CN104151433A - Mycobacterium tuberculosis fusion proteins and their preparation method and use - Google Patents

Abstract

The invention provides mycobacterium tuberculosis fusion proteins. The mycobacterium tuberculosis fusion proteins are shown in sequences 2 and 4 in the sequence table. The invention provides a preparation method and a use of the mycobacterium tuberculosis fusion proteins. The fusion protein LT70 and adjuvants of DDA and PolyI: C are combined and form a tuberculosis sigmasubunit vaccine. An experiment result shows that the vaccine can effectively induce immune response of tuberculosis antigen-specific cells and body fluid in mouse body and has a strong immune protection capacity, lasting immune protection time, immune protection effects better than that of BCG and BCG improvement effects in a mouse virulent strain attack protection efficiency test. The tuberculosis sigmasubunit vaccine is an effective tuberculosis vaccine candidate.

Description

A kind of mycobacterium tuberculosis fusion protein and its preparation method and application

Technical field

The present invention relates to a kind of mycobacterium tuberculosis fusion protein, the preparation method who the invention still further relates to this fusion rotein with and application in preparing tuberculosis subunit vaccine.

Background technology

Tuberculosis causes by m tuberculosis infection, be that global spread scope is the widest, the time length at most, the most serious transmissible disease of harm.Since the 1980s, appearance and popular and human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) (acquired immune deficiency syndrome (AIDS) along with the especially resistance to multiple medicines tubercule bacillus of Resistant strain, AIDS) spread and epidemic, all over the world, sickness rate lungy presents rise trend, and M & M can be in any more.Mycobacterium tuberculosis is as pathogenic agent lungy, one of reason that it is restive is that tubercule bacillus can exist for a long time with latent infection state in human immunocyte, with this, escape the removing of body specific immune response to it, and can when body immunity is low, develop into active tuberculosis.

Present stage, the vaccine bacille Calmette-Guerin vaccine (BCG) that uses of Tuberculosis Control Institute was a kind of attenuation Mycobacterium bovis living vaccine.Bacille Calmette-Guerin vaccine, can effectively prevent newborn infant and children to suffer from serious meningeal tuberculosis and whole body miliary tuberculosis, but can not effectively prevent the generation of adult pulmonary tuberculosis for mankind's inoculation from nineteen twenty-one.Therefore, the major objective of new generation vaccine and vaccine inoculation strategy is to extend vaccine guard time or just on foundation-free, carry out booster immunization at BCG.With take the vaccine that BCG and other microorganism be carrier and compare, tuberculosis subunit vaccine has the advantage of strengthening BCG immune effect, and has safe and reliable feature.Effective tuberculosis vaccine not only will have provide protection to active period tuberculosis, and wants to produce immunne response for tubercule bacillus in latent period.Therefore, desirable tuberculosis vaccine must merge mycobacterium tuberculosis active period and preclinical a plurality of antigen.

Research discovery, mycobacterium tuberculosis active period antigen ESAT-6 is Early insulin secretion albumen, can stimulate body T lymphopoiesis, secretion interferon-γ plays a significant role in protectiveness cellular immunization.Ag85 mixture is the main secreted protein in mycobacterium tuberculosis and bacille Calmette-Guerin vaccine, at mycobacterium tuberculosis H ₃₇r _vin strain, accounting for 30% of secretory protein total amount, is one group of important mycobacterium secretory protein, has the activity of mycolic acid transferring enzyme, in the final stage of mycobacterium Cell wall synthesis, plays a significant role, and can induce and produce special cellullar immunologic response.In early-stage Study, we are to the six kinds of fusion rotein ESAT6-Ag85B-MPT64190-198-Mtb8.4 (EAMM) that build, Mtb10.4-HspX (MH), ESAT6-Mtb8.4, Mtb10.4-Ag85B, ESAT6-Ag85B and ESAT6-RpfE have carried out immunoprotection evaluation, find that EAMM(is merged and formed by mycobacterium tuberculosis active period antigen) demonstrate stronger immune protective effect, especially when EAMM and MH(contain tubercule bacillus antigen in latent period HspX) while carrying out combined utilization (EAMM+MH vaccine), significantly reduced the bacterial loads of spleen and lung, reached the protection effect identical with BCG.The protein subunit vaccine immunoprotection that result shows the combined growth phase and the related antigen of hiding builds, can promote body to bacterium, and particularly effective identification of dormancy bacterium improves immunizing power, thereby removes kill bacteria.

Summary of the invention

The invention provides a kind of mycobacterium tuberculosis fusion protein, its combined vegetative period and latent period related antigen build and to form, can promote body to bacterium, particularly effective identification of dormancy bacterium, improves immunizing power, thereby removes kill bacteria.

Mycobacterium tuberculosis Hrp1 (Rv2626c) is at mycobacterium tuberculosis high expression level in latent period, and can be attached to the surface of scavenger cell, the remarkable expression of induction pro-inflammatory cytokine interleukin 12 and tumour necrosis factor, causing the immunne response that lunger is strong, is desirable resting stage antigen.

Therefore, the present invention is by the amino acid polypeptide (CD8+T cell epitope) of the 190-198 position of mycobacterium tuberculosis active period antigen ESAT6, Ag85B, Mtb8.4 and MPT64 and mycobacterium tuberculosis antigen in latent period Hrp1(Rv2626c) merge, build EAMM-Rv2626 fusion rotein (the about 70kD of molecular weight, therefore be called for short LT70).

The invention provides a kind of mycobacterium tuberculosis fusion protein, is the protein in sequence 2 and sequence 4 in sequence table.

In sequence table 2,1-95 position is the aminoacid sequence of ESAT6,96-97 position is the aminoacid sequence of restriction enzyme site, 98-382 position is the aminoacid sequence of Ag85B, 383-384 position is the aminoacid sequence of restriction enzyme site, the 190-198 amino acids sequence that 385-393 position is MPT64, the aminoacid sequence that 394-503 position is Mtb8.4,504-505 position is the aminoacid sequence of restriction enzyme site, the aminoacid sequence that 506-660 position is Rv2626c.

In sequence table 4,1-95 position is the aminoacid sequence of ESAT6,96-97 position is the aminoacid sequence of restriction enzyme site, 98-412 position is the aminoacid sequence that adds the Ag85B of linker, 413-414 position is the aminoacid sequence of restriction enzyme site, the 190-198 amino acids sequence that 415-423 position is MPT64, the aminoacid sequence that 424-533 position is Mtb8.4,534-535 position is the aminoacid sequence of restriction enzyme site, the aminoacid sequence that 536-690 position is Rv2626c.

Second object of the present invention is to provide the encoding gene of above-mentioned mycobacterium tuberculosis fusion protein.

Preferably, be following 1) or 2) described gene:

1) its nucleotide sequence is the sequence 1 or 3 in sequence table;

2) with sequence table in the nucleotide sequence of sequence 1 or 3 complementations;

3) under stringent condition with 1) the DNA sequence dna hybridization that limits and the DNA molecular of encoding said fusion protein.

In sequence table 1,1-285 position is the encoding gene of ESAT6,286-291 position is the encoding gene of restriction enzyme site, 292-1146 position is the encoding gene of Ag85B, 1147-1152 position is the encoding gene of restriction enzyme site, the encoding gene of the 190-198 amino acids that 1153-1179 position is MPT64, the encoding gene that 1180-1509 position is Mtb8.4,1510-1515 position is the encoding gene of restriction enzyme site, the encoding gene that 1516-1980 position is Rv2626c.

In sequence table 3,1-285 position is the encoding gene of ESAT6,286-291 position is the encoding gene of restriction enzyme site, 292-1236 position is the encoding gene of Ag85B, 1237-1242 position is the encoding gene of restriction enzyme site, the encoding gene of the 190-198 amino acids that 1243-1269 position is MPT64, the encoding gene that 1270-1599 position is Mtb8.4,1600-1605 position is the encoding gene of restriction enzyme site, the encoding gene that 1606-2070 position is Rv2626c.

The 3rd object of the present invention is to provide the recombinant expression vector that contains above-mentioned encoding gene.

The 4th object of the present invention is to provide the host cell that contains above-mentioned recombinant expression vector.

The 5th object of the present invention is to provide the preparation method of above-mentioned mycobacterium tuberculosis fusion protein, the expression and purification that comprises fusion rotein, it is characterized in that: being expressed as of described mycobacterium tuberculosis fusion protein: the thalline that contains fusion rotein is added to shaking culture in LB substratum, adding final concentration is the protein induced dose of IPTG of 0.5mmol/L, 29 ℃ of induction shaking culture 4h, obtain supernatant and are expression product after aftertreatment.

The 6th object of the present invention is to provide the application of above-mentioned mycobacterium tuberculosis fusion protein in preparing tuberculosis subunit vaccine.

The 7th object of the present invention is to provide a kind of tuberculosis subunit vaccine, and it contains above-mentioned mycobacterium tuberculosis fusion protein.

Preferably, described tuberculosis subunit vaccine is comprised of mycobacterium tuberculosis fusion protein claimed in claim 1, DDA and PolyI:C.

The 8th object of the present invention is to provide the preparation method of above-mentioned tuberculosis subunit vaccine, and the preparation method of every 200 μ L tuberculosis subunit vaccines is as follows: fusion rotein is diluted to 0.2mg/ml with PBS damping fluid; PolyI:C is dissolved to 1mg/ml with PBS damping fluid; DDA is mixed with 2.5mg/ml with PBS, is cooled to room temperature after water-bath; Get PolyI:C solution 50 μ l and equivalent fusion rotein solution and fully mix, room temperature is placed; In mixing solutions, dropwise add 100 μ L DDA solution, then fully emulsified, the creamy that makes vaccine be homogeneous obtains tuberculosis subunit vaccine.

The beneficial effect of mycobacterium tuberculosis of the present invention is as follows:

One, the present invention utilizes genetic engineering technique to build without any tag fusion protein LT70 expression plasmid pET30a (+)-LT70(ESAT6-Ag85B-MPT64 _190-198-Mtb8.4-Rv2626c) and pET30a (+)-LT70(ESAT6-linker-Ag85B-linker-MPT64 _190-198-Mtb8.4-Rv2626c), plasmid is proceeded to intestinal bacteria e. col-iin BL-21 (DE3) thalline, fusion rotein is expressed in supernatant with soluble form.The condition that target protein LT70 is expressed at supernatant soluble form is optimized.

Two, the present invention utilizes without label protein purification technique, by hydrophobic chromatography and two kinds of purification process of sieve chromatography, successfully LT70 albumen has been carried out to purifying.

Three, the present invention carries out joint mapping tuberculosis subunit vaccine by fusion rotein LT70 and dda adjuvant+PolyI:C, by C57BL/6 mouse model, carries out respectively immunological evaluation and protection efficiency rating, the immune protective effect of thoroughly evaluating LT70 vaccine.Experimental result shows that this vaccine can effectively induce the specific cell of tuberculosis antigen and humoral immunoresponse(HI) in Mice Body; and there is stronger immune protective efficiency in mouse virulent strain attack protection efficiency test; guard time is comparatively lasting; protection effect is better than BCG; also having the effect of strengthening BCG immunity, is an effective tuberculosis candidate vaccine.

Four, in order to strengthen the solubility expression of fusion rotein, be convenient to produce, we have done following optimization to this antigen-4 fusion protein gene sequence and antigenic structure: (1) has optimized the gene order of Ag85B and Rv2626c: according to homo sapiens, be optimized and use the codon of intestinal bacteria predominant expression, so that fusion rotein is expressed better at e. coli host cell; (2) in order to keep each antigen to be fully folded into native conformation separately, we introduce the joint linker that the amino acid by low hydrophobicity, low electrocharge effect forms before and after the Ag85B, by each antigen of fusion rotein separately, make each antigen fully folding separately, supernatant expression amount has lifting (referring to Fig. 1) when not adding linker, has strengthened the expressive function of LT70 fusion rotein.

Accompanying drawing explanation

Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, for explaining the present invention, is not construed as limiting the invention together with embodiments of the present invention.In the accompanying drawings:

Fig. 1 is that fusion rotein LT70 and LT70(add linker) expression of results in intestinal bacteria E. coli BL21 (DE3); Wherein, (a) be the expression of results of fusion rotein LT70.M, marker; The empty bacterium fragmentation of 1, BL21 is full bacterium night afterwards; Full bacterium liquid after 2, LT70 bacterial cell disruption; Supernatant after 3, LT70 bacterial cell disruption.(b) add linker for fusion rotein LT70 and LT70() expression of results.M, marker; Full bacterium liquid after 1, LT70 bacterial cell disruption; Supernatant after 2, LT70 bacterial cell disruption; 3, LT70 bacterial cell disruption postprecipitation; 4, LT70(adds linker) full bacterium liquid after bacterial cell disruption; 5, LT70(adds linker) supernatant after bacterial cell disruption; 6, LT70(adds linker) bacterial cell disruption postprecipitation.

Fig. 2 be fusion rotein LT70 when intestinal bacteria E.coli BL21 (DE3) expresses, different IP TG induced concentration is on the impact of expressing.Wherein, M, marker, 1,2,3 are respectively 0.5mM IPTG final concentration LT70 fragmentation full bacterium afterwards, supernatant after LT70 fragmentation, the broken postprecipitation of LT70; 4,5,6 are respectively 0.2mM IPTG final concentration LT70 fragmentation full bacterium afterwards, supernatant after LT70 fragmentation, the broken postprecipitation of LT70; 7,8,9 are respectively 0.1mM IPTG final concentration LT70 fragmentation full bacterium afterwards, supernatant after LT70 fragmentation, the broken postprecipitation of LT70.

Fig. 3 be fusion rotein LT70 when intestinal bacteria E.coli BL21 (DE3) expresses, the different temperature of expressing are on the impact of expressing.Wherein, (a) M, marker; 1,2,3 are respectively 37 ℃ of induction LT70 fragmentation full bacterium afterwards, supernatant after LT70 fragmentation, the broken postprecipitation of LT70; 4,5,6 are respectively 34 ℃ of induction LT70 fragmentation full bacterium afterwards, supernatant after LT70 fragmentation, the broken postprecipitation of LT70; 7,8 are respectively 25 ℃ of induction LT70 fragmentation full bacterium afterwards, supernatant after LT70 fragmentation.(b) M, marker; 1,2 is respectively supernatant after 31 ℃ of induction LT70 fragmentations, the broken postprecipitation of LT70; 3 is supernatant after 31 ℃ of induction LT70 fragmentation; 4,5 are respectively supernatant after 29 ℃ of induction LT70 fragmentations, the broken postprecipitation of LT70; 6 is 31 ℃ of broken postprecipitations of induction LT70; 7,8 are respectively supernatant after 27 ℃ of induction LT70 fragmentations, the broken postprecipitation of LT70.(c) M, marker; 1,2 is respectively supernatant after 31 ℃ of induction LT70 fragmentations, the broken postprecipitation of LT70; 3 is supernatant after 31 ℃ of induction LT70 fragmentation; 4 is 31 ℃ of broken postprecipitations of induction LT70; 5,6 are respectively supernatant after 29 ℃ of induction LT70 fragmentations, the broken postprecipitation of LT70; 7,8 are respectively supernatant after 27 ℃ of induction LT70 fragmentations, the broken postprecipitation of LT70.(d) M, marker; 1, LT70 fragmentation is full bacterium afterwards; The empty bacterium of 2, BL21; 3,4 are respectively 30 ℃ of broken postprecipitations of induction LT70, supernatant after LT70 fragmentation; 5,6 are respectively 29 ℃ of broken postprecipitations of induction LT70, supernatant after LT70 fragmentation; 7,8 are respectively 28 ℃ of broken postprecipitations of induction LT70, supernatant after LT70 fragmentation.

Fig. 4 is purifying and the analytical results of fusion rotein LT70 in intestinal bacteria E.coli BL21 (DE3).Wherein, (a) hydrophobic chromatography purifying.M, marker; 1, fusion rotein LT70 is after saturation ratio is 15% ammonium sulfate precipitation; 2, hydrophobic chromatography Butyl FF post Liu Chuan peak; 3, hydrophobic chromatography Butyl FF post elution peak; 4, supernatant after fusion rotein LT70 fragmentation; (b) sieve chromatography purifying.M, marker; 1, supernatant after fusion rotein LT70 fragmentation; 2, fusion rotein LT70 filters press proof product after saturation ratio is 15% ammonium sulfate precipitation; 3,4, gel Superdex 75 filtration chromatographies; 5 is fusion rotein LT70 sample after saturation ratio is 15% ammonium sulfate precipitation.(c) LT70 immunoblotting assay result: M, molecular weight of albumen standard; 1, anti-Rv2626c mouse serum; 2, mouse-anti Esat-6 monoclonal antibody; 3, negative control; 4, the anti-Ag85B monoclonal antibody of rabbit; 5, negative control.

Fig. 5 is the cell immunogenicity detected result of tuberculosis subunit vaccine LT70; * p<0.05 vspBS, BCG.

Fig. 6 is the humoral immunization originality detected result of tuberculosis subunit vaccine LT70, and wherein a is IgG1, and b is IgG2c; * p<0.05 vspBS, BCG.

Fig. 7 is the protection effect assessment of tuberculosis subunit vaccine: LT70 vaccine, respectively the 0th, 2,4 weeks immune mouses three times, is carried out to respiratory tract aerosol for after last immunity 30 weeks and attacks Mycobacterium tuberculosis H37Rv strain, after attacking, within 10 weeks, carry out internal organs CFU counting.* p<0.05 vs PBS, BCG，** p<0.01 vs PBS, BCG, EAMM+MH。

Fig. 8 is mouse lung histopathology reaction result after tuberculosis subunit vaccine immunity and strain are attacked.

Fig. 9 is the histogram that after tuberculosis subunit vaccine immunity and strain are attacked, mouse lung knot of tissue nuclearity pathology area accounts for the ratio of section area.

Figure 10 is the cell immune response result of tuberculosis subunit vaccine LT70 strengthening BCG; * p<0.05 vspBS, BCG.

Figure 11 is the humoral immune reaction result of tuberculosis subunit vaccine LT70 strengthening BCG, and wherein a is IgG1, and b is IgG2c ;* p<0.05 vspBS, BCG.

Figure 12 is the protection effect assessment that tuberculosis subunit vaccine LT70 strengthens BCG; * p<0.05 vspBS, * * p<0.01 vspBS.

Figure 13 is mouse lung histopathology reaction after tuberculosis subunit vaccine strengthening BCG immunity and strain are attacked; * p<0.01 vspBS.

Figure 14 is the histogram that after tuberculosis subunit vaccine strengthening BCG immunity and strain are attacked, mouse lung knot of tissue nuclearity pathology area accounts for section area ratio.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.

The present invention builds a kind of novel tag fusion protein that goes, and it is mixed to the novel tuberculosis subunit vaccine of structure with adjuvant.

Material:

Pre-coated ELISPOT test kit is purchased from Beijing Da Kewei Bioisystech Co., Ltd;

PET30a (+)-Rv2626c is gifted by Aeras company;

PolyI:C is purchased from opening the flat Biochem Pharma Inc of leading a cow;

Cationic-liposome DDA (dimo-thylidioctyl ammonium bromide, DDA) is purchased from Sigma's aldrich (Shanghai) trade Co., Ltd;

T4 ligase enzyme is purchased from Takara company.

embodiment 1

1. the structure of fusion rotein and purifying

1.1 construction of fusion protein LT70 expression plasmid pET30a (+)-LT70 and pET30a (+)-LT70(add linker)

(1) build recombination carrier pET30a (+)-LT69 (ESAT6-Ag85B-MPT64 _190-198-Mtb8.4-HspX).

A, pET30a (+)-EA(ESAT6-Ag85B) structure:

The clinical mycobacterium bacterium liquid of take is template, pcr amplification ESAT6, and Ag85B corresponding gene, and its clone is built up in plasmid pET30a.

Wherein, RCR reaction system:

Water 16.25 μ l

5×PCR buffer 5μl

dNTP 2μl

Upstream primer P1 0.5 μ l

Downstream primer P2 0.5 μ l

DNA profiling 0.5 μ l

Archaeal dna polymerase (Primer STAR, takara company) 0.25 μ l

Total system 25 μ l.

PCR reaction parameter: 98 ℃ of sex change 15s, 60 ℃ of annealing 15 s, 72 ℃ are extended 50 s, totally 30 circulations.

B, design primer amplification EA(ESAT6-Ag85B) gene fragment.Primer is respectively: upstream (5 '-3 ' CGG cATATgACAGAGCAGCAGTGGAAT, wherein underscore is partly ndei restriction enzyme site); Downstream (5 '-3 ' GA aGATCTgCCGGCGCCTAACGAACTCTGGAG, wherein underscore is partly bgliI restriction enzyme site).PET30a (+)-EA plasmid of take is carrier, and with above-mentioned two primers, pcr amplification goes out EA(ESAT6-Ag85B) fusion gene product.

Wherein, RCR reaction system:

Water 16.25 μ l

5×PCR buffer 5μl

dNTP 2μl

Upstream primer P1 0.5 μ l

Downstream primer P2 0.5 μ l

DNA profiling 0.5 μ l

Archaeal dna polymerase (Primer STAR, takara company) 0.25 μ l

Total system 25 μ l.

PCR reaction parameter: 98 ℃ of sex change 15s, 63 ℃ of annealing 15 s, 72 ℃ are extended 50 s, totally 35 circulations.

After this PCR product is purified, use ndei and bgliI carries out double digestion, and it is pET30a (+)-MPT64 that clone is structured in plasmid pET30a (+)-MMH( _190-198-Mtb8.4-HspX) upper, be built into pET30a (+)-LT69 (ESAT6-Ag85B-MPT64 _190-198-Mtb8.4-HspX) carrier.MPT64 _190-198refer to the aminoacid sequence of the 190-198 position of MPT64.

Wherein, the construction process of plasmid pET30a (+)-MMH is:

Use restriction enzyme site saci and hind at plasmid pET30a (+)-MH(, be pET30a (+)-Mtb8.4-Hspx) (construction process is open, patent of invention: a kind of tubercule bacillus fusion rotein preparation method and application.The patent No.: ZL 201010613320.6) on, cut Hspx and be pET30a (+)-MPT64 at plasmid pET30a (+)-MM( _190-198-Mtb8.4) after, cut, by T4 ligase enzyme, 16 ℃ of connections of spending the night.

Wherein, plasmid pET30a (+)-MM(is pET30a (+)-MPT64 _190-198-Mtb8.4) be directly synthetic.

(2) build recombination carrier pET30a (+)-LT70 (ESAT6-Ag85B-MPT64 _190-198-Mtb8.4-Rv2626c).

Design primer amplification Rv2626c gene fragment.Primer is respectively: upstream (5 '-3 ' AC gAGCTCaTGACCACGG, wherein underscore is partly saci restriction enzyme site); Downstream (5 '-3 ' CCC aAGCTTcTATGCATTTAG, wherein underscore is partly hind restriction enzyme site).PET30a (+)-Rv2626c plasmid of take is template, and with above-mentioned two primers, pcr amplification goes out Rv2626c gene order.After this PCR product is purified, use saci and hind carry out double digestion, it is upper that clone is structured in plasmid pET30a (+)-LT69, is built into pET30a (+)-LT70 (ESAT6-Ag85B-MPT64 _190-198-Mtb8.4-Rv2626c) carrier.

Wherein, RCR reaction system:

Water 16.25 μ l

5×PCR buffer 5μl

dNTP 2μl

Upstream primer P1 0.5 μ l

Downstream primer P2 0.5 μ l

DNA profiling 0.5 μ l

Archaeal dna polymerase (Primer STAR, takara company) 0.25 μ l

Total system 25 μ l

PCR reaction parameter: 98 ℃ of sex change 15s, 60 ℃ of annealing 15 s, 72 ℃ are extended 50 s, totally 30 circulations.

(3) build recombination carrier pET30a (+)-LT70 (ESAT6-Ag85B-MPT64 _190-198-Mtb8.4-Rv2626c) (add linker).

Design primer amplification Ag85B gene fragment.Primer is respectively: upstream primer (5 '-3 ' CGG gAATTC gGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTtTCTCC CGG CCGG, wherein single underscore is partly ecor I restriction enzyme site, double underline is partly linker sequence); Downstream primer (5 '-3 ' CCC aGATCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTG GTGGTGGTGGTTCTaCCAGCACCCAGAGAAG, wherein single underscore is partly bgliI restriction enzyme site, double underline is partly linker sequence).PET30a (+)-LT70 plasmid of take is carrier, and with above-mentioned two primers, pcr amplification goes out Ag85B (adding linker) gene order.

RCR reaction system:

Water 16.25 μ l

5×PCR buffer 5μl

dNTP 2μl

Upstream primer P1 0.5 μ l

Downstream primer P2 0.5 μ l

DNA profiling 0.5 μ l

Archaeal dna polymerase (Primer STAR, takara company) 0.25 μ l

Total system 25 μ l

PCR reaction parameter: 98 ℃ of sex change 15s, 60 ℃ of annealing 15 s, 72 ℃ are extended 50 s, totally 30 circulations.

After this PCR product is purified, use ecor I and bgliI carries out double digestion, and it is upper that clone is structured in plasmid pET30a (+)-LT70, is built into pET30a (+)-LT70 (ESAT6-Ag85B-MPT64 _190-198-Mtb8.4-Rv2626c) carrier that (adds linker).

1.2 fusion rotein LT70 and LT70(add linker) expression

By pET30a (+)-LT70 (ESAT6-Ag85B-MPT64 successfully constructing _190-198-Mtb8.4-Rv2626c) carrier proceeds to intestinal bacteria e. coliin BL21 (DE3).Expression of Activated has LT70 albumen e. colibL-21 (DE3) thalline, gets the rear thalline of 1ml activation and adds concussion in 200ml LB substratum to cultivate 4h; Adding protein induced dose of IPTG (1.0mol/L) 100 μ l(final concentrations is 0.5mmol/L), 29 ℃ of induction shaking culture 4h; 4 ℃, the centrifugal 30min of 10000rpm collects thalline; Thalline is resuspended in 20mM PB damping fluid (Na ₂hPO ₄12H ₂o 20mmol/L, Na ₂hPO ₄12H ₂o 20mmol/L, pH7.4), the 10ml/g bacterium that wets, ultrasonication bacterium 1h under ice bath (180～200 W, every ultrasonic 3s, stops 5s); After the centrifugal 30min of 10000rpm, collect respectively upper cleer and peaceful precipitation, upper cleer and peaceful precipitation is carried out respectively to polyacrylamide gel electrophoresis; Electrophorogram is referring to Fig. 1, through SDS-PAGE electrophoretic analysis, with e. colithe empty bacterium of BL21 (DE3) is compared, and molecular weight has obvious differential protein band of expression in 70KD left and right, with supernatant form, is expressed as master, and in precipitation, albumen is relatively less.

Fusion rotein LT70(adds linker) expression process identical with fusion rotein LT70 with condition.Electrophorogram is referring to Fig. 1.As shown in Figure 1, add after linker supernatant expression amount and have lifting when not adding linker.

Above-mentioned expressing fusion protein condition has been carried out the optimization of following aspect:

1.2.1 inductor IPTG concentration optimization

Expression of Activated has LT70 albumen e. colibL-21 (DE3) thalline, gets the rear thalline of 1ml activation and adds concussion in 200mlLB substratum to cultivate 4h.Add three kinds of final concentrations of protein induced dose of IPTG (1.0mol/L): a:100 μ l(final concentration is 0.5mmol/L); B:40 μ l(final concentration is 0.2mmol/L); C:20 μ l(final concentration is 0.1mmol/L), 37 ℃ of induction shaking culture 4h.4 ℃, the centrifugal 30min of 10000rpm collects respectively thalline.Thalline is resuspended in 20mM PB damping fluid (Na ₂hPO ₄12H ₂o 20mmol/L, Na ₂hPO ₄12H ₂o 20mmol/L, pH7.4) the wet bacterium of 10ml/g, ultrasonication bacterium 1h under ice bath (180～200 W, every ultrasonic 3s, stops 5s).10000rpm, collects respectively upper cleer and peaceful precipitation after 4 ℃ of centrifugal 30min, upper cleer and peaceful precipitation is carried out respectively to polyacrylamide gel electrophoresis.Analytical results shows, the expression that all can induce target protein LT70 when IPTG final concentration is three kinds of concentration, and when final concentration is 0.5mmol/L, target protein LT70 expression amount is maximum.

1.2.2 the optimization of LT70 abduction delivering temperature

Expression of Activated has LT70 albumen e. colibL-21 (DE3) bacterial strain, gets the rear thalline of 1ml activation and adds concussion in 200mlLB substratum to cultivate 4h; Add protein induced dose of IPTG (1.0mol/L) 20ul, respectively at (37 ℃/34 ℃/31 ℃/30 ℃/29 ℃/28 ℃/27 ℃/25 ℃) induction shaking culture 4h under condition of different temperatures; 4 ℃, the centrifugal 30min of 10000rpm collects thalline; Thalline is resuspended in 20mM PB damping fluid (Na ₂hPO ₄12H ₂o 20mmol/L, Na ₂hPO ₄12H ₂o 20mmol/L, pH7.4), the 10ml/g bacterium that wets, ultrasonication bacterium 1h under ice bath (180～200 W, every ultrasonic 3s, stops 5s); After the centrifugal 30min of 10000rpm, collect respectively upper cleer and peaceful precipitation, upper cleer and peaceful precipitation is carried out respectively to polyacrylamide gel electrophoresis; Through SDS-PAGE electrophoretic analysis, under different inducing temperature conditions, LT70 target protein all can be at supernatant solubility expression, the highest with inducing temperature supernatant expression amount when 29 ℃ of left and right.

The purification process of 1.3 fusion rotein LT70

LT70 thalline is resuspended in 20mM PB damping fluid, ultrasonication 1h left and right under ice bath, 4 ℃, the centrifugal 10min of 10000rpm, centrifugal rear collection is containing the supernatant of LT70 albumen.

1.3.1 saltout and slightly purify: this fusion rotein is saltoutd with the ammoniumsulphate soln that saturation ratio is 15%; The albumen salting out is carried out resuspended with 20mM PB damping fluid.

1.3.2 the fusion rotein after saltouing is carried out to chromatography purification

1.3.2.1 hydrophobic chromatography purifying: select hydrophobic prepacked column HiTrap Butyl FF to be further purified LT70 albumen after saltouing, collect different gradient eluents and carry out polyacrylamide gel (SDS-PAGE) electrophoretic analysis purified product.After the protein purification thing finally obtaining is measured to concentration with Lorry phenol reagent process, can use, before each use, all with PBS, be diluted to 0.2mg/ml.

1.3.2.2 sieve chromatography purifying: selecting gel filter medium Superdex 75 pre grade(superdex is the filler that meets of Sepharose and glucose; Separating ranges is: 3000～70000) carry out purifying, collect different gradient eluents and carry out polyacrylamide gel (SDS-PAGE) electrophoretic analysis and obtain final purified.After the protein purification thing finally obtaining is measured to concentration with Lorry phenol reagent process, can use, before each use, all with PBS, be diluted to 0.2mg/ml.

Fusion rotein LT70 adds linker with LT70() purge process is identical with method, through experimental verification, finds that both purification result are identical.

The activity of 1.4 Western Blot checking fusion rotein LT70

(1) albumen after purifying is run to SDS-PAGE electrophoresis;

(2) transferring film: first pvdf membrane is cut into 5.3 * 8.3cm size, filter paper is cut out 8 * 10cm size and immersed methyl alcohol 1-2min.Then, according to the order of blank/sponge/tetra-metafiltration paper/pvdf membrane/glue/tetra-metafiltration paper/sponge/blackboard, be arranged in order and make sandwich solid phase carrier.Connection electrode under ice bath state, in transfer groove, ice chest is placed on blackboard side, in interior water jacket, fills it up with transfering buffering liquid.Transferring film condition: 200V, 200mA(constant current turns soon), 1h;

(3) sealing: pvdf membrane is rinsed with TBST, rinse and get final product (1-2min).With confining liquid, film is covered to (2 pvdf membranes back-to-back, intersect and put into 5% skim-milk), the slow jog 4h of horizontal shaking table;

(4) wash film: the pvdf membrane after sealing is put into TBST, and the slow jog of horizontal shaking table, cleans 10min/ time 10 times;

(5) hatch primary antibodie (antigen immune mice serum): according to maker, show, the pvdf membrane of target protein position on pvdf membrane is cut out, note corner cut mark.The pvdf membrane bar of cutting out is put into the primary antibodie centrifuge tube that diluted (primary antibodie covers film bar, and a centrifuge tube can only add two, and back-to-back) is housed.The slow jog 12h of room temperature water yawing bed;

(6) wash film: the pvdf membrane after primary antibodie is hatched is put into TBST, the slow jog of horizontal shaking table, cleans 10min/ time 3 times;

(7) hatch two anti-(the anti-mouse polyclonal antibodies of rabbit): cleaned pvdf membrane is put into and contained the two large centrifuge tubes that resist (1:5000) that dilutes (identical with primary antibodie, a centrifuge tube can only add two, and back-to-back).Room temperature, the slow jog 3h of horizontal shaking table lucifuge;

(8) wash film: the pvdf membrane after two anti-hatching is put into TBST, and the slow jog of horizontal shaking table, cleans 10min/ time 5 times;

(9) develop (in darkroom, operating): the smooth preservative film of completing in exposure box, be placed on lower floor's preservative film pvdf membrane is smooth, every pvdf membrane dropwise adds the super quick luminescent solution of ECL, and upper strata preservative film is covered to pvdf membrane (bubble is driven in attention out of).In darkroom, observe fluorescence intensity, consider exposed plate thickness (number).Cut out 1 film, be pressed on pvdf membrane, avoid mobile, cover exposure box, lucifuge is placed 30min.Film is taken out and is positioned over successively developing solution, and ultrapure water, in stop bath.Observations after last tap water develops photographic film.

2.2 tuberculosis subunit vaccine LT70 and LT70(add linker) immunologic competence evaluation

Tuberculosis subunit vaccine LT70(adds linker) be to add linker from gene level, when amino acid is synthetic, increased by 15 amino acid whose expression.Due to this section of linker non-immunogenicity, can not impact the function of albumen, so can not change the protection effect of vaccine, can be used as vaccine and use.Meanwhile, through verification experimental verification, tuberculosis subunit vaccine LT70 adds linker with LT70() immune effect identical, below take tuberculosis subunit vaccine LT70 and its immunologic competence carried out to detailed assessment as example.

2.2.1 experiment material: tuberculosis subunit vaccine LT70-DDA/PolyI:C; Bacille Calmette-Guerin vaccine (BCG); Phosphate buffered saline buffer (PBS).

The preparation method of tuberculosis subunit vaccine LT70-DDA/PolyI:C is as follows: fusion rotein LT70 is diluted to 0.2mg/ml with PBS damping fluid; PolyI:C is dissolved to 1mg/ml with PBS damping fluid; Cationic-liposome DDA (dimo-thylidioctyl ammonium bromide, DDA) is mixed with 2.5mg/ml with PBS, and 80 ℃ of water-bath 10min, are cooled to room temperature.Get PolyI:C solution 50 μ l and equivalent fusion rotein LT70 solution fully mixes, room temperature is placed 1min.In mixing solutions, dropwise add 100 μ L DDA solution, then fully emulsified, make vaccine be the creamy of homogeneous, obtain tuberculosis subunit vaccine LT70-DDA/PolyI:C.When this vaccine is used for immunity, consumption is a 200 μ L/ mouse.

2.2.2 laboratory animal: C57BL/6 mouse;

2.2.3 laboratory animal grouping (totally four groups):

Control group: pBS; bCG;

Experimental group: (EAMM+MH)-DDA/PolyI:C; lT70-DDA/PolyI:C.

2.2.4 immune animal time point:

The 0th week: inguinal region subcutaneous inoculation control group PBS and BCG group (5 * 10 ⁶cFU/only), use subunit vaccine inguinal region subcutaneous inoculation experimental group (the simultaneously group and group) animal 200 μ l/ only;

2nd, 4 weeks: subunit vaccine inguinal region is subcutaneous with twice of same dose booster immunization experimental group animal.

2.2.5 immune index detection

2.2.5.1 ELISPOT method detects the level of mouse secretion antigen specificity IFN-γ

After last immune mouse 6 weeks, aseptic separating spleen lymphocyte, applies enzyme linked immunological Spot Jest (ELISPOT) technology for detection spleen lymphocyte at ESAT6, Ag85B, Mtb8.4, the secretion level of IFN-γ after Rv2626c and PPD (tuberculin) stimulate.

Concrete steps: aseptic excision spleen, grinds by 200 order nylon net filters, with the separated lymphocyte of lymphocyte separation medium.Isolated lymphocyte is added in the pre-coated ELISPOT plate of 96 hole IFN-γ, and final concentration is 5 * 10 ⁶/ hole, gives respectively ESAT6(10 ug/ml), Ag85B(5 ug/ml), Mtb8.4(200ug/ml, is our own synthetic peptide section), Rv2626c(10 ug/ml) and PPD (5ug/ml) stimulate.At 37 ℃, 5%CO ₂under condition, jointly hatch after 48 hours, by ELISPOT operation instructions, add successively reagent such as detecting antibody, wash plate, colour developing, counting spot number.Result is referring to Fig. 5.

2.2.5.2 ELISA method detects the level of mouse secretion antigen specific antibody (IgG1, IgG2c)

With ESAT6 (10ug/ml), Ag85B (5ug/ml) and Rv2626c (10ug/ml), being coated with respectively 4 ℃ of 96 orifice plates (100 μ l/well) spends the night; With PBST solution, 300 μ l/well wash plate 5 times * 1min/ time; Since 1: 100 doubling dilution to 1: 102400, add two-fold dilution's serum sample (serum that last immune mouse obtained after 6 weeks), place 1h for 37 ℃.Wash after plate, add the rabbit anti-mouse igg 1 of the dilution in 1: 15000 of 200 μ l/well, the rabbit anti-mouse igg 2c of dilution in 1: 10000, place 1h for 37 ℃.Wash after plate, add 100 μ l/well TMB nitrite ions, after room temperature lucifuge reaction colour developing in 15 minutes, add the 50 μ l/well stop buffer (H of 2N ₂sO ₄) termination reaction; At 450nm, detect OD value.Result is referring to Fig. 6.

2.3 the protection effect assessment of tuberculosis subunit vaccine LT70

By LT70 vaccine respectively the 0th, 2,4 weeks immune mouses three times, within after last immunity the 30th week, carry out respiratory tract aerosol and attack mycobacterium tuberculosis strain H37Rv strain, dosage is 50-100CFU/ mouse, within the 10th week, carries out the detection of lungs and splenic tuberculosis bacterium carrying capacity after attacking.This experiment is carried out in the ABSL-3 of Wuhan University laboratory.

Concrete grammar: aseptic mouse lungs and the spleen got, in mortar, internal organs are ground, add the PBS damping fluid of 2ml, then by five gradients of this liquid doubling dilution, each gradient liquid 0.1ml is inoculated on 7H11 substratum.37 ℃ of incubators are cultivated 18 days, carry out CFU counting.Result is referring to Fig. 7.

The histopathology damage check of 2.4 tuberculosis subunit vaccine LT70 to mouse

Experiment mice organs and tissues sections observation: attack after malicious mouse dissection, get right lung tissue, adopt the dyeing of paraffin section hematoxylin-eosin (HE) staining, observe lungs pathology damage degree; And tissue slice is done to acid-fast stain, in tissues observed, deposit tubercule bacillus number.Pathology detection entrusts the ABSL-3 of Wuhan University to complete.Result is referring to Fig. 8 and Fig. 9: PBS group: the mean value that Tuberculous pathology area accounts for section area is 17%, BCG group: the mean value that Tuberculous pathology area accounts for section area is 11%, EAMM+MH vaccine group: the mean value that Tuberculous pathology area accounts for section area is 14%, LT70 vaccine group: the mean value that Tuberculous pathology area accounts for section area is 12%.

2.5 tuberculosis subunit vaccine LT70 strengthen the immunocompetence evaluation of BCG

2.5.1 experiment material: subunit vaccine LT70-DDA/PolyI:C; Bacille Calmette-Guerin vaccine (BCG); Phosphate buffered saline buffer (PBS).

2.5.2 laboratory animal: C57BL/6 mouse;

2.5.3 laboratory animal grouping (totally three groups):

Control group pBS; bCG; Experimental group bCG just exempts from, LT70-DDA/PolyI:C vaccine reinforcement group.

2.5.4 immune animal time point

The 0th week: bacille Calmette-Guerin vaccine (BCG) 5 * 10 ⁶cFU inguinal region subcutaneous inoculation experimental group animal once; Immune control group PBS of while and BCG group (5 * 10 ⁶cFU/only).

18th, 21 weeks: subunit vaccine LT70 inguinal region is subcutaneous with twice of 200ul/ booster immunization experimental group animal.

2.5.5 immune indexes measuring method

2.5.5.1 ELISPOT method detects the level of mouse secretion antigen specificity IFN-γ

Last immunity, after 6 weeks, detects mouse boosting cell at antigen ESAT6, under the stimulation of Ag85B and Rv2626c, and the level of secrete cytokines.The same 2.2.5.1 of concrete grammar and step.Result is referring to Figure 10.

2.5.5.2 ELISA method detects the level of mouse secretion antigen specific antibody (IgG1, IgG2c)

After last immunity, after 6 weeks, detect mouse boosting cell at antigen ESAT6, under the stimulation of Ag85B and Rv2626c, the level of antibody.The same 2.2.5.2 of concrete grammar and step .result is referring to Figure 11.

2.6 tuberculosis subunit vaccine LT70 strengthen the protection effect assessment of BCG

Mouse carried out BCG at the 0th week and just exempts from, LT70 vaccine is twice of the 18th, 21 weeks booster immunization, after last immunity, at the 12nd week, carry out respiratory tract aerosol and attack mycobacterium tuberculosis strain H37Rv strain, dosage is 50-100CFU/ mouse, within the 10th week, carries out the detection of lungs and splenic tuberculosis bacterium carrying capacity after attacking.

Concrete grammar: aseptic mouse lungs and the spleen got, in mortar, internal organs are ground, add the PBS damping fluid of 2ml, then by five gradients of this liquid doubling dilution, each gradient liquid 0.1ml is inoculated on 7H11 substratum.37 ℃ of incubators are cultivated 18 days, carry out CFU counting.Result is referring to Figure 12.

Histopathology damage check to mouse after 2.7 tuberculosis subunit vaccine LT70 strengthening BCG immunity and strain attack

Experiment mice organs and tissues sections observation: attack after malicious mouse dissection, get right lung tissue, adopt the dyeing of paraffin section hematoxylin-eosin (HE) staining, observe lungs pathology damage degree; And tissue slice is done to acid-fast stain, in tissues observed, deposit tubercule bacillus number.Result is referring to Figure 13 and Figure 14: PBS group: the mean value that Tuberculous pathology area accounts for section area is 17%, BCG group: the mean value that Tuberculous pathology area accounts for section area is 11%, LT70 vaccine strengthening BCG group: the mean value that Tuberculous pathology area accounts for section area is 7%.

Finally it should be noted that: the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.

sequence table

<110> Lanzhou University

<120> mycobacterium tuberculosis fusion protein and its preparation method and application

<210> 1

<211> 1980

<212> DNA

<213> artificial sequence

<400> 1

atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga 60

aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120

gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180

acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240

caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcagaatt cttctcccgg 300

ccggggctgc cggtcgagta cctgcaggtg ccgtcgccgt cgatgggccg cgacatcaag 360

gttcagttcc agagcggtgg gaacaactca cctgcggttt atctgctcga cggcctgcgc 420

gcccaagacg actacaacgg ctgggatatc aacaccccgg cgttcgagtg gtactaccag 480

tcgggactgt cgatagtcat gccggtcggc gggcagtcca gcttctacag cgactggtac 540

agcccggcct gcggtaaggc tggctgccag acttacaagt gggaaacctt cctgaccagc 600

gagctgccgc aatggttgtc cgccaacagg gccgtgaagc ccaccggcag cgctgcaatc 660

ggcttgtcga tggccggctc gtcggcaatg atcttggccg cctaccaccc ccagcagttc 720

atctacgccg gctcgctgtc ggccctgctg gacccctctc aggggatggg gcctagcctg 780

atcggcctcg cgatgggtga cgccggcggt tacaaggccg cagacatgtg gggtccctcg 840

agtgacccgg catgggagcg caacgaccct acgcagcaga tccccaagct ggtcgcaaac 900

aacacccggc tatgggttta ttgcgggaac ggcaccccga acgagttggg cggtgccaac 960

atacccgccg agttcttgga gaacttcgtt cgtagcagca acctgaagtt ccaggatgcg 1020

tacaacgccg cgggcgggca caacgccgtg ttcaacttcc cgcccaacgg cacgcacagc 1080

tgggagtact ggggcgctca gctcaacgcc atgaagggtg acctgcagag ttcgttaggc 1140

gccggcagat ctttcgcagt cacgaacgac ggggtgatta tgaggctgtc gttgaccgca 1200

ttgagcgccg gtgtaggcgc cgtggcaatg tcgttgaccg tcggggccgg ggtcgcctcc 1260

gcagatcccg tggacgcggt cattaacacc acctgcaatt acgggcaggt agtagctgcg 1320

ctcaacgcga cggatccggg ggctgccgca cagttcaacg cctcaccggt ggcgcagtcc 1380

tatttgcgca atttcctcgc cgcaccgcca cctcagcgcg ctgccatggc cgcgcaattg 1440

caagctgtgc cgggggcggc acagtacatc ggccttgtcg agtcggttgc cggctcctgc 1500

aacaactatg agctcatgac cacggcgcgt gatatcatga atgcgggtgt cacctgtgtt 1560

ggcgagcacg aaacgttgac cgcagcagca cagtacatgc gcgaacatga tatcggcgca 1620

ttgccgattt gcggcgacga tgatcgtctg cacggtatgc tgaccgaccg cgatatcgtt 1680

atcaagggtc tggccgcagg cttggacccg aacaccgcga ccgccggtga actggcacgt 1740

gacagcatct attacgtcga cgcgaacgcc agcattcaag agatgctgaa cgtgatggaa 1800

gagcatcagg tgcgtcgtgt cccggttatc agcgaacatc gtctggttgg tatcgttacc 1860

gaagccgaca tcgcacgtca cctgccggag cacgcgattg ttcagttcgt gaaagcgatt 1920

tgcagcccga tggcgttggc gtctcgtcaa aagggcgaca caaaatttat tctaaatgca 1980

<210> 2

<211> 660

<212> PRT

<213> artificial protein

<400> 2

Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser

1 5 10 15

Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly

20 25 30

Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser

35 40 45

Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu

50 55 60

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

65 70 75 80

Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Glu

85 90 95

Phe Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser

100 105 110

Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Asn

115 120 125

Asn Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp

130 135 140

Tyr Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Tyr Gln

145 150 155 160

Ser Gly Leu Ser Ile Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr

165 170 175

Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr

180 185 190

Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gln Trp Leu Ser Ala

195 200 205

Asn Arg Ala Val Lys Pro Thr Gly Ser Ala Ala Ile Gly Leu Ser Met

210 215 220

Ala Gly Ser Ser Ala Met Ile Leu Ala Ala Tyr His Pro Gln Gln Phe

225 230 235 240

Ile Tyr Ala Gly Ser Leu Ser Ala Leu Leu Asp Pro Ser Gln Gly Met

245 250 255

Gly Pro Ser Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys

260 265 270

Ala Ala Asp Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Glu Arg Asn

275 280 285

Asp Pro Thr Gln Gln Ile Pro Lys Leu Val Ala Asn Asn Thr Arg Leu

290 295 300

Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu Gly Gly Ala Asn

305 310 315 320

Ile Pro Ala Glu Phe Leu Glu Asn Phe Val Arg Ser Ser Asn Leu Lys

325 330 335

Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn Ala Val Phe Asn

340 345 350

Phe Pro Pro Asn Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu

355 360 365

Asn Ala Met Lys Gly Asp Leu Gln Ser Ser Leu Gly Ala Gly Arg Ser

370 375 380

Phe Ala Val Thr Asn Asp Gly Val Ile Met Arg Leu Ser Leu Thr Ala

385 390 395 400

Leu Ser Ala Gly Val Gly Ala Val Ala Met Ser Leu Thr Val Gly Ala

405 410 415

Gly Val Ala Ser Ala Asp Pro Val Asp Ala Val Ile Asn Thr Thr Cys

420 425 430

Asn Tyr Gly Gln Val Val Ala Ala Leu Asn Ala Thr Asp Pro Gly Ala

435 440 445

Ala Ala Gln Phe Asn Ala Ser Pro Val Ala Gln Ser Tyr Leu Arg Asn

450 455 460

Phe Leu Ala Ala Pro Pro Pro Gln Arg Ala Ala Met Ala Ala Gln Leu

465 470 475 480

Gln Ala Val Pro Gly Ala Ala Gln Tyr Ile Gly Leu Val Glu Ser Val

485 490 495

Ala Gly Ser Cys Asn Asn Tyr Glu Leu Met Thr Thr Ala Arg Asp Ile

500 505 510

Met Asn Ala Gly Val Thr Cys Val Gly Glu His Glu Thr Leu Thr Ala

515 520 525

Ala Ala Gln Tyr Met Arg Glu His Asp Ile Gly Ala Leu Pro Ile Cys

530 535 540

Gly Asp Asp Asp Arg Leu His Gly Met Leu Thr Asp Arg Asp Ile Val

545 550 555 560

Ile Lys Gly Leu Ala Ala Gly Leu Asp Pro Asn Thr Ala Thr Ala Gly

565 570 575

Glu Leu Ala Arg Asp Ser Ile Tyr Tyr Val Asp Ala Asn Ala Ser Ile

580 585 590

Gln Glu Met Leu Asn Val Met Glu Glu His Gln Val Arg Arg Val Pro

595 600 605

Val Ile Ser Glu His Arg Leu Val Gly Ile Val Thr Glu Ala Asp Ile

610 615 620

Ala Arg His Leu Pro Glu His Ala Ile Val Gln Phe Val Lys Ala Ile

625 630 635 640

Cys Ser Pro Met Ala Leu Ala Ser Arg Gln Lys Gly Asp Thr Lys Phe

645 650 655

Ile Leu Asn Ala

660

<210> 3

<211> 2070

<212> DNA

<213> artificial sequence

<400> 3

atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga 60

aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120

gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180

acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240

caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcagaatt cggtggtggt 300

ggttctggtg gtggtggttc tggtggtggt ggttctttct cccggccggg gctgccggtc 360

gagtacctgc aggtgccgtc gccgtcgatg ggccgcgaca tcaaggttca gttccagagc 420

ggtgggaaca actcacctgc ggtttatctg ctcgacggcc tgcgcgccca agacgactac 480

aacggctggg atatcaacac cccggcgttc gagtggtact accagtcggg actgtcgata 540

gtcatgccgg tcggcgggca gtccagcttc tacagcgact ggtacagccc ggcctgcggt 600

aaggctggct gccagactta caagtgggaa accttcctga ccagcgagct gccgcaatgg 660

ttgtccgcca acagggccgt gaagcccacc ggcagcgctg caatcggctt gtcgatggcc 720

ggctcgtcgg caatgatctt ggccgcctac cacccccagc agttcatcta cgccggctcg 780

ctgtcggccc tgctggaccc ctctcagggg atggggccta gcctgatcgg cctcgcgatg 840

ggtgacgccg gcggttacaa ggccgcagac atgtggggtc cctcgagtga cccggcatgg 900

gagcgcaacg accctacgca gcagatcccc aagctggtcg caaacaacac ccggctatgg 960

gtttattgcg ggaacggcac cccgaacgag ttgggcggtg ccaacatacc cgccgagttc 1020

ttggagaact tcgttcgtag cagcaacctg aagttccagg atgcgtacaa cgccgcgggc 1080

gggcacaacg ccgtgttcaa cttcccgccc aacggcacgc acagctggga gtactggggc 1140

gctcagctca acgccatgaa gggtgacctg cagagttcgt taggcgccgg cggtggtggt 1200

ggttctggtg gtggtggttc tggtggtggt ggttctagat ctttcgcagt cacgaacgac 1260

ggggtgatta tgaggctgtc gttgaccgca ttgagcgccg gtgtaggcgc cgtggcaatg 1320

tcgttgaccg tcggggccgg ggtcgcctcc gcagatcccg tggacgcggt cattaacacc 1380

acctgcaatt acgggcaggt agtagctgcg ctcaacgcga cggatccggg ggctgccgca 1440

cagttcaacg cctcaccggt ggcgcagtcc tatttgcgca atttcctcgc cgcaccgcca 1500

cctcagcgcg ctgccatggc cgcgcaattg caagctgtgc cgggggcggc acagtacatc 1560

ggccttgtcg agtcggttgc cggctcctgc aacaactatg agctcatgac cacggcgcgt 1620

gatatcatga atgcgggtgt cacctgtgtt ggcgagcacg aaacgttgac cgcagcagca 1680

cagtacatgc gcgaacatga tatcggcgca ttgccgattt gcggcgacga tgatcgtctg 1740

cacggtatgc tgaccgaccg cgatatcgtt atcaagggtc tggccgcagg cttggacccg 1800

aacaccgcga ccgccggtga actggcacgt gacagcatct attacgtcga cgcgaacgcc 1860

agcattcaag agatgctgaa cgtgatggaa gagcatcagg tgcgtcgtgt cccggttatc 1920

agcgaacatc gtctggttgg tatcgttacc gaagccgaca tcgcacgtca cctgccggag 1980

cacgcgattg ttcagttcgt gaaagcgatt tgcagcccga tggcgttggc gtctcgtcaa 2040

aagggcgaca caaaatttat tctaaatgca 2070

<210> 4

<211> 690

<212> PRT

<213> artificial protein

<400> 4

Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser

1 5 10 15

Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly

20 25 30

Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser

35 40 45

Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu

50 55 60

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

65 70 75 80

Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Glu

85 90 95

Phe Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

100 105 110

Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro

115 120 125

Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Asn Asn

130 135 140

Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Tyr

145 150 155 160

Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Tyr Gln Ser

165 170 175

Gly Leu Ser Ile Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser

180 185 190

Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys

195 200 205

Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gln Trp Leu Ser Ala Asn

210 215 220

Arg Ala Val Lys Pro Thr Gly Ser Ala Ala Ile Gly Leu Ser Met Ala

225 230 235 240

Gly Ser Ser Ala Met Ile Leu Ala Ala Tyr His Pro Gln Gln Phe Ile

245 250 255

Tyr Ala Gly Ser Leu Ser Ala Leu Leu Asp Pro Ser Gln Gly Met Gly

260 265 270

Pro Ser Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala

275 280 285

Ala Asp Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Glu Arg Asn Asp

290 295 300

Pro Thr Gln Gln Ile Pro Lys Leu Val Ala Asn Asn Thr Arg Leu Trp

305 310 315 320

Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu Gly Gly Ala Asn Ile

325 330 335

Pro Ala Glu Phe Leu Glu Asn Phe Val Arg Ser Ser Asn Leu Lys Phe

340 345 350

Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn Ala Val Phe Asn Phe

355 360 365

Pro Pro Asn Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn

370 375 380

Ala Met Lys Gly Asp Leu Gln Ser Ser Leu Gly Ala Gly Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Ser Phe Ala

405 410 415

Val Thr Asn Asp Gly Val Ile Met Arg Leu Ser Leu Thr Ala Leu Ser

420 425 430

Ala Gly Val Gly Ala Val Ala Met Ser Leu Thr Val Gly Ala Gly Val

435 440 445

Ala Ser Ala Asp Pro Val Asp Ala Val Ile Asn Thr Thr Cys Asn Tyr

450 455 460

Gly Gln Val Val Ala Ala Leu Asn Ala Thr Asp Pro Gly Ala Ala Ala

465 470 475 480

Gln Phe Asn Ala Ser Pro Val Ala Gln Ser Tyr Leu Arg Asn Phe Leu

485 490 495

Ala Ala Pro Pro Pro Gln Arg Ala Ala Met Ala Ala Gln Leu Gln Ala

500 505 510

Val Pro Gly Ala Ala Gln Tyr Ile Gly Leu Val Glu Ser Val Ala Gly

515 520 525

Ser Cys Asn Asn Tyr Glu Leu Met Thr Thr Ala Arg Asp Ile Met Asn

530 535 540

Ala Gly Val Thr Cys Val Gly Glu His Glu Thr Leu Thr Ala Ala Ala

545 550 555 560

Gln Tyr Met Arg Glu His Asp Ile Gly Ala Leu Pro Ile Cys Gly Asp

565 570 575

Asp Asp Arg Leu His Gly Met Leu Thr Asp Arg Asp Ile Val Ile Lys

580 585 590

Gly Leu Ala Ala Gly Leu Asp Pro Asn Thr Ala Thr Ala Gly Glu Leu

595 600 605

Ala Arg Asp Ser Ile Tyr Tyr Val Asp Ala Asn Ala Ser Ile Gln Glu

610 615 620

Met Leu Asn Val Met Glu Glu His Gln Val Arg Arg Val Pro Val Ile

625 630 635 640

Ser Glu His Arg Leu Val Gly Ile Val Thr Glu Ala Asp Ile Ala Arg

645 650 655

His Leu Pro Glu His Ala Ile Val Gln Phe Val Lys Ala Ile Cys Ser

660 665 670

Pro Met Ala Leu Ala Ser Arg Gln Lys Gly Asp Thr Lys Phe Ile Leu

675 680 685

Asn Ala

690

Claims (10)

1. a mycobacterium tuberculosis fusion protein is the protein in sequence 2 and sequence 4 in sequence table.

2. the encoding gene of mycobacterium tuberculosis fusion protein claimed in claim 1.

3. encoding gene according to claim 2, is characterized in that: be following 1) or 2) described gene:

1) its nucleotide sequence is the sequence 1 or 3 in sequence table;

2) with sequence table in the nucleotide sequence of sequence 1 or 3 complementations;

3) under stringent condition with 1) the DNA sequence dna hybridization that limits and the DNA molecular of encoding said fusion protein.

4. the recombinant expression vector that contains encoding gene described in claim 2 or 3.

5. the host cell that contains recombinant expression vector described in claim 4.

6. the preparation method of mycobacterium tuberculosis fusion protein claimed in claim 1, the expression and purification that comprises fusion rotein, it is characterized in that: being expressed as of described mycobacterium tuberculosis fusion protein: the thalline that contains fusion rotein is added to shaking culture in LB substratum, adding final concentration is the protein induced dose of IPTG of 0.5mmol/L, 27 ℃-31 ℃ (29 ℃ is optimum) induction shaking culture 4h, obtain supernatant and are expression product after aftertreatment.

7. the application of mycobacterium tuberculosis fusion protein claimed in claim 1 in preparing tuberculosis subunit vaccine.

8. a tuberculosis subunit vaccine, it contains mycobacterium tuberculosis fusion protein claimed in claim 1.

9. tuberculosis subunit vaccine according to claim 8, is characterized in that: described tuberculosis subunit vaccine is comprised of mycobacterium tuberculosis fusion protein claimed in claim 1, DDA and PolyI:C; As preferably, the mass ratio of mycobacterium tuberculosis fusion protein claimed in claim 1, DDA and PolyI:C is: 1:25:5.

10. the preparation method of tuberculosis subunit vaccine claimed in claim 8, is characterized in that: the preparation method of every 200 μ L tuberculosis subunit vaccines is as follows: fusion rotein is diluted to 0.2mg/ml with PBS damping fluid; PolyI:C is dissolved to 1mg/ml with PBS damping fluid; DDA is mixed with 2.5mg/ml with PBS, is cooled to room temperature after water-bath; Get PolyI:C solution 50 μ l and equivalent fusion rotein solution and fully mix, room temperature is placed; In mixing solutions, dropwise add 100 μ L DDA solution, then fully emulsified, the creamy that makes vaccine be homogeneous obtains tuberculosis subunit vaccine.