CN105567660A - Escherichia coli recombinate expression method of mycobacterium tuberculosis Rv 2837c active protein and applications thereof - Google Patents

Abstract

The invention relates to an escherichia coli recombinate expression method of mycobacterium tuberculosis Rv 2837c active protein and applications thereof. The mycobacterium tuberculosis Rv 2837c protein gene is cloned into a prokaryotic expression vector Pet28a+, and the gene is named as pET-CnpB. A Ni ion affinity chromatography method is adopted to obtain purified Rv2837c active protein. The recombinant Rv2837c protein can be used to detect the serum of animals and patients affected by mycobacterium tuberculosis. The recombinant Rv2837c protein can induce high level body fluid and cellular immunologic response of mouse, and can be used to develop novel subunit vaccine of tuberculosis. So Rv2837c protein has a good application prospect. The invention relates to the applications of the active recombinant protein in the preparation of drugs or preparations for diagnosing and preventing tuberculosis.

Description

A kind of method of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein and application thereof

Technical field

The invention belongs to Diagnosis of Tuberculosis reagent and vaccines arts.The present invention relates to a kind of Recombinant protein expression and obtain the method for mycobacterium tuberculosis Rv2837c activated protein.The invention still further relates to this recombinant protein and prepare the application in diagnosis of tuberculosis and preventing preparation or medicine.

Background technology

TB endemic present situation

Tuberculosis (Tuberculosis, TB) infects by mycobacterium tuberculosis (Mycobacteriumtuberculosis, Mtb) chronic infectious disease caused, and remains the transmissible disease that in worldwide, harm is the most serious at present.According to WHO report, existing about 1/3 population in the whole world infects mycobacterium tuberculosis, and within 2013, newly send out patient about 9,000,000, in all transmissible diseases, tuberculosis mortality rate is only second to acquired immune deficiency syndrome (AIDS).TB is classified as the topmost infectivity killer of the mankind by WHO together with acquired immune deficiency syndrome (AIDS), malaria.China belongs to one of 22 TB high burden countries in the whole world, in China's 27 kinds of notifiable infectious diseases, ranked first position.The Ministry of Health of China is one of major disease being classified as national priority control by TB, and tuberculosis is all classified as one of primary study object by national Eleventh Five-Year Plan and the special problem of " 12 " serious infectious diseases.

Diagnosis of tuberculosis present Research

Sediments microscope inspection, Sputum culturing are the Main Means of pathogen detection, but the diagnostic method based on the detecting of pathogenic agent exists that experimental period is long, susceptibility is low, specificity is undesirable and the shortcoming such as length consuming time.Molecular biology method such as PCR, hybridization and gene and protein chip have also been applied to quick diagnosis lungy, but because of its recall rate undesirable, and there is the reasons such as technical requirements is high, expensive, be not suitable for clinical Large-scale Screening.The diagnostic method that active research is novel and reagent contribute to control lungy.

The fast development of immunological technique facilitates the development of diagnostic techniques.After human infection's mycobacterium tuberculosis, while producing immunizing power, there is delayed type hypersensitivity.Tuberculin test is that whether judgement experimenter has against mycobacterium tuberculosis cellular immunization, to judge whether experimenter infects a kind of method of this pathogenic bacteria by measuring body to mycobacterium tuberculosis with or without allergy.The tuberculin test (PPD) developed with this principle still widely uses clinical at present, but due to the extensive inoculation of bacille Calmette-Guerin vaccine, the results of tuberculin test positive can not be diagnosed as tuberculosis, and is more used for vaccine effect monitoring.

After tuberculosis infection, the memory t cell of long-term existence antigen-specific in body, when again running into antigenic stimulation, can breed by prompt activation, producing cytokine profiles, wherein IFN-γ is crucial cytokine.IFN-γ release test (interferongammareleaseassay, IGRAs) be the novel method that a kind of ion vitro immunization for m tuberculosis infection detects, in discriminating active tuberculosis with tuberculosis infection of hiding, prediction tuberculosis onset risk, there is definite meaning, but clinical Diagnosis of Tuberculosis value ratio is comparatively limited to, in addition, T-SPOT.TB experimental technique operational requirement is high, and test kit is expensive, also limit its application in the not high countries and regions of economic level.

Serodiagnosis, by detecting infected person anteserum tuberculosis specific antibody level, indirect diagnosis of tuberculosis, has specificity good, consuming time short and be easy to the significant advantage of batch detection.Select mycobacterium tuberculosis and bacille Calmette-Guerin vaccine genome difference district antigen at present, adopt the methods such as ELISA to detect specific antibody in patients serum and carry out the research of auxiliary diagnosis, study more deep as Early insulin secretion antigen target 6kD albumen (earlysecretaryantigenictarget6kuprotein, ESAT-6), culturing filtrate albumen (culturefiltrateprotein, CFP-10), LAM antigen, Ag85 mixture, 38KD albumen, A60 antigen, 16KD albumen, MPT64, MTB48 etc.These antigens differ from one another, although domestic and international multiple antitubercle sera diagnostic kit listing, its clinical report result differs, and therefore continues the specific antigen of sieve series and disease-related, combine existing dominant antigen, contribute to developing more effective serological diagnostic method.

Tuberculosis subunit vaccine present Research

Bacille Calmette-Guerin vaccine (BacillusCalmette-Gu é rinvaccine, BCG) is at present for unique vaccine of tuberculosis prophylaxis, but it is short to there is the protection period; the problems such as protective immune response is more weak; in addition, as a kind of attenuated live vaccine, the inoculation of immunocompromised crowd can not be used for.The TB new generation vaccine developed at present mainly comprises subunit vaccine, gene vaccine, recombinant BCG vaccine, the whole cell living vaccine of attenuation or enhancing, auxotroph living vaccine, vaccine etc. in conjunction with dendritic cell.Wherein subunit vaccine is grouped into by one or more one-tenth that can induce protective immunity, and because moiety is clear and definite, structure is simple, safe and special and receive the concern of researchist.So far; in tuberculosis candidate vaccine; nearly 50% is subunit vaccine, adopts the drive member (as albumen, polypeptide, mycolic acid, glycolipid etc.) of mycobacterium tuberculosis to make vaccine component, body can be induced to produce immunoprotection or reach the effect of immunotherapy.

Through for many years to the research of mycobacterium tuberculosis protective antigen, now identify and multiple there is stronger immunogenic albumen, mainly comprised secretory protein, as ESAT-6, CFP10, Ag85, TB10.4 etc.; The T cell antigen target point protein (as MTB39 and MTB32 combines the fusion rotein MTB72f formed) of mouse and the mankind; The specific antigen that latent infection is expressed is as Hsp65, Hsp70, HspX etc.Ag85B research wherein in Ag85 family comparatively early, Horwitzetal (rotectiveimmunityagainsttuberculosisinducedbyvaccination withmajorextracellularproteinsofMycobacteriumtuberculosi s.ProceedingsoftheNationalAcademyofSciencesoftheUnitedSt atesofAmerica, 1995, 92 (5): 1530-4.) injected s. c is adopted to attack with strain with after Ag85B protein immunization cavy 3 times, compared with control group, after immunity, cavy can produce stronger cellullar immunologic response and show certain immunizing power to strain, survival time increases.The rBCG expressing Ag85B has gone through to enter clinical trial.Ag85B and the TB10.4 amalgamation protein vaccine of AERAS, SSI, Sanofi and Intercell joint research and development, carries out I clinical trial phase at present.Therefore, it is good anti-TB vaccine component that multinomial research all shows Ag85B albumen, has good Development volue.

Although above-mentioned protective antigen all demonstrates certain vaccine potential, so far, also do not obtain the tuberculosis new generation vaccine that can be used for alternative BCG, show the tuberculosis antigen limited use based on secretory protein.To the develop rapidly of mycobacterium tuberculosis physiologic characteristic research, provide more candidate molecules.The acting in conjunction of the antigen of multipath, multimachine, just likely obtains more effective immune effect, is therefore also the research direction of following novel vaccines against tuberculosis.

Mycobacterium tuberculosis Rv2837c progress

2008, a kind of new micromolecule nucleotide class signaling molecule ring two adenosine (Cyclicdi-adenosinemonophosphate, c-di-AMP) be found, it mainly participates in bacterial spore formation, cell walls formation, cell walls pressure sensitive and controls the physiological processs such as bacterium size, its level is relevant to bacterial virulence, thus causes investigator's interest widely.JunYang, etal. (Deletionofthecyclicdi-AMPphosphodiesterasegene (cnpB) inMycobacteriumtuberculosisleadstoreducedvirulenceinamou semodelofinfection.MolMicrobiol.201493 (1): 65-79.) finds that Rv2837c is c-di-AMP degrading enzyme, and by its called after CnpB.Rv2837c contains DHH-DHHAI structural domain, and the albumen containing this structural domain is extensively present in bacterium, as streptococcus aureus, Bacillus subtilus etc.ManikandanKetal. (Two-stepsynthesisandhydrolysisofcyclicdi-AMPinMycobacter iumtuberculosis.PLoSOne.201423; 9 (1): e86096.) find that c-di-AMP first can be degraded to pApA by Rv2837c, be degraded to 5'-AMP further.PosticGetal (CharacterizationofNrnAhomologsfromMycobacteriumtuberculo sisandMycoplasmapneumoniae.RNA.201218 (1): 155-65.) finds that Rv2837c has RNA enzymic activity, degradable nanoRNA, and dephosphorylation pAp, infer, in bacterium pathogenic course, there is important regulating effect.CronLEetal. (TwoDHHsubfamily1proteinscontributetopneumococcalvirulenc eandconferprotectionagainstpneumococcaldisease.InfectImm un.201179 (9): 3697-710.) finds that the albumen containing DHH structural domain is relevant with the virulence of bacterium; after this protein immunization mouse; the infection of a certain amount of bacterium can be resisted; thus have immune protective efficiency, the Rv2837c of prompting DHH structural domain family has similar biology and immunological characteristic.

Summary of the invention

The object of this invention is to provide a kind of method of Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein.The restructuring Rv2837c activated protein that the method obtains can be used for the development of tuberculosis new diagnostic and/or vaccine (preparation).

The present invention is achieved in that

A method for Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein, the genome Rv2837c gene order design primer according to Mycobacterium tuberculosis H37Rv strain:

P1:5 '-CGGCATATGGTGACGACGATCGACCCAAG-3 ', NdeI site

P2:5 '-TTTAAGCTTGCCAGCTGCGCGATCTGACG-3 ', HindIII site

With pcr amplification c-di-AMP lytic enzyme Rv2837c gene from Mycobacterium tuberculosis H37Rv genome, reaction parameter: 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 57 DEG C of renaturation 30s, 72 DEG C extend 100s, 35 circulations, and latter 72 DEG C of last circulation extends 5min, the 1023bp object fragment that pcr amplification goes out, with NdeI and HindIII double digestion, T4DNA ligase enzyme connects same double digestion and the pET28a+ carrier reclaimed, Transformed E .coliDH5 α competent cell, use kalamycin resistance plate screening, picking positive colony is inoculated in liquid nutrient medium 37 DEG C of shaking culture and spends the night, test kit extracts plasmid DNA, double digestion qualification positive plasmid carries out sequencing, sequencing shows, success builds Rv2837c prokaryotic expression carrier, called after pET-CnpB, its complete genome sequence is as shown in SEQIDNO:1, pET-CnpB Transformed E .coliBL21, IPTG induces destination gene expression, anti-His tag monoclonal antibody Western-blot method is used to identify target protein, this expression strain culture presevation number is: CCTCCNO:M2015703, the mode inducible protein of 0.05mmoL/L lower concentration IPTG and 16 DEG C Low-temperature culture is adopted to express, Ni ion affinity chromatography method purifying is adopted to obtain target protein that is solvable, high yield, detecting through HPLC of purifying protein, have the activity of degraded c-di-AMP, its amino acid complete genome sequence is as shown in SEQIDNO:2.

Described restructuring mycobacterium tuberculosis Rv2837c albumen is for diagnosing and preventing the application in preparation lungy.

Described recombinant protein detects the result of m tuberculosis infection serum: the Rv2837c protein 10 μ g/mL of purifying of the present invention wraps by elisa plate, detects different infection period mice serum and clinical tubercular's serum.Detected result shows, in infecting mouse body, Rv2837c antibody increases with infection time remaining, and its level is higher than Ag85B; Draw ROC curve according to tuberculosis patient serum Rv2837c antibody horizontal, area under curve is greater than 0.5, shows to have diagnostic.

Described recombinant protein stimulates the result of immune response: the Rv2837c albumen dorsal sc immune mouse 50 μ g/ of purifying of the present invention only, 2 weeks, interval, immune three times; Detect humoral immunoresponse(HI) level and comprise antibody titers and Subclass of antibody mensuration; Detect cellullar immunologic response level, comprise spleen lymphocyte proliferation, CD4 ⁺/ CD8 ⁺the expression level that ratio, RT-PCR detect cytokine transcriptional level, ELISA detects cytokine.Detected result shows, the Rv2837c protein immunization mouse of restructuring, can induce higher humoral and cellular immune response response level, and partial immunity response level is higher than the strong immunizing antigen Ag85B of existing mycobacterium tuberculosis.The immunne response that body is stronger can be stimulated after restructuring Rv2837c protein immunization; improve the immune protective efficiency of body; because its mechanism of action and existing tuberculosis antigen are all not identical, alone or in combination for the development of tuberculosis novel subunit vaccine, thus can have a good application prospect.

Our research finds, at expression in escherichia coli, and by the Rv2837c degradable c-di-AMP of affinity chromatography purifying, therefore has c-di-AMP lytic enzyme active.In infection animal body, Rv2837c antibody titer raises gradually along with the prolongation of infection time, and in tubercular's body, the antibody titer of this antigen is significantly higher than non-tuberculosis patient, shows that it can be used for the development of tuberculosis serological diagnosis test kit.After Rv2837c protein immunization mouse, detect and find that it can produce the titre of high antibody by induction of immunity mouse, and antibody titers is higher; Recombinant protein significantly can induce spleen lymphocyte proliferation, particularly stimulates CD8 ⁺cell increases; More cytokine can be discharged by induction of lymphocyte, the especially rising of key cytokines IFN-γ, be conducive to body opposing m tuberculosis infection.Therefore, Rv2837c albumen can be used as the component of candidate of tuberculosis subunit vaccine.

In sum, Rv2837c, as newfound c-di-AMP lytic enzyme, has the mechanism of action that proteantigen existing from other is different in Bacterial Physiological.Our research obtains tool activated mycobacterium tuberculosis c-di-AMP lytic enzyme Rv2837c, and can be used as the component of development diagnostic reagent of tuberculosis and vaccine, thus has good market using value.

Accompanying drawing illustrates:

Fig. 1: Rv2837c gene amplification PCR result figure.

Fig. 2: Rv2837c prokaryotic expression carrier enzyme cuts qualification result figure.

Fig. 3: Rv2837c expression of recombinant proteins SDS-PAGE analytical results figure.

Fig. 4: Rv2837c expression of recombinant proteins Western-blot analytical results figure

Fig. 5: Rv2837c recombinant protein purification SDS-PAGE analysis chart.

Fig. 6: Rv2837c recombinant protein enzymic activity HPLC analytical results schematic diagram.

Fig. 7: Rv2837c recombinant protein detects infecting mouse specific antibody result schematic diagram.

Fig. 8: Rv2837c recombinant protein detects tuberculosis patient serum specific antibody ROC curve.

Fig. 9: Rv2837c recombinant protein immune mouse specific antibody level analytical results schematic diagram.

Figure 10: Rv2837c recombinant protein immune mouse spleen lymphocyte proliferation analytical results schematic diagram.

Figure 11: Rv2837c recombinant protein immune mouse splenic lymphocyte flow cytomery result figure.

Figure 12: Rv2837c recombinant protein immune mouse splenic lymphocyte levels of cytokine secretion analytical results schematic diagram.

Figure 13: Rv2837c recombinant protein immune mouse lung cell cytokine secretion horizontal analysis result schematic diagram.

Depositary institution's title: China typical culture collection center, depositary institution address: Wuhan, China Wuhan University, preservation date: on November 26th, 2015, Classification And Nomenclature: e. coli bl21/pET-CnpBEscherichiacoliBL21/pET-CnpB.

Embodiment

The structure of Rv2837c prokaryotic expression carrier:

Genome Rv2837c gene order design primer according to Mycobacterium tuberculosis H37Rv strain:

P1:5 '-CGGCATATGGTGACGACGATCGACCCAAG-3 ', NdeI site

P2:5 '-TTTAAGCTTGCCAGCTGCGCGATCTGACG-3 ', HindIII site

By Sani bio tech ltd, Shanghai synthetic primer.With Mycobacterium tuberculosis H37Rv pnca gene group DNA for template PCR amplifications goal gene, reaction parameter: 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 57 DEG C of renaturation 30s, 72 DEG C extend 100s, 35 circulations, and latter 72 DEG C of last circulation extends 5min.1% agarose gel electrophoresis analysis shows to amplify 1023bp object band (see Fig. 1).Gel reclaims test kit and reclaims object fragment, and with NdeI and HindIII double digestion, 1% agarose gel electrophoresis reclaims digestion products.T4DNA ligase enzyme connects same double digestion and the pET28a+ carrier reclaimed, and 16 DEG C are spent the night, and Transformed E .coliDH5 α competent cell, uses kalamycin resistance plate screening.Picking positive colony is inoculated in liquid nutrient medium 37 DEG C of shaking culture and spends the night, and test kit extracts plasmid DNA, and restriction enzymes double zyme cutting qualification positive plasmid carries out sequencing (see Fig. 2).Sequencing shows, successfully build Rv2837c prokaryotic expression carrier, called after pET-CnpB, its complete genome sequence is as shown in SEQIDNO:1.

Rv2837c activated protein purifying and qualification:

Get the recombinant plasmid that sequencing is correct, Transformed E .coliBL21 (DE3), the LB coating kalamycin resistance is dull and stereotyped, and 37 DEG C are cultured to bacterium colony and generate.On picking flat board, single bacterium colony transfers 5mL containing in the LB liquid nutrient medium of kantlex, and 37 DEG C of shaking culture are spent the night, and overnight culture is forwarded to fresh in antibiotic LB liquid nutrient medium, continues 37 DEG C of shaking culture to OD with the ratio of 1:100 ₆₀₀=0.6, add IPTG to final concentration to 1mmol/L, continue 37 DEG C and cultivate 4h.Collect culture, the centrifugal 5min of 6000rpm, collect thalline, 12%SDS-PAGE gel electrophoresis analysis shows, band of expression is about 36kDa (see Fig. 3 wherein M:Marker; 1: induction bacterium; 2: do not induce bacterium).After SDS-PAGE gel electrophoresis, half-dried electrotransfer electrophoresis is used to go on pvdf membrane by albumen, 5%BSA37 DEG C of closed 1h, PBST wash three times, add the anti-His tag antibody of 1:1000, hatch 1h for 37 DEG C, after PBST washing, add HRP and mark anti-37 DEG C of goat anti-mouse igg two and hatch 45min, add Chemoluminescent substrate colour developing, BIO-RAD chemoluminescence fluoroscopic imaging systems is analyzed, and there is specific band at about 36kDa place (see Fig. 4 wherein M:Marker as seen; 1:DH5 α; 2: do not induce bacterium) successful expression Rv2837c albumen, is shown in intestinal bacteria.

For obtaining the target protein of great amount of soluble, transferred by overnight culture 1:100 into the fresh LB of 500mL, 37 DEG C are cultured to OD ₆₀₀=0.6, culture temperature is down to room temperature by water-bath, adds the IPTG that final concentration is 0.05mmoL/L, 16 DEG C of overnight incubation; Collected by centrifugation bacterial precipitation ,-80 DEG C are frozen more than 2 hours; Take out bacterial precipitation, take weight, add the ratio of 20ml lysate A (50mMTris-HCl, 150mM sodium-chlor, 10mM imidazoles, 10% glycerine, proteinase inhibitor) according to the 1g bacterium that wets, resuspended bacterial precipitation, hatches 30min on ice; Bacterial suspension is placed in ice bath with the ultrasonic 10min of 40% cleavage rate (cracking in 5 seconds, 10 seconds intervals); By ultrasonic degradation thing in 4 DEG C of 12000rpm high speed centrifugations, leave and take supernatant; Get 2mLNi ion affinity chromatography column packing, balance, be placed in appropriate containers, add supernatant with the lysate of 5 times of volumes, 4 DEG C slowly vibrate in conjunction with 2 hours; Mixture is transferred to chromatography column, use the washing lotion B (50mMTris-HCl of 20 times of column volumes respectively, 150mM sodium-chlor, 20mM imidazoles), washing lotion C (50mMTris-HCl, 150mM sodium-chlor, 50mM imidazoles) and washing lotion D (50mMTris-HCl, 150mM sodium-chlor, 75mM imidazoles) clean Ni post; Finally add elutriant E ((50mMTris-HCl, 150mM sodium-chlor, 300mM imidazoles), the effluent liquid after Fractional Collections wash-out; Sample from Fractional Collections liquid, 12%SDS-PAGE electrophoresis detection purifying protein; Collect all collection liquid containing target protein, proceed to dialysis tubing and put into 500mLPBS, 4 DEG C are stirred dialysis 30min, proceed in the 500mLPBS containing 10% glycerine precooling the 30min that again dialyses ,-80 DEG C of preservations after packing.Ni ion affinity chromatography purifying is adopted to obtain the soluble recombinant protein (see Fig. 5) of purity >95%.Purifying protein BCA standard measure, yield is 17.4mg/L culture.

Get purifying protein, form following reaction system: 50mMTris-HCl (pH7.5), 1mMMnCl2,10mMNaCl, 1mMc-di-AMP, 3 μMs of Rv2837c, totally 10 μ L.Hatch 1h for 37 DEG C, add 1 μ L0.5MEDTA termination reaction, add 40 μ L deionized waters, after mixing, add isopyknic HPLC level methyl alcohol.Preserve standby inspection for 4 DEG C.Get 20 μ L samples, be injected into C-18 chromatographic column (250 × 4.6mm, Vydac) and carry out reverse phase HPLC chromatogram HPLC and analyze.Flow visualizing is: bufferA (100mMKH ₂pO ₄, 4mM 4-butyl ammonium hydrogen sulfate, pH5.9) and bufferB (75%bufferA, 25% methyl alcohol).Separable programming: time (minute) and moving phase (per-cent of bufferB) are respectively: 0.0,0; 2.5,0; 5.0,30; 10.0,60; 14.0,100; 21.0,100; 22.0,50; 23.0,0.Flow velocity is 0.7mL/min.254nm length ultraviolet light detects product and generates.HPLC detected result shows, c-di-AMP can be degraded (see Fig. 6) by Rv2837c.

Rv2837c Protein Detection infection animal and tuberculosis patient serum:

Get purifying protein 5 μ g/mL to wrap by 96 hole elisa plates, after PBS washing, add 5%BSA-PBS room temperature and close 1h, after infected mouse sera, tuberculosis patient and consumptive's dilution, add μ L/ hole, each hole 100 respectively, hatch 1h for 37 DEG C, add HRP subsequently and mark anti-37 DEG C of goat anti-mouse igg two and hatch 45min, add substrate (TMB) nitrite ion 100 μ L/ hole, after colour developing, add 50 μ L2MH ₂sO ₄stop buffer, measures OD ₄₅₀value, and draw corresponding chart.

Detected result shows, after mouse infection 2w, specific antibody (P<0.05) can be detected in serum, with the prolongation of infection time, serum antibody titer is in significantly increasing trend, after transferring chronic infection to (12w), Rv2837c specific antibody titres is the highest, and higher than Ag85B antibody horizontal (see Fig. 7).With purifying protein bag by elisa plate, detect tuberculosis patient and consumptive's serum, according to OD ₄₅₀value draws ROC curve, and area under curve is 0.65, shows to have diagnostic (see Fig. 8).

Rv2837c protein immunization response level detection:

1, Rv2837c protein immunization humoral immunization measures

The result that ELISA method detects specific antibody in immune serum shows, Rv2837c immunity just can detect antibody after two weeks, after booster immunization, antibody horizontal raises (P<0.01) gradually, and antibody horizontal and strong immunogen Ag85B are quite (see Fig. 9).

2, Rv2837c protein immunization mouse cell immunoassay

(1) spleen lymphocyte proliferation situation detects

After mouse immune completes 4 weeks, get the aseptic separation splenic lymphocyte of immune mouse, with the RPMI1640 complete culture solution containing 10%FCS, the lymphocyte concentration of separation is adjusted to 1 × 10 respectively ⁶individual/mL, add in (100 μ l/ hole) in 96 orifice plates, experimental group add respectively 100 μ LRPMI1640 complete culture solutions dilution PPD (25mg/L) and recombinant protein (25mg/L), negative control group adds 100 μ L nutrient solutions, the blank control wells not adding splenic lymphocyte is set simultaneously, all establishes 3 multiple holes for all groups.5%CO ₂incubator 37 DEG C adds the MTS (5mg/mL) in 20 μ L/ holes after cultivating 72h, continues to cultivate 4h, adds 10%SDS25 μ L/ hole, measures A490 value.Result proliferation index SI represents: SI=A490 (experimental group-blank)/A490 (negative control-blank).Detected result shows, and recombinant protein immunity significantly can promote spleen lymphocyte proliferation (P<0.05 is shown in Figure 10).

(2) splenic lymphocyte CD4 ⁺/ CD8 ⁺cells ratio detects:

After mouse immune completes 4 weeks, get the aseptic separation splenic lymphocyte of immune mouse, add 5mL erythrocyte cracked liquid effect 5min, add 5mL not containing the RPMI1640 nutrient solution termination reaction of serum, centrifugal 10min, the PBS washed cell of 1500rpm 3 times (5mL/ pipe).Cell precipitation is resuspended with the RPMI1640 nutrient solution not containing serum, FITC: CD4/RPE: CD8 two mark lucifuge dyeing 30min.The same washed cell 3 times, adds cell stationary liquid 0.5mL/ and manages to be checked.Flow cytomery cell sample, result shows: protein immunization group mouse, CD4 ⁺/ CD8 ⁺cells ratio in increasing trend, and with the CD8 be even more important to Intracellular Infection ⁺cell number increases to master, and this removes the entozoic tubercule bacillus of born of the same parents favourable (see Figure 11) to immunity of organism.

(3) immune mouse splenic lymphocyte secrete cytokines detects

Mouse spleen lymphocyte suspension concentration is adjusted to 2 × 10 ⁶/ ml, joins in 96 orifice plates by 100 μ L/ holes respectively, and often group establishes 3 multiple holes, 2 positive controls and 2 negative controls, establishes the blank control wells and reagent controls hole that do not add cell simultaneously; Experimental port add respectively 100 μ LRPMI1640 complete culture solutions dilution PPD (25mg/L) and recombinant protein (25mg/L), negative control group adds 100 μ L nutrient solutions.Put 37 DEG C of 5%CO ₂hatch 72h, collecting cell culture supernatant, ELISA method detects the concentration of cytokine in supernatant, and result shows, recombinant protein can induce splenic lymphocyte Th1 cytokines IFN-γ and IL-2 to discharge to be increased, and does not improve Th2 cytokines IL-10 secretion level (see Figure 12).

(4) immune mouse lungs secrete cytokines detects

After mouse immune completes 4 weeks, put to death mouse, get lung tissue segment, grind after adding liquid nitrogen freezing, Trizol method extracts total serum IgE, and ultraviolet spectrophotometer is quantitative.Getting 500ngRNA reverse transcription is cDNA, real-time quantitative PCR detects cytokine transcriptional level, result shows, recombinant protein immunity can inducing mouse secretion of gamma-IFN and IL-10 cytokine all increase, wherein IFN-γ secretion level is significantly higher than Ag85B immunity (P<0.05, see Figure 13), show that recombinant protein immunity can induce lung's lymphocyte release cells factor to increase, contribute to the immunity of body resisting tuberculosis infection.

Claims (2)

1. a method for Recombinant protein expression mycobacterium tuberculosis Rv2837c activated protein, is characterized in that: the genome Rv2837c gene order design primer according to Mycobacterium tuberculosis H37Rv strain:

P1:5 '-CGGCATATGGTGACGACGATCGACCCAAG-3 ', NdeI site

P2:5 '-TTTAAGCTTGCCAGCTGCGCGATCTGACG-3 ', HindIII site

With pcr amplification c-di-AMP lytic enzyme Rv2837c gene from Mycobacterium tuberculosis H37Rv genome, reaction parameter: 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 57 DEG C of renaturation 30s, 72 DEG C extend 100s, 35 circulations, and latter 72 DEG C of last circulation extends 5min, the 1023bp object fragment that pcr amplification goes out, with NdeI and HindIII double digestion, T4DNA ligase enzyme connects same double digestion and the pET28a+ carrier reclaimed, Transformed E .coliDH5 α competent cell, use kalamycin resistance plate screening, picking positive colony is inoculated in liquid nutrient medium 37 DEG C of shaking culture and spends the night, test kit extracts plasmid DNA, double digestion qualification positive plasmid carries out sequencing, sequencing shows, success builds Rv2837c prokaryotic expression carrier, called after pET-CnpB, its complete genome sequence is as shown in SEQIDNO:1, pET-CnpB Transformed E .coliBL21, IPTG induces destination gene expression, anti-His tag monoclonal antibody Western-blot method is used to identify target protein, this expression strain culture presevation number is: CCTCCNO:M2015703, the mode inducible protein of 0.05mmoL/L lower concentration IPTG and 16 DEG C Low-temperature culture is adopted to express, Ni ion affinity chromatography method purifying is adopted to obtain target protein that is solvable, high yield, detecting through HPLC of purifying protein, have the activity of degraded c-di-AMP, its amino acid complete genome sequence is as shown in SEQIDNO:2.

2. by this restructuring mycobacterium tuberculosis Rv2837c albumen described in claim 1 for diagnosing and preventing the application in preparation lungy.