized mycolic acids.

Preparation method is as follows

DDA 2.5mg, MPL 0.25mg, TDB 0.5mg accurately are weighed, it is dissolved in chloroform and in methyl alcohol mixed liquor (9:1, V/v), then it is slowly introducing nitrogen to dry up organic solvent, until forming one layer of white film.By lipid membrane mistake at room temperature Night dries, to remove remaining organic solvent.It is dissolved with 1mlPBS, (per 10min, vortex keeps it fully molten to 60 DEG C of heating water bath 1h Solution), the DMT vaccine adjuvants are prepared into after cooling, are liposomal form.

Embodiment 7

A kind of tuberculosis DNA vaccination, containing fusion dna pKtCMFO prepared by embodiment 2, concentration is respectively 0.2mg/ Ml, 0.5mg/ml, 0.8mg/ml, isometric to mix respectively with DDA adjuvants, conventional mechanical oscillation or stirring are mixed into uniform Suspension.

The preparation method of the tuberculosis DNA vaccination is as follows：

The DNA solution that 100 μ l working concentrations are 0.5mg/ml and DDA adjuvants prepared by 100 μ l are drawn in sterile EP tube, In interval concussion 2 to 3 minutes in vortex oscillator, uniform emulsion is ultimately formed, the tuberculosis DNA vaccination is obtained pKtCMFO/DDA。

The DDA vaccine adjuvants contain 2.5mg/ml dimethyl dioctadecyl ammoniums (DDA), are liposomal form.

Preparation method is as follows：

DDA 2.5mg accurately are weighed, are dissolved in chloroform and in methyl alcohol mixed liquor (9:1, v/v), then it is slowly introducing nitrogen Organic solvent is dried up, until forming one layer of white film.Lipid membrane is dried overnight at room temperature, remaining is had to remove Solvent.It is dissolved with 1ml PBS, 60 DEG C of heating water bath 1h (vortex makes it fully dissolve per 10min) are prepared into institute after cooling DDA vaccine adjuvants are stated, are liposomal form.

Embodiment 8

A kind of tuberculosis DNA vaccination, containing fusion dna pKtCMFO prepared by embodiment 2, concentration is respectively 0.2mg/ Ml, 0.5mg/ml, 0.8mg/ml, isometric to mix respectively with DMT adjuvants, conventional mechanical oscillation or stirring are mixed into uniform Suspension.

The preparation method of the tuberculosis DNA vaccination is as follows：

The DNA solution that 100 μ l working concentrations are 0.2mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP tube, In interval concussion 2 to 3 minutes in vortex oscillator, uniform emulsion is ultimately formed, the tuberculosis DNA vaccination is obtained pKtCMFO/DMT。

The DMT vaccine adjuvants contain 2.5mg/ml dimethyl dioctadecyl ammoniums (DDA), 0.25mg/ml monophosphoryl lipids The trehalose (TDB) of matter (MPLA) and 0.5mg/ml is liposomal form.The trehalose is artificial synthesized 6,6 '-two Mycolic acid.

Preparation method is as follows：

DDA 2.5mg, MPL 0.25mg, TDB 0.5mg accurately are weighed, is dissolved in chloroform and in methyl alcohol mixed liquor (9:1, V/v), then it is slowly introducing nitrogen to dry up organic solvent, until forming one layer of white film.By lipid membrane mistake at room temperature Night dries, to remove remaining organic solvent.It is dissolved with 1ml PBS, (per 10min, vortex makes it fully to 60 DEG C of heating water bath 1h Dissolving), the DMT vaccine adjuvants are prepared into after cooling, are liposomal form.

Embodiment 9

A kind of tuberculosis DNA vaccination, which is characterized in that contain fusion dna pKtCMFO prepared by embodiment 2, concentration point Not Wei 0.2mg/ml, 0.5mg/ml, 0.8mg/ml, isometric to mix respectively with DMT adjuvants, conventional mechanical oscillation or stirring mix Synthesize uniform suspension.

The preparation method of the tuberculosis DNA vaccination is as follows：

The DNA solution that 100 μ l working concentrations are 0.5mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP tube, In interval concussion 2 to 3 minutes in vortex oscillator, uniform emulsion is ultimately formed, that is, is prepared into the tuberculosis DNA vaccination pKtCMFO/DMT。

The DMT vaccine adjuvants contain 2.5mg/ml dimethyl dioctadecyl ammoniums (DDA), 0.25mg/ml monophosphoryl lipids The trehalose of matter A and 0.5mg/ml are liposomal form.The trehalose is 6,6 '-two artificial synthesized mycolic acids.

Preparation method is as follows

DDA2.5mg, MPL 0.25mg, TDB 0.5mg accurately are weighed, is dissolved in chloroform and in methyl alcohol mixed liquor (9:1, v/ V), it is then slowly introducing nitrogen to dry up organic solvent, until forming one layer of white film.At room temperature overnight by lipid membrane It dries, to remove remaining organic solvent.It is dissolved with 1mlPBS.(per 10min, vortex keeps it fully molten to 60 DEG C of heating water bath 1h Solution), the DMT vaccine adjuvants are prepared into after cooling, are liposomal form.

Embodiment 10

The preparation method of the tuberculosis DNA vaccination is as follows：

The fusion dna solution that 100 μ l working concentrations are 0.8mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP Guan Zhong ultimately forms uniform emulsion, that is, is prepared into the tuberculosis in interval concussion 2 to 3 minutes in vortex oscillator DNA vaccination pKtCMFO/DMT.

Preparation method is as follows

Embodiment 11

A kind of tuberculosis DNA vaccination, which is characterized in that contain fusion dna pCMFO prepared by embodiment 3, concentration difference It is isometric to mix respectively with DMT adjuvants for 0.2mg/ml, 0.5mg/ml, 0.8mg/ml, conventional mechanical oscillation or stirring, mixing As uniform suspension.

The preparation method of the tuberculosis DNA vaccination is as follows：

The DNA solution that 100 μ l working concentrations are 0.5mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP tube, In interval concussion 2 to 3 minutes in vortex oscillator, uniform emulsion is ultimately formed, that is, is prepared into the tuberculosis DNA vaccination pCMFO/DMT。

Preparation method is as follows

Embodiment 12

A kind of tuberculosis DNA vaccination, which is characterized in that contain fusion dna pKCMFO prepared by embodiment 3, concentration point Not Wei 0.2mg/ml, 0.5mg/ml, 0.8mg/ml, isometric to mix respectively with DMT adjuvants, conventional mechanical oscillation or stirring mix Synthesize uniform suspension.

The preparation method of the tuberculosis DNA vaccination is as follows：

The DNA solution that 100 μ l working concentrations are 0.5mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP tube, In interval concussion 2 to 3 minutes in vortex oscillator, uniform emulsion is ultimately formed, that is, is prepared into the tuberculosis DNA vaccination pKCMFO/DMT。

Preparation method is as follows

Embodiment 13

A kind of tuberculosis DNA vaccination, which is characterized in that contain fusion dna ptCMFO prepared by embodiment 3, concentration point Not Wei 0.2mg/ml, 0.5mg/ml, 0.8mg/ml, isometric to mix respectively with DMT adjuvants, conventional mechanical oscillation or stirring mix Synthesize uniform suspension.

The preparation method of the tuberculosis DNA vaccination is as follows：

The DNA solution that 100 μ l working concentrations are 0.5mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP tube, In interval concussion 2 to 3 minutes in vortex oscillator, uniform emulsion is ultimately formed, that is, is prepared into the tuberculosis DNA vaccination ptCMFO/DMT。

Preparation method is as follows

Embodiment 14

A kind of tuberculosis DNA vaccination, which is characterized in that contain fusion dna pKt0577 prepared by embodiment 3, concentration point Not Wei 0.2mg/ml, 0.5mg/ml, 0.8mg/ml, isometric to mix respectively with DMT adjuvants, conventional mechanical oscillation or stirring mix Synthesize uniform suspension.

The preparation method of the tuberculosis DNA vaccination is as follows：

The DNA solution that 100 μ l working concentrations are 0.5mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP tube, In interval concussion 2 to 3 minutes in vortex oscillator, uniform emulsion is ultimately formed, that is, is prepared into the tuberculosis DNA vaccination pKt0577/DMT。

Preparation method is as follows

Embodiment 15

A kind of tuberculosis DNA vaccination, which is characterized in that contain fusion dna pKt2875 prepared by embodiment 3, concentration point Not Wei 0.2mg/ml, 0.5mg/ml, 0.8mg/ml, isometric to mix respectively with DMT adjuvants, conventional mechanical oscillation or stirring mix Synthesize uniform suspension.

The preparation method of the tuberculosis DNA vaccination is as follows：

The DNA solution that 100 μ l working concentrations are 0.5mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP tube, In interval concussion 2 to 3 minutes in vortex oscillator, uniform emulsion is ultimately formed, that is, is prepared into the tuberculosis DNA vaccination p Kt2875/DMT。

Preparation method is as follows

Embodiment 16

A kind of tuberculosis DNA vaccination, which is characterized in that contain fusion dna pKt3044 prepared by embodiment 3, concentration point Not Wei 0.2mg/ml, 0.5mg/ml, 0.8mg/ml, isometric to mix respectively with DMT adjuvants, conventional mechanical oscillation or stirring mix Synthesize uniform suspension.

The preparation method of the tuberculosis DNA vaccination is as follows：

The DNA solution that 100 μ l working concentrations are 0.5mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP tube, In interval concussion 2 to 3 minutes in vortex oscillator, uniform emulsion is ultimately formed, that is, is prepared into the tuberculosis DNA vaccination p Kt3044/DMT。

Preparation method is as follows

Embodiment 17

A kind of tuberculosis DNA vaccination, which is characterized in that contain fusion dna pKt2073c prepared by embodiment 3, concentration Respectively 0.2mg/ml, 0.5mg/ml, 0.8mg/ml, isometric to mix respectively with DMT adjuvants, conventional mechanical oscillation or stirring, It is mixed into uniform suspension.

The preparation method of the tuberculosis DNA vaccination is as follows：

The DNA solution that 100 μ l working concentrations are 0.5mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP tube, In interval concussion 2 to 3 minutes in vortex oscillator, uniform emulsion is ultimately formed, that is, is prepared into the tuberculosis DNA vaccination pKt2073c/DMT。

Preparation method is as follows

Embodiment 18

A kind of tuberculosis subunit protein vaccine, which is characterized in that a concentration of to be respectively containing CMFO fusion proteins 0.1mg/ml, 0.2mg/ml, 0.5mg/ml are mixed with DMT adjuvants in equal volume respectively, and conventional mechanical oscillation or stirring are mixed into For uniform suspension.

The preparation method of the tuberculosis subunit vaccine is as follows：

The fusion protein solution that 100 μ l working concentrations are 0.2mg/ml and DMT adjuvants prepared by 100 μ l are drawn in sterile EP Guan Zhong ultimately forms uniform emulsion, that is, it is sub- to be prepared into the tuberculosis in interval concussion 2 to 3 minutes in vortex oscillator Unit-protein vaccine CMFO/DMT.The DMT vaccine adjuvants contain 2.5mg/ml dimethyl dioctadecyl ammoniums (DDA), The trehalose of 0.25mg/ml monophosphoryl lipid As and 0.5mg/ml is liposomal form.The trehalose is artificial synthesized 6,6 '-two mycolic acids.

Preparation method is as follows：

Embodiment 19

The structure of recombinant plasmid pcDNA3.1-ESAT6-Ag85A (pcD685A)

1, target gene obtains：The coded sequence of the ESAT-6 and Ag85A of M.tb H37Rv are searched by Genbank.

(1) design of primers：According to the polyclonal position in the sequence of ESAT-6 and Ag85A genes and carrier pcDNA3.1 (+) Point design primer, and GeneSOEing technologies are utilized, it is held with Ag85AF 5 ' at the ends ESAT-6R 5 ' and introduces complementary coding respectively The linker sequences of 15 amino acid, to form new fusion ESAT6-Ag85A, primer sequence is public by Shanghai English fine horse biology Department's synthesis, and primer is diluted to 100pmol/ μ l according to explanation, -20 DEG C save backup.Primer sequence is shown in SEQ ID No18, SEQ ID No.19, SEQ ID No.20 and SEQ ID No.21.

(2) PCR reaction systems：

The template for expanding ESAT-6 segments is mycobacterium tuberculosis gene group, and upstream and downstream primer sequence is respectively P1：SEQID No.18；P2：SEQ ID No.19；

The template for expanding Ag85A segments is mycobacterium tuberculosis gene group, and upstream and downstream primer sequence is respectively P1：SEQID No.20；P2：SEQ ID No.21；

(3) PCR reaction conditions：

98℃30s；98℃10s,68℃20s and 72℃50s,30cycles；72℃10min；4℃forever

(4) fusion DNA vaccine reaction system

The upstream and downstream primer sequence for expanding ESAT6-Ag85APCR segments is respectively P1：SEQ ID No.18；P2：SEQID No.21；

(5) PCR reaction conditions：

98℃30s；98℃10s,68℃1min and 72℃90s,30cycles；72℃10min；4℃forever

2, the structure of recombinant plasmid：

The PCR product and carrier pcDNA3.1 (+) that are recycled by DNA gel electrophoresis are subjected to double digestion, digestion body respectively System is：

The ESAT6-Ag85A segments that digestion is recycled are attached with carrier pcDNA3.1 (+) according to following condition and are reacted：

3, the identification of conversion and recombinant plasmid

PcD685A is through low temperature CaCl₂Method and heat shock are transferred to E.coli DH5 α, and positive gram is obtained after resistance screening It is grand.Recombinant plasmid pcD685A is through digestion and sequencing identification, correctly.

Embodiment 20

A kind of tuberculosis DNA vaccination, the fusion dna pcD685A, a concentration of 0.5mg/ml prepared containing embodiment 19.

Embodiment 21

The immunological effect of DNA vaccination pCFMO/DMT, pKCFMO/DMT, ptCFMO/DMT, pKtCFMO/DMT.Tuberculosis DNA vaccination pCFMO/DMT, pKCFMO/DMT, ptCFMO/DMT, pKtCMFO/DMT are respectively adopted in embodiment 11,12,13,9 The tuberculosis DNA vaccination of preparation,.

1, experimental animal is grouped and is immunized

SPF grades of C57BL/6 female mices, 6-8 week old are purchased from Wuhan University's Experimental Animal Center.The animal quality certification No.42000500005564.According to experiment needs, experimental animal is divided into following five groups：PBS groups；PCFMO/DMT groups； PKCFMO/DMT groups；PtCFMO/DMT groups；PKtCFMO/DMT groups；Every group 6.

Mouse is all made of muscle injection mode and is immunized, and volume injected is 200 μ l.Repeat immune secondary, every minor tick 3 Week.

2, ELISA detects immune serum specific antibody

1) mouse after being immunized 9 weeks collects serum after eyeball takes blood, and is frozen in -20 DEG C after dispensing；

2) fusion protein CMFO is diluted according to 5 μ g/ml of working concentration, 100 holes μ l/ carry out 96 holes of conventional coating Plate；

3) each group mice serum sample presses 1 with sterile 1 × PBS:400 times of dilutions, 200 holes μ l/ add to the first hole of each column, often The second hole of row to octal is separately added into 100 μ l PBS, and each sample does multiple holes, then carries out doubling dilution to 1 from the first hole: 51200,100 μ l of PBS are added in blank control wells；

4) HRP labels sheep anti mouse secondary antibody dilution is respectively：IgG 1:5000, IgG11:10000, IgG21:10000；

5) result treatment：After each each sample OD values are subtracted blank control wells OD values, taken with portion blood serum sample multiple holes Mean OD value.Wherein using the OD values of PBS negative control groups as negative control (N), immune group be sample (P), when blood-serum P/N values >= 2.1 can be judged as the positive.Antibody titer is indicated with the inverse for the highest extension rate of positive findings occur；

6) result calculates：Each sample result is expressed as log10 (antibody titer), be averaged between each sample multiple holes as The actual value of the mouse, same group of interior calculating average value and standard error, it is for statistical analysis；IgG2a：IgG1 is with every mouse Antibody titer is compared, and result is indicated with average value ± standard error in same group.

3, it is horizontal to detect mouse boosting cell culture supernatant antigentic specificity IFN-γ by ELISA

1) 100 μ l of extracting spleen cell suspension are inoculated in 96 orifice plates (cell number 2.5 × 10⁶)；

2) stimulant is CMFO albumen, 10 holes μ g/ of final concentration；

3) after cultivating 72 hours, liquid in hole is collected respectively and is marked, 2000rpm, 10min is centrifuged, collects supernatant And be sub-packed in EP pipes, it is frozen after label for use in -80 DEG C；

4) content of cell factor IFN-γ in supernatant is detected according to Mouse ELISAkit operation manuals：

5) it is returned to zero with blank control wells, double UV check light absorption value is read using microplate reader：450nm is Detection wavelength, 630nm is reference wavelength；

6) establishing criteria sample wells data draw standard curve, and surveying OD values according to sample measures corresponding cytokine concentrations. Results are averaged for every mouse multiple holes, and after subtracting RPMI1640 values of control groups, average value and mark are calculated separately in same group It is accurate poor, it is for statistical analysis.

Experimental result is shown in Fig. 2.

From the experimental result of Fig. 2A, Fig. 2 B, Fig. 2 C：With pCFMO/DMT, ptCFMO/DMT, pKCFMO/DMT is compared, PKtCFMO/DMT inducing mouses generate IgG, IgG1 and the IgG2a antibody of higher levels of antigen-specific.By Fig. 2 D it is found that PKtCFMO/DMT group IgG2a/IgG1 ratios are more than 1, and are higher than other three groups of (p<0.05).After 9 weeks immune, PBS group CMFO eggs The IFN-γ level of Bai Teyi is minimum.In addition, by Fig. 2 E it is found that compared with other three groups, pKtCFMO/DMT group inducing mouses produce IFN-γ (the p of raw high-caliber antigen-specific<0.05).TH1 is tended in immune response vaccine-induced prompt pKtCFMO/DMT Type immune response.

Embodiment 22

The immunological evaluation for single subfraction antigen of DNA vaccination pKtCFMO/DMT

Tuberculosis DNA vaccination pKtCMFO/DMT uses the tuberculosis DNA vaccination prepared in embodiment 9.

1, experimental animal immune

2, ELISA detects immune serum specific antibody

2) four kinds of subfraction antigen proteins are diluted according to 5 μ g/ml of working concentration respectively, 100 holes μ l/ carry out conventional It is coated with 96 orifice plates；

3) mice serum sample presses 1 with sterile 1 × PBS:400 times of dilutions, 200 holes μ l/ add to the first hole of each column, each column the Two holes to octal is separately added into 100 μ l PBS, and each sample does multiple holes, then carries out doubling dilution to 1 from the first hole: 51200,100 μ l of PBS are added in blank control wells；

2) stimulant is four kinds of subfraction antigen proteins, 10 holes μ g/ of final concentration；

Experimental result is shown in Fig. 3.

From experimental result：After pKtCFMO/DMT is immune, produced in mice serum for four kinds of subfraction antigen Specific antibody, and IgG2a/IgG1 ratios are all higher than 1.After 9 weeks immune, four kinds of subfraction antigen protein thorns of mouse boosting cell pair Swash, generates IFN-γ secretion.Wherein, the horizontal highest of the IFN-γ stimulation of 3044 albumen generated.These results prompt, four kinds Subfraction plays a significant role in pKtCFMO/DMT vaccines.

Embodiment 23

DNA vaccination pKt0577/DMT, pKt2875/DMT, pKt3044/DMT, pKt2073c/DMT, pKtCFMO/DMT's Immunological effect and anti-infective protectiveness

Tuberculosis DNA vaccination pKtCMFO/DMT, pKt0577/DMT, pKt2875/DMT, pKt3044/DMT, The tuberculosis DNA vaccination prepared in embodiment 9,14,15,16,17 is respectively adopted in pKt2073c/DMT.

1, experimental animal is grouped and is immunized

According to experiment needs, experimental animal is divided into following five groups：PBS groups；PKt0577/DMT groups；pKt2875/DMT Group；PKt3044/DMT groups；PKt2073c/DMT groups；PKtCFMO/DMT groups；Every group 12.

2, ELISA detects immune serum specific antibody

1) mouse after being immunized 9 weeks, every group takes 6 mouse, collects serum after eyeball takes blood, and frozen after dispensing in- 20℃；

2) 0577,2875,3044,2073c and fusion protein CMFO is diluted respectively according to 5 μ g/ml of working concentration, 100 holes μ l/ carry out 96 orifice plates of conventional coating；

3) each DNA vaccination group mice serum sample corresponds to respective antigen coat hole；PBS groups corresponding 0577,2875,3044, 2073c and fusion protein CMFO are coated with hole, the negative control as each DNA vaccination group.Sterile 1 × PBS presses 1:400 times of diluted bloods Clearly, 200 holes μ l/ add to the first hole of each column, and the second hole of each column to octal is separately added into 100 μ lPBS, and each sample does multiple holes, Then doubling dilution is carried out to 1 from the first hole:51200, PBS100 μ l are added in blank control wells；

1) 6 mouse, 100 μ l of extracting spleen cell suspension are taken to be inoculated in 96 orifice plates (cell number 2.5 × 10 for every group⁶)；

2) stimulant is that each DNA vaccination group corresponds to respective antigen, PBS and BCG groups fusion protein CMFO, 10 μ of final concentration The holes g/；

Experimental result is shown in Fig. 4.

From Fig. 4 A, 4B, 4C experimental result：With pKt0577/DMT, pKt2875/DMT, pKt3044/DMT, PKt2073c/DMT is compared, and pKtCFMO/DMT can induce IgG, IgG1 and the IgG2a that mouse generates higher levels of antigen-specific Antibody.In addition by Fig. 4 D it is found that pKtCFMO/DMT group IgG2a/IgG1 ratios be more than 1, and be higher than other DNA vaccination group (p< 0.05).By Fig. 4 E it is found that after 9 weeks immune, compared with other DNA vaccination groups, pKtCFMO/DMT group inducing mouses generate higher IFN-γ (the p of horizontal antigen-specific<0.05).

4, infection due to Mycobacterium tuberculosis mouse

It is every group 6, (practical using M.tb H37Rv standard strains through collunarium infection immunity mouse after initial immunity 9 weeks Infection dosage is about 100CFU/).

5, the lotus bacterium amount analysis of lungs

After H37Rv infects 4 weeks, mouse is put to death, sterile taking-up lungs are separately added into the sterile 1 × PBS of 2ml according to every internal organs, It is fully ground, is transferred in 5ml sterile tubes, oscillator mixes well in mortar.100 μ l stostes are taken to be added to 900 μ l PBS In do doubling dilution, after oscillation mixes well, each 100 μ l coatings 7H11 culture dishes of the bacterium solution of 4 dilutions is taken (to be added when preparation Cycloheximide inhibits the growth of fungi, and TCH is added can remain the growth of BCG with selective depression).Plate is placed in 37 DEG C of cultures Case culture carries out bacterium colony counting after 3~4 weeks.With Log₁₀(CFU) lotus bacterium amount analysis is carried out.Calculate the mean value and standard error of each group.

Experimental result is shown in Fig. 5, each group mouse lung lotus bacterium amounts.The lungs lotus bacterium amount highest of PBS negative control group mouse. The lotus bacterium amount of DNA vaccination group mouse lung significantly reduces (p relative to PBS groups<0.05).With pKt0577/DMT, pKt2875/ DMT, pKt3044/DMT, pKt2073c/DMT are compared, the lower (p of lotus bacterium amount of pKtCMFO/DMT groups<0.05).These result tables Bright, relative to single antigen dna vaccine, pKtCMFO/DMT vaccines preferably can inhibit mycobacterium tuberculosis in Mice Body Proliferation has the ability of stronger resistance mycobacterium tuberculosis infection.

Embodiment 24

The infection effect of the anti-M.tb H37Rv of mouse is immunized in DNA vaccination pKtCFMO/DMT

DDA, DMT adjuvant are respectively adopted the adjuvant prepared in embodiment 5,6, tuberculosis DNA vaccination pKtCMFO, The tuberculosis DNA vaccination prepared in embodiment 4,7,9 is respectively adopted in pKtCMFO/DDA, pKtCMFO/DMT.

1, experimental animal is grouped and is immunized

According to experiment needs, experimental animal is divided into following seven groups：PBS groups；DDA adjuvant groups；DMT adjuvant groups；pKtCMFO Group；PKtCMFO/DDA groups；PKtCMFO/DMT groups；BCG groups；Every group of 6 mouse.

PBS, adjuvant, DNA vaccination group mouse are all made of muscle injection mode and are immunized, and BCG is carried out using injected s.c. Immune, volume injected is 200 μ l.DDA adjuvant groups；DMT adjuvant groups；PKtCMFO groups；PKtCMFO/DDA groups；pKtCMFO/ DMT groups repeat immune secondary, every minor tick 3 weeks.BCG immunizing doses are 1 × 10⁶CFU/ is only.PBS groups and BCG groups immune 1 It is secondary.

2, infection due to Mycobacterium tuberculosis mouse

After initial immunity 9 weeks, using M.tb H37Rv standard strains through collunarium infection immunity mouse.

5, the lotus bacterium amount analysis of lungs

After H37Rv infects 4 weeks, mouse is put to death, sterile taking-up lungs are separately added into the sterile 1 × PBS of 2ml according to every internal organs, It is fully ground, is transferred in 5ml sterile tubes, oscillator mixes well in mortar.100 μ l stostes are taken to be added to 900 μ l PBS In do doubling dilution, after oscillation mixes well, each 100 μ l coatings 7H11 culture dishes of the bacterium solution of 4 dilutions is taken (to be added when preparation Cycloheximide inhibits the growth of fungi, and TCH is added can remain the growth of BCG with selective depression).Plate is placed in 37 DEG C of cultures Case culture carries out bacterium colony counting after 3~4 weeks.Lotus bacterium amount analysis is carried out with Log10 (CFU).Calculate the mean value and standard error of each group.

Experimental result is shown in Fig. 6, each group mouse lung lotus bacterium amounts.The lungs lotus bacterium amount highest of PBS negative control group mouse. DDA adjuvants and PBS group indifferences.The lotus bacterium amount of DMT adjuvant group mouse lungs is less than PBS groups (p<0.05), prompt DMT adjuvants can Generate certain non-specific protective effect.PKtCMFO vaccines can generate better protecting effect (p relative to DMT adjuvants< 0.05).DNA joint adjuvants enhance the effect of vaccine.Also, pKtCMFO/DMT, compared with BCG groups, lotus bacterium amount is without statistics Difference.These results indicate that pKtCMFO/DMT vaccines can preferably inhibit proliferation of the mycobacterium tuberculosis in Mice Body, support The ability of Killing Mycobacterium Tuberculosis infection is suitable with BCG.

Embodiment 25

The response to treatment of DNA vaccination

The tuberculosis prepared in embodiment 18,20,9 is respectively adopted in Vaccinum Calmette-Guerini CMFO/DMT, pcD685A, pKtCMFO/DMT Sick DNA vaccination.

1, infection due to Mycobacterium tuberculosis mouse

Using M.tb H37Rv standard strains through collunarium infection immunity mouse.

2, animal packet

After M.tb infects 18 days, start to treat.Experimental animal is divided into following eight groups：1. infecting non-treatment group；② PcD685A treatment groups；3. CMFO/DMT treatment groups；4. pKtCMFO/DMT treatment groups；5. chemotherapeutics group；6. chemotherapeutics+ PcD685A treatment groups；7. chemotherapeutics+CMFO/DMT treatment groups；8. chemotherapeutics+pKtCMFO/DMT treatment groups；Every group 6.

DNA vaccination group mouse is inoculated with after M.tb infects 18 days using muscle injection mode, is inoculated with again after three weeks.Chemotherapy The therapeutic scheme that medicine group uses for：2 months Rimactazids and pyrazinamide add 2 gentle isoniazid of monthly interest good fortune to control again It treats；It is administered by mouse weight, daily each drug dose is rifampin 10mg/kg, isoniazid 25mg/kg, pyrazinamide 150mg/ kg.Chemotherapeutics adds vaccine group to be inoculated with using muscle injection mode after M.tb infects 18 days, is inoculated with again after three weeks, is administered Scheme is consistent with chemotherapeutics group.

3, response to treatment is analyzed

After treatment 4 months, mouse is put to death, sterile taking-up lungs are separately added into the sterile 1 × PBS of 2ml according to every internal organs, It is fully ground, is transferred in 5ml sterile tubes, oscillator mixes well in mortar.It takes 100 μ l stostes to be added in 900 μ lPBS to do Doubling dilution after oscillation mixes well, takes each 100 μ l coatings 7H11 culture dishes of the bacterium solution of 4 dilutions.Plate is placed in 37 DEG C of trainings Bacterium colony counting is carried out after supporting case culture 3~4 weeks.Lotus bacterium amount analysis is carried out with Log10 (CFU).Calculate the mean value and standard of each group Accidentally.

Experimental result is shown in Fig. 7.The lungs lotus bacterium amount highest of non-treatment group mouse.PcD685A treatments are with non-treatment group without bright Significant difference is other.The lotus bacterium amount of CMFO/DMT groups and pKtCMFO/DMT group mouse lungs is below non-treatment group (p<0.05), but PKtCMFO/DMT group mouse lotus bacterium amounts are lower.

Table 1 is the C57BL/6 mouse that H37Rv infects in the present embodiment, receives vaccine Cocktail treatment after 4 months, lung Dirty lotus bacterium amount.

1 embodiment of table, 25 infecting mouse receives the lungs lotus bacterium amount table after different vaccine therapies

1 result of table is shown：Chemotherapy in 4 months makes the mouse of 50% (3/6) remove the bacteriums of lungs；Drug Joint pcD685A 33% (2/6) does not increase the mouse quantity of bacteria removal；CMFO/DMT enhances the therapeutic effect of drug, The bacterium of lungs that kept the mouse of 83% (5/6) fully erased；PKtCMFO/DMT vaccine adjuvant chemotherapy effect of drugs is most apparent, The mouse of 100% (6/6) is set to remove the bacterium of lungs.These results indicate that pKtCMFO/DMT vaccine energy adjuvant chemotherapy drugs Pulmonary tuberculosis is treated, treatment cycle is shortened.

Embodiment 26

Express packaging and the purifying of the recombined adhenovirus (rAdKtCMFO) of CMFO

1, by plasmid pDC316-KtCMFO and skeleton plasmid pBHGlox Δs E1,3Cre (1:1) cotransfection HEK293 cells, Pack recombinant virus rAdKtCMFO.

(1) the inoculation good HEK293 cells of growth conditions are in six orifice plates, per hole 5 × 10⁵A cell, 37 DEG C, 5%CO₂Carefully It is cultivated 18-24 hours in born of the same parents' incubator, replaces not antibiotic DMEM complete mediums.

(2)DNA-Lipofectamine^TMThe preparation of 2000 compounds：The DMEM culture mediums of serum are free of with 125 μ L, point 2 μ g plasmids pDC316-CMFO and 2 μ g plasmid pBHGlox Δ E1,3Cre are not diluted, and the two is blown and beaten into mixing, 250 μ of final volume L,；10 μ L LipofectamineTM 2000 are taken to be added in DMEM culture mediums of the 240 μ L not comprising serum, 250 μ of total volume L blows and beats mixing, is incubated at room temperature 5 minutes；The plasmid of mixed diluting is added to the diluted Lipofectamine of equivalent^TM2000 In, mixing is blown and beaten, is incubated at room temperature 20 minutes, to allow the formation of compound.

(3) by 2000 compounds of plasmid-LipofectamineTM after mixing totally 500 μ L be added dropwise to it is celliferous In culture medium, it is mixed evenly.37 DEG C, CO₂Incubator is incubated overnight.

After (4) 24 hours, the fresh DMEM complete mediums of 2mL are replaced.

After (5) 48 hours, trypsin digestion cell, the culture dish for being transferred to bigger expands culture.At this moment playing cell will generate Cytopathic effect, and virus is generated, and the more cells of infection.Fresh DMEM complete mediums were replaced per 2-3 days later.

(6) the tenth day after transfecting, whether observation cell monolayer has hole, if any cell death, the area cytopathic effect (CPE) Domain is up to 80%, you can collects.Such as without apparent CPE, fresh culture can be supplemented, continues to observe, until CPE is up to 80%.

(7) virus is collected, lesion will partly occur but even adherent cell is lightly blown and beaten, collects cell.- 80 DEG C of placements are placed on 37 DEG C of water-bath solutions in 30 minutes and melt 15 minutes, repeat freeze thawing 2 times.3000rpm room temperatures centrifuge 15 minutes, will be upper Sorting is filled in 1.5mL sterile EP tubes and freezes for -80 DEG C.

2, a large amount of Prepare restructuring adenovirus rAdKtCMFO

(1) in 75cm²Expand culture HEK293 cells in culture bottle, its density is made to reach 70%-100%.

(2) culture medium is removed, primary package rAdKtCMFO viral suspension 2mL/ culture bottles are slowly added to.Infection 1 hour Afterwards, DMEM complete mediums are added.

(3) after lesion phenomenon all occur in most cells (about 3-4 days) cell and culture medium, are collected.It is put by -80 DEG C It sets and is placed within 30 minutes 37 DEG C of water-bath solutions and melts 15 minutes, repeat 3 abundant lytic cell releasing virus particles of freeze thawing.3000rpm, 4 DEG C centrifuge 10 minutes, collect the supernatant containing virion, -80 DEG C save backup.

(4) CsCl density gradient centrifugations purified virus particles

1) 50mL viral pellets liquid (20%EG8000,2.5M NaCl), ice is added in the viral supernatants collected per 100mL 1 hour is bathed with precipitate virus.12000rpm is centrifuged 20 minutes, abandons supernatant, and it is 1.1g/mL's that sediment, which is suspended in 10mL density, In CsCl solution (solvent is 20mM Tris-HCl, pH 8.0), 4 DEG C, 7000rpm is centrifuged 5 minutes, collects viral suspension.

2) the 1.4g/mL CsCl solution (solvent is same as above) of 2mL is added in ultracentrifugation pipe, adds 3mL 1.3g/mL CsCl solution.It is eventually adding the viral suspension of 5mL, 22800rpm, 4 DEG C centrifuge 2.5 hours.Density is collected in 1.3- In virus band to bag filter between 1.4g/mL.In elution buffer (50g sucrose, the Tris- that 10mL 1M pH are 8.0 HCl liquid, 2mL 1M MgCl₂Solution is settled to 1000mL) in, 4 DEG C of dialysed overnights, it is primary that dialyzate is replaced in centre.

3) virus is collected, virus is added and preserves liquid, corresponding genomic medicine and vaccine is made in packing.It is stored in -80 ℃。

4) recombined adhenovirus titre detects

1. 1mL 5.0 × 10 is added in 24 orifice plates⁵The cell suspension of a/mL, 37 DEG C, 5%CO2 cultures.

2. the viral sample of 10 times of gradient dilutions is got out, then successively by 10^-5To 10^-8Diluted virus liquid is added 24 In orifice plate, 100 μ L are added per hole.

3. infection 48 hours removes culture solution, the 500 μ L of methanol of precooling are added per hole, -20 DEG C are fixed 20 minutes.It is added PBS rinses cell 3 times, every time 5 minutes.

4. 37 DEG C of 200 μ L 1%BSA are added to close 1 hour.PBS is added and rinses cell 3 times, every time 5 minutes.

5. being added in the primary antibody solution to each hole of 200 μ L, 37 DEG C are incubated 1 hour.PBS is added and rinses cell 3 times, every time 5 minutes.

6. the secondary antibody of 200 μ L is added to every hole, 37 DEG C are incubated 1 hour.PBS is added and rinses cell 3 times, every time 5 minutes.

7. working solution that 200 μ L are newly configured is added to every hole, it is incubated at room temperature 5-10 minutes.Working solution is abandoned, PBS is added and rinses 1mLPBS is added per hole for cell 3 times.

8. randomly choosing 5 visuals field per hole, calculating positive cell number under 10 × object lens of light microscope is used.

9. calculating the mean number and virus titer per hole positive cell.

The structure of rAdKtCMFO is as shown in Figure 8.

Embodiment 27

The immunological effect of Vaccinum Calmette-Guerini CMFO/DMT, pKtCFMO/DMT, rAdKtCMFO based on CMFO antigens and anti-sense Contaminate protectiveness

Tuberculosis vaccine pKtCFMO/DMT, CMFO/DMT, rAdKtCMFO are respectively adopted to be prepared in embodiment 9,18,26 Tuberculosis DNA vaccination.

1, experimental animal is grouped and is immunized

According to experiment needs, experimental animal is divided into following five groups：PBS groups；PKtCFMO/DMT groups；CMFO/DMT groups； RAdKtCMFO groups；BCG groups；Every group 12.

PKtCFMO/DMT groups mouse is immune using muscle injection mode, 200 μ l of volume injected；RAdKtCMFO groups are using drop Nose mode is immune, and dosage of inoculation is 1 × 10⁸PFU；PBS groups；CMFO/DMT groups；BCG groups are all made of injected s.c. and are immunized, Volume injected is that 200 μ l, BCG dosages of inoculation are 1 × 10⁶CFU.Wherein pKtCFMO/DMT, CMFO/DMT repeat immune two It is secondary, every minor tick 3 weeks；PBS groups；RAdKtCMFO groups；BCG groups are immune primary.

2, ELISA detects immune serum specific antibody

2) stimulant is fusion protein CMFO, 10 holes μ g/ of final concentration；

Experimental result is shown in Fig. 9.

From the experimental result of Fig. 9 A, 9B, 9C：Three kinds of vaccines based on CMFO antigens, can stimulate mouse to produce IgG, IgG1 and the IgG2a antibody of raw high-caliber antigen-specific.In addition, by 9D it is found that three kinds of vaccine group IgG2a/IgG1 ratios Value is all higher than 1, and is higher than BCG groups (p<0.05).Also, three kinds of equal inducing mouses of vaccine generate high-caliber antigen-specific IFN-γ。

4, infection due to Mycobacterium tuberculosis mouse

5, the lotus bacterium amount analysis of lungs

Experimental result is shown in Figure 10, each group mouse lung lotus bacterium amounts.The lungs lotus bacterium amount highest of PBS negative control group mouse. The lotus bacterium amount of three kinds of vaccine group mouse lungs based on CMFO antigens significantly reduces (p relative to PBS groups<0.05)；And And with the lotus bacterium indifference (p of BCG groups<0.05).These results indicate that three kinds of vaccines based on CMFO antigens, With the ability with the comparable resistance mycobacterium tuberculosis infection of BCG.

SEQUENCE LISTING

<110>The Central China University of Science and Technology

<120>A kind of fusion dna and its vaccine of preparation

<130>Nothing

<160> 21

<170> PatentIn version 3.3

<210> 1

<211> 3171

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

gccaccatgg atgcaatgaa gagagggctc tgctgtgtgc tgctgctgtg tggagcagtc 60

ttcgtttcgc ccatgcccaa gagaagcgaa tacaggcaag gcacgccgaa ctgggtcgac 120

cttcagacca ccgatcagtc cgccgccaaa aagttctaca catcgttgtt cggctggggt 180

tacgacgaca acccggtccc cggaggcggt ggggtctatt ccatggccac gctgaacggc 240

gaagccgtgg ccgccatcgc accgatgccc ccgggtgcac cggaggggat gccgccgatc 300

tggaacacct atatcgcggt ggacgacgtc gatgcggtgg tggacaaggt ggtgcccggg 360

ggcgggcagg tgatgatgcc ggccttcgac atcggcgatg ccggccggat gtcgttcatc 420

accgatccga ccggcgctgc cgtgggccta tggcaggcca atcggcacat cggagcgacg 480

ttggtcaacg agacgggcac gctcatctgg aacgaactgc tcacggacaa gccggatttg 540

gcgctagcgt tctacgaggc tgtggttggc ctcacccact ccagcatgga gatagctgcg 600

ggccagaact atcgggtgct caaggccggc gacgcggaag tcggcggctg tatggaaccg 660

ccgatgcccg gcgtgccgaa tcattggcac gtctactttg cggtggatga cgccgacgcc 720

acggcggcca aagccgccgc agcgggcggc caggtcattg cggaaccggc tgacattccg 780

tcggtgggcc ggttcgccgt gttgtccgat ccgcagggcg cgatcttcag tgtgttgaag 840

cccgcaccgc agcaaggcga tctggtgggc ccgggctgcg cggaatacgc ggcagccaat 900

cccactgggc cggcctcggt gcagggaatg tcgcaggacc cggtcgcggt ggcggcctcg 960

aacaatccgg agttgacaac gctgacggct gcactgtcgg gccagctcaa tccgcaagta 1020

aacctggtgg acaccctcaa cagcggtcag tacacggtgt tcgcaccgac caacgcggca 1080

tttagcaagc tgccggcatc cacgatcgac gagctgaaga ccaattcgtc actgctgacc 1140

agcatcctga cctaccacgt agtggccggc caaaccagcc cggccaacgt cgtcggcacc 1200

cgtcagaccc tccagggcgc cagcgtgacg gtgaccggtc agggtaacag cctcaaggtc 1260

ggtaacgccg acgtcgtctg tggtggggtg tctaccgcca acgcgacggt gtacatgatt 1320

gacagcgtgc taatgcctcc ggcgatgcga tccactgttg ctgtcgccgt agcggcagcc 1380

gtgatcgcag cgtccagtgg ttgcggctcc gatcaaccgg cccataaggc gtcacaatcg 1440

atgatcacgc ccaccaccca gatcgccggc gccggggtgc tgggaaacga cagaaagccg 1500

gatgagtcgt gcgcgcgtgc ggcggccgcg gccgatccgg ggccaccgac ccgaccagcg 1560

cacaatgcgg cgggagtcag cccggagatg gtgcaggtgc cggcggaggc gcagcgcatc 1620

gtggtgctct ccggtgacca gctcgacgcg ctgtgcgcgc tgggcctgca atcgcggatc 1680

gtcgccgccg cgttgccgaa cagctcctca agtcaacctt cctatctggg cacgaccgtg 1740

catgatctgc ccggtgtcgg tactcgcagc gcccccgacc tgcgcgccat tgcggcggct 1800

cacccggatc tgatcctggg ttcgcagggt ttgacgccgc agttgtatcc gcagctggcg 1860

gcgatcgccc cgacggtgtt taccgcggca ccgggcgcgg actgggaaaa taacctgcgt 1920

ggtgtcggtg ccgccacggc ccgtatcgcc gcggtggacg cgctgatcac cgggttcgcc 1980

gaacacgcca cccaggtcgg gaccaagcat gacgcgaccc acttccaagc gtcgatcgtg 2040

cagctgaccg ccaacaccat gcgggtatac ggcgccaaca acttcccggc cagcgtgctg 2100

agcgcggtcg gcgtcgaccg accgccgtct caacggttca ccgacaaggc ctacatcgag 2160

atcggcacca cggccgccga cctggcgaaa tcaccggact tctcggcggc cgacgccgat 2220

atcgtctacc tgtcgtgcgc gtcggaagca gccgcggaac gcgcggccgt catcctggat 2280

agcgacccat ggcgcaagct gtccgccaac cgtgacaacc gggtcttcgt cgtcaacgac 2340

caggtatggc agaccggcga gggtatggtc gctgcccgcg gcattgtcga tgatctgcgc 2400

tgggtcgacg cgccgatcaa catggacgac acgggcgctg ctccggtagt aattttcggc 2460

ggccgcagcc agatcggcgg cgaactcgcg cgacgcctgg ctgccggggc gacgatggtg 2520

ctggccgcgc ggaacgccga tcaactcgcc gaccaggccg ccgcactccg cgcagctggc 2580

gctatagcgg tgcacacccg ggagttcgac gccgacgacc tggccgcaca cggcccgttg 2640

gtcgcttcgc tcgttgccga gcacggcccc atcggcaccg cggtgctggc cttcgggata 2700

ctcggcgacc aggcccgcgc cgagacagac gcggcgcacg cggtggccat cgtgcacacc 2760

gactacgtcg cccaggtcag cctgctgact catctggcag cggcgatgcg caccgccgga 2820

cggggatcgc tggtggtgtt ctcctcggtc gccgggattc gggtgcgccg cgccaactat 2880

gtctacggat cggccaaagc cggcctggac ggcttcgcca gcggcctggc cgatgcgttg 2940

cacggcaccg gggtgcggtt actgatcgcg cggccgggat tcgtcatcgg gcgcatgacc 3000

gagggcatga cgcccgcacc cctgtcggtc accccggagc gggtggccgc cgcgaccgcg 3060

cgtgcgctgg tcaacggtaa gcgcgtggtg tggattccgt gggcgctgcg gccaatgttt 3120

gttgcgctgc ggttgcttcc ccggttcgtc tggcgcagga tgccgcgata a 3171

<210> 2

<211> 31

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

cccaagcttg ccaccatgga tgcaatgaag a 31

<210> 3

<211> 36

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

cgggatcctt atcgcggcat cctgcgccag acgaac 36

<210> 4

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

cccaagctta tgcccaagag aagcgaa 27

<210> 5

<211> 29

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

cccaagcttg ccaccatgcc caagagaag 29

<210> 6

<211> 29

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

cccaagctta tggatgcaat gaagagagg 29

<210> 7

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

cgggatccct attgctgcgg tgcgggct 28

<210> 8

<211> 29

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

cgggatcctt acgccggagg cattagcac 29

<210> 9

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

cgggatccct agttgatcgg cgcgtcg 27

<210> 10

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

cgggatcctc atcgcggcat cctgcgcc 28

<210> 11

<211> 3099

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

atgcccaaga gaagcgaata caggcaaggc acgccgaact gggtcgacct tcagaccacc 60

gatcagtccg ccgccaaaaa gttctacaca tcgttgttcg gctggggtta cgacgacaac 120

ccggtccccg gaggcggtgg ggtctattcc atggccacgc tgaacggcga agccgtggcc 180

gccatcgcac cgatgccccc gggtgcaccg gaggggatgc cgccgatctg gaacacctat 240

atcgcggtgg acgacgtcga tgcggtggtg gacaaggtgg tgcccggggg cgggcaggtg 300

atgatgccgg ccttcgacat cggcgatgcc ggccggatgt cgttcatcac cgatccgacc 360

ggcgctgccg tgggcctatg gcaggccaat cggcacatcg gagcgacgtt ggtcaacgag 420

acgggcacgc tcatctggaa cgaactgctc acggacaagc cggatttggc gctagcgttc 480

tacgaggctg tggttggcct cacccactcc agcatggaga tagctgcggg ccagaactat 540

cgggtgctca aggccggcga cgcggaagtc ggcggctgta tggaaccgcc gatgcccggc 600

gtgccgaatc attggcacgt ctactttgcg gtggatgacg ccgacgccac ggcggccaaa 660

gccgccgcag cgggcggcca ggtcattgcg gaaccggctg acattccgtc ggtgggccgg 720

ttcgccgtgt tgtccgatcc gcagggcgcg atcttcagtg tgttgaagcc cgcaccgcag 780

caaggcgatc tggtgggccc gggctgcgcg gaatacgcgg cagccaatcc cactgggccg 840

gcctcggtgc agggaatgtc gcaggacccg gtcgcggtgg cggcctcgaa caatccggag 900

ttgacaacgc tgacggctgc actgtcgggc cagctcaatc cgcaagtaaa cctggtggac 960

accctcaaca gcggtcagta cacggtgttc gcaccgacca acgcggcatt tagcaagctg 1020

ccggcatcca cgatcgacga gctgaagacc aattcgtcac tgctgaccag catcctgacc 1080

taccacgtag tggccggcca aaccagcccg gccaacgtcg tcggcacccg tcagaccctc 1140

cagggcgcca gcgtgacggt gaccggtcag ggtaacagcc tcaaggtcgg taacgccgac 1200

gtcgtctgtg gtggggtgtc taccgccaac gcgacggtgt acatgattga cagcgtgcta 1260

atgcctccgg cgatgcgatc cactgttgct gtcgccgtag cggcagccgt gatcgcagcg 1320

tccagtggtt gcggctccga tcaaccggcc cataaggcgt cacaatcgat gatcacgccc 1380

accacccaga tcgccggcgc cggggtgctg ggaaacgaca gaaagccgga tgagtcgtgc 1440

gcgcgtgcgg cggccgcggc cgatccgggg ccaccgaccc gaccagcgca caatgcggcg 1500

ggagtcagcc cggagatggt gcaggtgccg gcggaggcgc agcgcatcgt ggtgctctcc 1560

ggtgaccagc tcgacgcgct gtgcgcgctg ggcctgcaat cgcggatcgt cgccgccgcg 1620

ttgccgaaca gctcctcaag tcaaccttcc tatctgggca cgaccgtgca tgatctgccc 1680

ggtgtcggta ctcgcagcgc ccccgacctg cgcgccattg cggcggctca cccggatctg 1740

atcctgggtt cgcagggttt gacgccgcag ttgtatccgc agctggcggc gatcgccccg 1800

acggtgttta ccgcggcacc gggcgcggac tgggaaaata acctgcgtgg tgtcggtgcc 1860

gccacggccc gtatcgccgc ggtggacgcg ctgatcaccg ggttcgccga acacgccacc 1920

caggtcggga ccaagcatga cgcgacccac ttccaagcgt cgatcgtgca gctgaccgcc 1980

aacaccatgc gggtatacgg cgccaacaac ttcccggcca gcgtgctgag cgcggtcggc 2040

gtcgaccgac cgccgtctca acggttcacc gacaaggcct acatcgagat cggcaccacg 2100

gccgccgacc tggcgaaatc accggacttc tcggcggccg acgccgatat cgtctacctg 2160

tcgtgcgcgt cggaagcagc cgcggaacgc gcggccgtca tcctggatag cgacccatgg 2220

cgcaagctgt ccgccaaccg tgacaaccgg gtcttcgtcg tcaacgacca ggtatggcag 2280

accggcgagg gtatggtcgc tgcccgcggc attgtcgatg atctgcgctg ggtcgacgcg 2340

ccgatcaaca tggacgacac gggcgctgct ccggtagtaa ttttcggcgg ccgcagccag 2400

atcggcggcg aactcgcgcg acgcctggct gccggggcga cgatggtgct ggccgcgcgg 2460

aacgccgatc aactcgccga ccaggccgcc gcactccgcg cagctggcgc tatagcggtg 2520

cacacccggg agttcgacgc cgacgacctg gccgcacacg gcccgttggt cgcttcgctc 2580

gttgccgagc acggccccat cggcaccgcg gtgctggcct tcgggatact cggcgaccag 2640

gcccgcgccg agacagacgc ggcgcacgcg gtggccatcg tgcacaccga ctacgtcgcc 2700

caggtcagcc tgctgactca tctggcagcg gcgatgcgca ccgccggacg gggatcgctg 2760

gtggtgttct cctcggtcgc cgggattcgg gtgcgccgcg ccaactatgt ctacggatcg 2820

gccaaagccg gcctggacgg cttcgccagc ggcctggccg atgcgttgca cggcaccggg 2880

gtgcggttac tgatcgcgcg gccgggattc gtcatcgggc gcatgaccga gggcatgacg 2940

cccgcacccc tgtcggtcac cccggagcgg gtggccgccg cgaccgcgcg tgcgctggtc 3000

aacggtaagc gcgtggtgtg gattccgtgg gcgctgcggc caatgtttgt tgcgctgcgg 3060

ttgcttcccc ggttcgtctg gcgcaggatg ccgcgataa 3099

<210> 12

<211> 3105

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

gccaccatgc ccaagagaag cgaatacagg caaggcacgc cgaactgggt cgaccttcag 60

accaccgatc agtccgccgc caaaaagttc tacacatcgt tgttcggctg gggttacgac 120

gacaacccgg tccccggagg cggtggggtc tattccatgg ccacgctgaa cggcgaagcc 180

gtggccgcca tcgcaccgat gcccccgggt gcaccggagg ggatgccgcc gatctggaac 240

acctatatcg cggtggacga cgtcgatgcg gtggtggaca aggtggtgcc cgggggcggg 300

caggtgatga tgccggcctt cgacatcggc gatgccggcc ggatgtcgtt catcaccgat 360

ccgaccggcg ctgccgtggg cctatggcag gccaatcggc acatcggagc gacgttggtc 420

aacgagacgg gcacgctcat ctggaacgaa ctgctcacgg acaagccgga tttggcgcta 480

gcgttctacg aggctgtggt tggcctcacc cactccagca tggagatagc tgcgggccag 540

aactatcggg tgctcaaggc cggcgacgcg gaagtcggcg gctgtatgga accgccgatg 600

cccggcgtgc cgaatcattg gcacgtctac tttgcggtgg atgacgccga cgccacggcg 660

gccaaagccg ccgcagcggg cggccaggtc attgcggaac cggctgacat tccgtcggtg 720

ggccggttcg ccgtgttgtc cgatccgcag ggcgcgatct tcagtgtgtt gaagcccgca 780

ccgcagcaag gcgatctggt gggcccgggc tgcgcggaat acgcggcagc caatcccact 840

gggccggcct cggtgcaggg aatgtcgcag gacccggtcg cggtggcggc ctcgaacaat 900

ccggagttga caacgctgac ggctgcactg tcgggccagc tcaatccgca agtaaacctg 960

gtggacaccc tcaacagcgg tcagtacacg gtgttcgcac cgaccaacgc ggcatttagc 1020

aagctgccgg catccacgat cgacgagctg aagaccaatt cgtcactgct gaccagcatc 1080

ctgacctacc acgtagtggc cggccaaacc agcccggcca acgtcgtcgg cacccgtcag 1140

accctccagg gcgccagcgt gacggtgacc ggtcagggta acagcctcaa ggtcggtaac 1200

gccgacgtcg tctgtggtgg ggtgtctacc gccaacgcga cggtgtacat gattgacagc 1260

gtgctaatgc ctccggcgat gcgatccact gttgctgtcg ccgtagcggc agccgtgatc 1320

gcagcgtcca gtggttgcgg ctccgatcaa ccggcccata aggcgtcaca atcgatgatc 1380

acgcccacca cccagatcgc cggcgccggg gtgctgggaa acgacagaaa gccggatgag 1440

tcgtgcgcgc gtgcggcggc cgcggccgat ccggggccac cgacccgacc agcgcacaat 1500

gcggcgggag tcagcccgga gatggtgcag gtgccggcgg aggcgcagcg catcgtggtg 1560

ctctccggtg accagctcga cgcgctgtgc gcgctgggcc tgcaatcgcg gatcgtcgcc 1620

gccgcgttgc cgaacagctc ctcaagtcaa ccttcctatc tgggcacgac cgtgcatgat 1680

ctgcccggtg tcggtactcg cagcgccccc gacctgcgcg ccattgcggc ggctcacccg 1740

gatctgatcc tgggttcgca gggtttgacg ccgcagttgt atccgcagct ggcggcgatc 1800

gccccgacgg tgtttaccgc ggcaccgggc gcggactggg aaaataacct gcgtggtgtc 1860

ggtgccgcca cggcccgtat cgccgcggtg gacgcgctga tcaccgggtt cgccgaacac 1920

gccacccagg tcgggaccaa gcatgacgcg acccacttcc aagcgtcgat cgtgcagctg 1980

accgccaaca ccatgcgggt atacggcgcc aacaacttcc cggccagcgt gctgagcgcg 2040

gtcggcgtcg accgaccgcc gtctcaacgg ttcaccgaca aggcctacat cgagatcggc 2100

accacggccg ccgacctggc gaaatcaccg gacttctcgg cggccgacgc cgatatcgtc 2160

tacctgtcgt gcgcgtcgga agcagccgcg gaacgcgcgg ccgtcatcct ggatagcgac 2220

ccatggcgca agctgtccgc caaccgtgac aaccgggtct tcgtcgtcaa cgaccaggta 2280

tggcagaccg gcgagggtat ggtcgctgcc cgcggcattg tcgatgatct gcgctgggtc 2340

gacgcgccga tcaacatgga cgacacgggc gctgctccgg tagtaatttt cggcggccgc 2400

agccagatcg gcggcgaact cgcgcgacgc ctggctgccg gggcgacgat ggtgctggcc 2460

gcgcggaacg ccgatcaact cgccgaccag gccgccgcac tccgcgcagc tggcgctata 2520

gcggtgcaca cccgggagtt cgacgccgac gacctggccg cacacggccc gttggtcgct 2580

tcgctcgttg ccgagcacgg ccccatcggc accgcggtgc tggccttcgg gatactcggc 2640

gaccaggccc gcgccgagac agacgcggcg cacgcggtgg ccatcgtgca caccgactac 2700

gtcgcccagg tcagcctgct gactcatctg gcagcggcga tgcgcaccgc cggacgggga 2760

tcgctggtgg tgttctcctc ggtcgccggg attcgggtgc gccgcgccaa ctatgtctac 2820

ggatcggcca aagccggcct ggacggcttc gccagcggcc tggccgatgc gttgcacggc 2880

accggggtgc ggttactgat cgcgcggccg ggattcgtca tcgggcgcat gaccgagggc 2940

atgacgcccg cacccctgtc ggtcaccccg gagcgggtgg ccgccgcgac cgcgcgtgcg 3000

ctggtcaacg gtaagcgcgt ggtgtggatt ccgtgggcgc tgcggccaat gtttgttgcg 3060

ctgcggttgc ttccccggtt cgtctggcgc aggatgccgc gataa 3105

<210> 13

<211> 3165

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 13

atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60

tcgcccatgc ccaagagaag cgaatacagg caaggcacgc cgaactgggt cgaccttcag 120

accaccgatc agtccgccgc caaaaagttc tacacatcgt tgttcggctg gggttacgac 180

gacaacccgg tccccggagg cggtggggtc tattccatgg ccacgctgaa cggcgaagcc 240

gtggccgcca tcgcaccgat gcccccgggt gcaccggagg ggatgccgcc gatctggaac 300

acctatatcg cggtggacga cgtcgatgcg gtggtggaca aggtggtgcc cgggggcggg 360

caggtgatga tgccggcctt cgacatcggc gatgccggcc ggatgtcgtt catcaccgat 420

ccgaccggcg ctgccgtggg cctatggcag gccaatcggc acatcggagc gacgttggtc 480

aacgagacgg gcacgctcat ctggaacgaa ctgctcacgg acaagccgga tttggcgcta 540

gcgttctacg aggctgtggt tggcctcacc cactccagca tggagatagc tgcgggccag 600

aactatcggg tgctcaaggc cggcgacgcg gaagtcggcg gctgtatgga accgccgatg 660

cccggcgtgc cgaatcattg gcacgtctac tttgcggtgg atgacgccga cgccacggcg 720

gccaaagccg ccgcagcggg cggccaggtc attgcggaac cggctgacat tccgtcggtg 780

ggccggttcg ccgtgttgtc cgatccgcag ggcgcgatct tcagtgtgtt gaagcccgca 840

ccgcagcaag gcgatctggt gggcccgggc tgcgcggaat acgcggcagc caatcccact 900

gggccggcct cggtgcaggg aatgtcgcag gacccggtcg cggtggcggc ctcgaacaat 960

ccggagttga caacgctgac ggctgcactg tcgggccagc tcaatccgca agtaaacctg 1020

gtggacaccc tcaacagcgg tcagtacacg gtgttcgcac cgaccaacgc ggcatttagc 1080

aagctgccgg catccacgat cgacgagctg aagaccaatt cgtcactgct gaccagcatc 1140

ctgacctacc acgtagtggc cggccaaacc agcccggcca acgtcgtcgg cacccgtcag 1200

accctccagg gcgccagcgt gacggtgacc ggtcagggta acagcctcaa ggtcggtaac 1260

gccgacgtcg tctgtggtgg ggtgtctacc gccaacgcga cggtgtacat gattgacagc 1320

gtgctaatgc ctccggcgat gcgatccact gttgctgtcg ccgtagcggc agccgtgatc 1380

gcagcgtcca gtggttgcgg ctccgatcaa ccggcccata aggcgtcaca atcgatgatc 1440

acgcccacca cccagatcgc cggcgccggg gtgctgggaa acgacagaaa gccggatgag 1500

tcgtgcgcgc gtgcggcggc cgcggccgat ccggggccac cgacccgacc agcgcacaat 1560

gcggcgggag tcagcccgga gatggtgcag gtgccggcgg aggcgcagcg catcgtggtg 1620

ctctccggtg accagctcga cgcgctgtgc gcgctgggcc tgcaatcgcg gatcgtcgcc 1680

gccgcgttgc cgaacagctc ctcaagtcaa ccttcctatc tgggcacgac cgtgcatgat 1740

ctgcccggtg tcggtactcg cagcgccccc gacctgcgcg ccattgcggc ggctcacccg 1800

gatctgatcc tgggttcgca gggtttgacg ccgcagttgt atccgcagct ggcggcgatc 1860

gccccgacgg tgtttaccgc ggcaccgggc gcggactggg aaaataacct gcgtggtgtc 1920

ggtgccgcca cggcccgtat cgccgcggtg gacgcgctga tcaccgggtt cgccgaacac 1980

gccacccagg tcgggaccaa gcatgacgcg acccacttcc aagcgtcgat cgtgcagctg 2040

accgccaaca ccatgcgggt atacggcgcc aacaacttcc cggccagcgt gctgagcgcg 2100

gtcggcgtcg accgaccgcc gtctcaacgg ttcaccgaca aggcctacat cgagatcggc 2160

accacggccg ccgacctggc gaaatcaccg gacttctcgg cggccgacgc cgatatcgtc 2220

tacctgtcgt gcgcgtcgga agcagccgcg gaacgcgcgg ccgtcatcct ggatagcgac 2280

ccatggcgca agctgtccgc caaccgtgac aaccgggtct tcgtcgtcaa cgaccaggta 2340

tggcagaccg gcgagggtat ggtcgctgcc cgcggcattg tcgatgatct gcgctgggtc 2400

gacgcgccga tcaacatgga cgacacgggc gctgctccgg tagtaatttt cggcggccgc 2460

agccagatcg gcggcgaact cgcgcgacgc ctggctgccg gggcgacgat ggtgctggcc 2520

gcgcggaacg ccgatcaact cgccgaccag gccgccgcac tccgcgcagc tggcgctata 2580

gcggtgcaca cccgggagtt cgacgccgac gacctggccg cacacggccc gttggtcgct 2640

tcgctcgttg ccgagcacgg ccccatcggc accgcggtgc tggccttcgg gatactcggc 2700

gaccaggccc gcgccgagac agacgcggcg cacgcggtgg ccatcgtgca caccgactac 2760

gtcgcccagg tcagcctgct gactcatctg gcagcggcga tgcgcaccgc cggacgggga 2820

tcgctggtgg tgttctcctc ggtcgccggg attcgggtgc gccgcgccaa ctatgtctac 2880

ggatcggcca aagccggcct ggacggcttc gccagcggcc tggccgatgc gttgcacggc 2940

accggggtgc ggttactgat cgcgcggccg ggattcgtca tcgggcgcat gaccgagggc 3000

atgacgcccg cacccctgtc ggtcaccccg gagcgggtgg ccgccgcgac cgcgcgtgcg 3060

ctggtcaacg gtaagcgcgt ggtgtggatt ccgtgggcgc tgcggccaat gtttgttgcg 3120

ctgcggttgc ttccccggtt cgtctggcgc aggatgccgc gataa 3165

<210> 14

<211> 858

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 14

gccaccatgg atgcaatgaa gagagggctc tgctgtgtgc tgctgctgtg tggagcagtc 60

ttcgtttcgc ccatgcccaa gagaagcgaa tacaggcaag gcacgccgaa ctgggtcgac 120

cttcagacca ccgatcagtc cgccgccaaa aagttctaca catcgttgtt cggctggggt 180

tacgacgaca acccggtccc cggaggcggt ggggtctatt ccatggccac gctgaacggc 240

gaagccgtgg ccgccatcgc accgatgccc ccgggtgcac cggaggggat gccgccgatc 300

tggaacacct atatcgcggt ggacgacgtc gatgcggtgg tggacaaggt ggtgcccggg 360

ggcgggcagg tgatgatgcc ggccttcgac atcggcgatg ccggccggat gtcgttcatc 420

accgatccga ccggcgctgc cgtgggccta tggcaggcca atcggcacat cggagcgacg 480

ttggtcaacg agacgggcac gctcatctgg aacgaactgc tcacggacaa gccggatttg 540

gcgctagcgt tctacgaggc tgtggttggc ctcacccact cgagcatgga gatagctgcg 600

ggccagaact atcgggtgct caaggccggc gacgcggaag tcggcggctg tatggaaccg 660

ccgatgcccg gcgtgccgaa tcattggcac gtctactttg cggtggatga cgccgacgcc 720

acggcggcca aagccgccgc agcgggcggc caggtcattg cggaaccggc tgacattccg 780

tcggtgggcc ggttcgccgt gttgtccgat ccgcagggcg cgatcttcag tgtgttgaag 840

cccgcaccgc agcaatag 858

<210> 15

<211> 564

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 15

gccaccatgg atgcaatgaa gagagggctc tgctgtgtgc tgctgctgtg tggagcagtc 60

ttcgtttcgc ccggcgatct ggtgggcccg ggctgcgcgg aatacgcggc agccaatccc 120

actgggccgg cctcggtgca gggaatgtcg caggacccgg tcgcggtggc ggcctcgaac 180

aatccggagt tgacaacgct gacggctgca ctgtcgggcc agctcaatcc gcaagtaaac 240

ctggtggaca ccctcaacag cggtcagtac acggtgttcg caccgaccaa cgcggcattt 300

agcaagctgc cggcatccac gatcgacgag ctcaagacca attcgtcact gctgaccagc 360

atcctgacct accacgtagt ggccggccaa accagcccgg ccaacgtcgt cggcacccgt 420

cagaccctcc agggcgccag cgtgacggtg accggtcagg gtaacagcct caaggtcggt 480

aacgccgacg tcgtctgtgg tggggtgtct accgccaacg cgacggtgta catgattgac 540

agcgtgctaa tgcctccggc gtaa 564

<210> 16

<211> 1152

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 16

gccaccatgg atgcaatgaa gagagggctc tgctgtgtgc tgctgctgtg tggagcagtc 60

ttcgtttcgc ccatgcgatc cactgttgct gtcgccgtag cggcagccgt gatcgcagcg 120

tccagtggtt gcggctccga tcaaccggcc cataaggcgt cacaatcgat gatcacgccc 180

accacccaga tcgccggcgc cggggtgctg ggaaacgaca gaaagccgga tgagtcgtgc 240

gcgcgtgcgg cggccgcggc cgatccgggg ccaccgaccc gaccagcgca caatgcggcg 300

ggagtcagcc cggagatggt gcaggtgccg gcggaggcgc agcgcatcgt ggtgctctcc 360

ggtgaccagc tcgacgcgct gtgcgcgctg ggcctgcaat cgcggatcgt cgccgccgcg 420

ttgccgaaca gctcctcaag tcaaccttcc tatctgggca cgaccgtgca tgatctgccc 480

ggtgtcggta ctcgcagcgc ccccgacctg cgcgccattg cggcggctca cccggatctg 540

atcctgggtt cgcagggttt gacgccgcag ttgtatccgc agctggcggc gatcgccccg 600

acggtgttta ccgcggcacc gggcgcggac tgggaaaata acctgcgtgg tgtcggtgcc 660

gccacggccc gtatcgccgc ggtggacgcg ctgatcaccg ggttcgccga acacgccacc 720

caggtcggga ccaagcatga cgcgacccac ttccaagcgt cgatcgtgca gctgaccgcc 780

aacaccatgc gggtatacgg cgccaacaac ttcccggcca gcgtgctgag cgcggtcggc 840

gtcgaccgac cgccgtctca acggttcacc gacaaggcct acatcgagat cggcaccacg 900

gccgccgacc tggcgaaatc accggacttc tcggcggccg acgccgatat cgtctacctg 960

tcgtgcgcgt cggaagcagc cgcggaacgc gcggccgtca tcctggatag cgacccatgg 1020

cgcaagctgt ccgccaaccg tgacaaccgg gtcttcgtcg tcaacgacca ggtatggcag 1080

accggcgagg gtatggtcgc tgcccgcggc attgtcgatg atctgcgctg ggtcgacgcg 1140

ccgatcaact ag 1152

<210> 17

<211> 822

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 17

gccaccatgg atgcaatgaa gagagggctc tgctgtgtgc tgctgctgtg tggagcagtc 60

ttcgtttcgc ccatggacga cacgggcgct gctccggtag taattttcgg cggccgcagc 120

cagatcggcg gcgaactcgc gcgacgcctg gctgccgggg cgacgatggt gctggccgcg 180

cggaacgccg atcaactcgc cgaccaggcc gccgcactcc gcgcagctgg cgctatagcg 240

gtgcacaccc gggagttcga cgccgacgac ctggccgcac acggcccgtt ggtcgcttcg 300

ctcgttgccg agcacggccc catcggcacc gcggtgctgg ccttcgggat actcggcgac 360

caggcccgcg ccgagacaga cgcggcgcac gcggtggcca tcgtgcacac cgactacgtc 420

gcccaggtca gcctgctgac tcatctggca gcggcgatgc gcaccgccgg acggggatcg 480

ctggtggtgt tctcctcggt cgccgggatt cgggtgcgcc gcgccaacta tgtctacgga 540

tcggccaaag ccggcctgga cggcttcgcc agcggcctgg ccgatgcgtt gcacggcacc 600

ggggtgcggt tactgatcgc gcggccggga ttcgtcatcg ggcgcatgac cgagggcatg 660

acgcccgcac ccctgtcggt caccccggag cgggtggccg ccgcgaccgc gcgtgcgctg 720

gtcaacggta agcgcgtggt gtggattccg tgggcgctgc ggccaatgtt tgttgcgctg 780

cggttgcttc cccggttcgt ctggcgcagg atgccgcgat ga 822

<210> 18

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 18

ggatccatga cagagcagca gtg 23

<210> 19

<211> 72

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 19

gctgccgcca ccgccgcttc cgccaccgcc gcttccaccg ccacctgcga acatcccagt 60

gacgttgcct tc 72

<210> 20

<211> 66

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 20

ggtggcggtg gaagcggcgg tggcggaagc ggcggtggcg gcagcgcatt ttcccggccg 60

ggcttg 66

<210> 21

<211> 30

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 21

ggaattctgt tcggagctag gcgccctggg 30

Claims

1. a kind of fusion dna, it is characterised in that：The encoding gene of the fusion dna be Kozak sequences, tPA sequences, Rv0577, Rv2875, Rv3044 and Rv2073c are according to Kozak sequence-tPA sequences-Rv0577-Rv2875-Rv3044-Rv2073c's Sequential series.

2. fusion dna according to claim 1, it is characterised in that：The nucleotides sequence of the fusion dna is classified as：

(1) nucleotide sequence shown in SEQ ID No.1；Or

(2) nucleotide sequence homology limited with sequence SEQ ID No.1 encodes identical function protein 80% to 100% Nucleotide sequence.

3. a kind of DNA vaccination for tuberculosis prophylaxis and immunization therapy, it is characterised in that：The vaccine contains claim 1 The fusion dna.

4. a kind of DNA vaccination for tuberculosis prophylaxis and immunization therapy, it is characterised in that：The vaccine is to contain claim 1 The pVAXI plasmids of the fusion dna.

5. the DNA vaccination according to claim 3 or 4 for tuberculosis prophylaxis and immunization therapy, it is characterised in that：It is described The concentration of fusion dna is in 0.2mg/ml between 0.8mg/ml.

6. the DNA vaccination according to claim 3 or 4 for tuberculosis prophylaxis and immunization therapy, it is characterised in that：It is described DNA vaccination contain 2mg/ml to 3mg/ml dimethyl dioctadecyl ammoniums, 0.2mg/ml to 0.3mg/ml monophosphoryl lipid As, with And the trehalose of 0.4mg/ml to 0.6mg/ml, the vaccine are liposomal form.

7. the DNA vaccination according to claim 6 for tuberculosis prophylaxis and immunization therapy, it is characterised in that：The sea Algae sugar is 6,6 '-two mycolic acids.

8. a kind of recombinant adenoviral vector vaccine for latent infection and tuberculosis prophylaxis of being grown up, it is characterised in that：The epidemic disease Seedling contains fusion dna as claimed in claim 1 or 2.

9. recombinant adenoviral vector vaccine according to claim 8, it is characterised in that：The concentration of the recombined adhenovirus 1 × 10⁷To 1 × 10⁹Between.

10. a kind of application of fusion dna as claimed in claim 1 or 2 is applied to prepare tuberculosis DNA vaccination and recombination Adenovirus carrier vaccine.