CN104292315A - Preparation for Rv2645 protein, and application thereof on aspects of tuberculosis diagnosis and reorganized BCG vaccines - Google Patents

Abstract

The invention discloses preparation for Rv2645 protein, and the application thereof on aspects of tuberculosis diagnosis and recombination BCG vaccines. According to the preparation for Rv2645 protein and the application thereof, the Rv2645 protein in a tuberculosis gene RD10-14 zone is found to have the ability of inducing high-level gamma interferon, and when used for the diagnosis of the serum antibodies, especially IgG4 subtype antibodies, of a tuberculosis patient, has prominent statistics difference compared with the serum antibodies of healthy people; as the dominant antigen of the mycobacterium tuberculosis, the Rv2645 protein has the function of inducing high-level cellular immunity and humoral immunity, and can be used as a novel diagnostic reagent for tuberculosis; the Rv2645 protein can further be used for novel recombination BCG vaccines or protein vaccines for tuberculosis, the constructed recombination BCG capable of expressing the Rv2645 protein has the function of antituberculous immunity protection. According to the preparation for the Rv2645 protein and the application thereof provided by the invention, the Rv2645 protein is proved to have potential application value when used for tuberculosis diagnosis and reorganized BCG vaccines, so that the preparation and the application thereof has significant economic benefits.

Description

A kind of preparation of Rv2645 albumen and the application in Diagnosis of Tuberculosis and recombinant BCG vaccine thereof

Technical field

The invention belongs to molecular microbiology and immunology of infection field, relate to a kind of preparation of Rv2645 albumen and the application in Diagnosis of Tuberculosis and recombinant BCG vaccine thereof.

Background technology

The tuberculosis (Tuberculosis, TB) that mycobacterium tuberculosis (Mycobacterium tuberculosis, M.tb) causes be a kind of popular wide, endanger large transmissible disease, with acquired immune deficiency syndrome (AIDS), malaria be called the large transmissible disease in the world three.The whole world has the people of 1/3rd to infect tuberculosis, and dying from tuberculosis patient every year has more than 170 ten thousand people, for control TB exhausts a large amount of human and material resources.

For a long time, diagnosis lungy depends on Tuberculin skin experiment, but this method can not be used for the Healthy People distinguishing tuberculosis patient and inoculated Vaccinum Calmette-Guerini BCG.Although Sputum culturing is the gold standard of Diagnosis of Tuberculosis, its culture cycle is long, at least need a week to obtain result, and this experiment must be carried out in strict biocontainment laboratory.Although the acid-fast stain of Sputum smears is simple, its sensitivity low (about 34% recall rate).Uniquely get permission for preventing tuberculosis and the bacille Calmette-Guerin vaccine (BCG, bacillus Calmette-Gue ' rin) prepared of the Mycobacterium bovis that widely used vaccine is attenuation.Practice for many years shows, BCG only has certain protective role to children, widely different to grownup's protected effect, and from 0% ~ 80% not etc., the effect of this traditional vaccine is subject to severe challenge to its prophylactic effect.BCG is that the method beginning through cultivation from the sixties in 20th century carries out going down to posterity and preserving.Repeatedly go down to posterity and some important immune protective antigen genes of BCG are changed, the phthisical BCG bacterial strain of prevention good for the year of issue only has not existed.(the Behr MA such as Behr, Wilson MA, Anderson P, et al.Comparative genomic of BCG vaccines by whole genome DNA microarray [J] .Sciernce, 1996,284 (541): 1520-1523) with biochip technology find BCG comparatively M.tb lacked RD1-RD16 (regions of difference, RD) these 16 gene regions.Many research reports display in recent years, the albumen exactly expressed by these lost regions has very strong immunogenicity.For example, the loss gene ESAT-6 widely reported in RD1 district gene, albumen expressed by this gene has been proved has very strong immunogenicity.Follow-up research confirms; turn technology by electricity and ESAT-6 gene insertion BCG postgenome can be strengthened immanoprotection action; this research has been published in Nature (Pym AS; Brodin P; Mailessi L, et al.Recombinant BCG exporting ESAT-6confers enhanced protection against tuberculosis.Nature, 2003; 5,9 (5): 533-9.) on.But this research is only limitted to RD1 district, also may there is the dominant antigen being similar to ESAT-6 or being more better than ESAT-6 in other RD districts.But because RD1-16 gene number is numerous, make this research comparatively difficult.

Summary of the invention

The first object of the present invention is to screen and prepares the dominant antigen albumen in tubercule bacillus RD10-14 district.

The second object of the present invention is the using value of advantage object antigen Rv2645 albumen in diagnosis of tuberculosis in the RD13 district that Evaluating and screening goes out.By ELISA antibody sandwich, detect Healthy People and the difference of tuberculosis patient on Rv2645 antigen-specific antibodies IgG, IgM, IgG2, IgG4.Filter out Rv2645 further for most specific antibody subtype during tuberculosis clinical diagnosis.

The third object of the present invention be evaluation advantage object antigen Rv2645 tuberculosis vaccine research in potentiality and using value, especially for the tuberculosis protectiveness effect of recombinant BCG.

For achieving the above object, the present invention is achieved through the following technical solutions:

Research contents of the present invention adopts classical molecular biosciences clone technology, from GenBank Zhong Cha RD district (RD10-14) gene order, design primer and from M.tb bacterial strain H37Rv (ATCC 25618) genome PCR obtain RD district PCR primer, PCR primer is building up on the prokaryotic expression carrier pET28a (Invitrogen) of His label, to recombinate successful Plastid transformation protein expression engineering bacteria intestinal bacteria-BL21 (Invitrogen), IPTG (isopropylthiogalactoside) is utilized to induce and Ni-NTA (agarose-nickel bead) affinity purification acquisition expression and purification albumen, and the concentration of each purifying protein is measured by the Bradford method of BSA (bovine serum albumin) production standard curve.

First by the H37Rv bacterial strain tail vein injection BABL/c mouse of deactivation, pre-immune mouse 7 days, immunity terminates to get spleen cell after 7 days, the H37Rv bacterial strain RD albumen of purifying is stimulated the Mouse spleen cells through immunity respectively, utilizes ELISPOT (enzyme-linked immunospot assay) to detect its INF-γ emission levels.In addition, the IFN-γ that RD albumen stimulates tuberculosis patient PBMC (peripheral blood mononuclear cell) to discharge detects horizontally through ELISA (enzyme linked immunosorbent assay).The detected result of comprehensive mouse and people, assessment stimulates INF-γ release to produce the RD albumen of higher level.Classical ELISA method is utilized to detect its antibody horizontal in tuberculosis patient by 96 orifice plates this RD albumen bag.Above-mentioned experimental result finds that Rv2645 is not only the highest in IFN-γ emission levels, and the significant difference shown in 24 tuberculosis patients and 24 Healthy Human Serum IgG and IgM antibody detect is maximum.In order to confirm the purposes of this Rv2645 albumen in Diagnosis of Tuberculosis and value further, a large amount of preparation purifying Rv2645 albumen, detection sample size is increased to 104 tuberculosis patients and 104 routine Healthy Peoples, the Antibody types detected comprises IgG, IgM, IgG2, IgG4, add simultaneously have been reported can the fusion rotein of diagnostic CFP-10 and ESAT6 and CE albumen as positive control.Experimental data is analyzed through SPASS software, finds that CE albumen is little all compared with Rv2645 of the significant difference of these three indexs of IgG, IgM and IgG4 in tuberculosis patient and Healthy People.And the tuberculosis patient of Rv2645 and Healthy Human Serum IgG2 detected result also have Variant statistical meaning, and CE albumen is without the difference of IgG2.Thus prove that the using value of Rv2645 at serology Diagnosis of Tuberculosis is higher than the CE albumen had been reported.

A series of research of the present invention proves that Rv2645 has stronger immunogenicity, and this research builds the recombinant BCG Vaccinum Calmette-Guerini of recombinant expressed Rv2645 albumen further.By gene constructed for Rv2645 to shuttle plasmid pMV361 (Biovector Science Lab, Inc) on, turning technology by electricity proceeds in BCG (ATCC 35734) competence by recombinant plasmid pMV361-Rv2645, verifies that electricity changes into merit by that resistance screening of card, PCR, Western-blot (immunoblot experiment), Southern blot (hybridization of the gene probe marking).This gene will be integrated in BCG karyomit(e) with pMV361, in BCG, obtain stable expression and long-time in can not go down to posterity along with the BCG after recombinating and lose.By recombinant BCG and rBCG-Rv2645, BCG and PBS (phosphate buffered saline buffer blank group) immune BALB/c mouse respectively, BCG as negative blank group as positive controls, PBS, then carries out tubercule bacillus reference culture H37Rv (ATCC 25618) aerosol and attacks poison infection.Attack that poison terminates that rear dissection mouse gets lungs, spleen does enumeration, acid-fast stain and HE dyeing observes pathology damage degree.Net result shows, and rBCG-Rv2645 enumeration is lower than BCG group, and BCG is again lower than PBS blank group, and each difference has statistical significance.The interpretation of result of comprehensive acid-fast stain, HE dyeing finds that the immanoprotection action of rBCG-Rv2645 is really higher than BCG group, confirms that Rv2645 albumen has higher using value in prevention tuberculosis.

A kind of mycobacterium tuberculosis dominant antigen albumen, be RD13 district Rv2645 albumen, this molecular weight of albumen size is about 15.6KD, and its aminoacid sequence is as shown in SEQ ID NO.1; The long 432bp of encoding gene segment of this albumen, its nucleotide sequence is as shown in SEQ ID NO.2.Confirm that Rv2645 albumen energy significant stimulation mouse and people produce the specific cell of high-caliber tuberculosis and humoral immunization through screening experiment.So far, there is no the research report of the preparation of its albumen and function.

As the preparation method of the Rv2645 albumen of mycobacterium tuberculosis dominant antigen, comprise the steps:

(1) with the genome of mycobacterium tuberculosis for template, use following primer amplification Rv2645 gene;

Upstream primer: 5'-ATTGGATCCATGACCACCACGC-3',

Downstream primer: 5'-AATAAGCTTCCGCCGGTGTTCGC-3';

(2) Rv2645 gene is building up on prokaryotic expression carrier pET28a by restriction enzyme BamH I and Hind III obtains recombinant plasmid pET28a-Rv2645;

(3) again pET28a-Rv2645 is transformed in e. coli bl21, obtains Rv2645 albumen by IPTG induction and Ni-NTA affinity purification.

Rv2645 albumen as mycobacterium tuberculosis dominant antigen can be used for diagnosis lungy.

Rv2645 albumen can, for the preparation of the Novel diagnosis reagent of tuberculosis T-SPOT, may be used for distinguishing tuberculosis infection or BCG inoculation.The Rv2645 albumen prepared as stated above is removed intracellular toxin and quantitatively after can be used as the Novel diagnosis reagent of T-SPOT, stimulate patient P BMC to utilize ELISPOT method to detect the emission levels of INF-γ with it.Its ESAT6-CFP10 protein immunogenic reported comparatively is at present strong, stimulates INF-γ higher level.

Rv2645 albumen also can for the preparation of the Novel diagnosis reagent of the special IgG4 antibody of tuberculosis, and when Rv2645 is for detecting tuberculosis specific antibody IgG4, its specific degree and sensitivity are up to more than 80%.Rv2645 is that BCG lacks albumen, can be used for distinguishing tuberculosis infection or BCG inoculation when its serodiagnosis for tuberculosis patient, comprises the steps:

(1) will wrap by 96 orifice plates after Rv2645 protein quantification, 100 μ g/mL, 100 μ L/ holes.4 DEG C, wrap and spent the night or 37 DEG C, 1h.

The BSA of (2) 2% closes: wrap every hole after being washed twice by plate PBS and add the BSA of 2%, every hole 200 μ L, 37 DEG C, 1h.Close PBS after terminating and wash 2 times, pat dry, elisa plate 4 DEG C preservation is stand-by.

(3) sample incubation: after serum sample PBS carries out 200 times of dilutions, every hole adds 100 μ L, 37 DEG C, 1h.Wash plate 6 times with PBST after end, pat dry.

(4) antibody is hatched: the human IgG 4 antibody by specification adding band HRP mark dilutes.Every hole 100 μ L, 37 DEG C, 1h.Wash plate 6 times with PBST after end, pat dry.

(5) substrate colour developing is added: by tmb substrate 100 μ L, 37 DEG C, 1-5min; Wait for and become blue.

(6) stop: the H adding stop buffer 2M after color stability ₂sO ₄solution 50 μ L.

(7) detect: after termination turns yellow, elisa plate is placed in microplate reader and reads OD value, determined wavelength is 450nm.Result carries out statistical study, distinguishes tuberculosis and non-tuberculosis people according to cut off value.

Rv2645 albumen can be used for the novel recombinant BCG vaccine of tuberculosis or protein vaccine as the dominant antigen of mycobacterium tuberculosis.

A kind of tuberculosis protein vaccine is the Rv2645 albumen prepared by aforesaid method.

The novel recombinant BCG vaccine of a kind of tuberculosis, for expressing the recombinant BCG of mycobacterium tuberculosis dominant antigen Rv2645 albumen, it is preferably by the method preparation comprising following steps:

(1) with the genome of mycobacterium tuberculosis for template, use following primer amplification Rv2645 gene;

Upstream primer: 5'-ATTGAATTCATGACCACCACGC-3',

Downstream primer: 5'-AATAAGCTTCCGCCGGTGTTCGC-3';

(2) Rv2645 gene is building up in shuttle plasmid pMV361 by restriction enzyme EcoR I and Hind III obtains recombinant plasmid pMV361-Rv2645;

(3) again by pMV361-Rv2645 electricity forward in BCG the recombinant BCG obtaining expressing Rv2645 albumen to, be proved to be successful after thus as improve novel recombinant BCG vaccine.

Advantage of the present invention and effect:

(1) Rv2645 utilizes prokaryotic expression expression vector to express, and Ni-NTA affinity chromatography carries out purifying, and method is simple, and output is high, and purity is higher.Rv2645 albumen comparatively other part RD protein immunogenic is strong, stimulates INF-γ level the highest.

(2) the BCG vaccine of recombinant expressed Rv2645 albumen confirms in mouse tubercule bacillus aerosol challenge viral dosage, and its immanoprotection action than BCG strengthens, and its tuberculosis provide protection obtains confirmation.

(3) when being integrated on BCG by Rv2645, make use of comparatively advanced electricity and turn technology and shuttle plasmid pMV361, this plasmid can, by Rv2645 gene integration on BCG karyomit(e), make it be different from ordinary plasmids, can not to go down to posterity interior loss in short-term.Be beneficial to the long-term stability of this gene in BCG to express.

(4) simultaneously the patients serum of large sample and the experiment of Healthy Human Serum corresponding antibodies confirm that Rv2645 has higher using value in tuberculosis serum diagnosis.When particularly Rv2645 is used for detection specificity IgG antibody 4, its specific degree and sensitivity are up to more than 80%, and simultaneously itself and clinical T-Spot diagnose the rate that conforms to reach 72% rate that conforms to (70%) when diagnosing for tuberculosis patient IgG antibody 4 a little more than T-Spot autoantigen protein composition (CFP-10 and ESAT-6).In addition, Rv2645 is that BCG lacks albumen, can distinguish come when its serodiagnosis for tuberculosis patient with the Healthy People of inoculated BCG.

Accompanying drawing explanation

Fig. 1 is the concrete scheme schematic diagram of the pre-immune mouse of deactivation H37Rv.

Its corresponding IFN-γ level produced is detected by ELISPOT and ELISA method after Tu2Shi RD10-14 district albumen stimulates pre-immune mouse spleen cell and human PBMC.A figure detects its INF-γ emission levels by ELISPOT method after each RD purifying protein stimulates the spleen cell 48h of the pre-immune mouse of deactivation H37Rv, and wherein shown in arrow, place is the RD albumen of emission levels front three, is respectively Rv2645, Rv1768 and Rv1773.PHA (phaseolus vulgaris agglutinin) is lymphocyte positive stimulus thing appended by test kit, and Ag85B is the albumen of the stimulation INF-γ high level release had been reported, in this as the free positive control of this experiment; In figure, gene code name is for writing a Chinese character in simplified form.B figure is the real figure of part spot in A result.C figure is that after Rv2645, Rv1768 and Rv1773 albumen stimulates tuberculosis patient and Healthy People PBMC, ELISA method detects its IFN-γ emission levels, ConA (canavaline) is the subsidiary lymphocyte positive stimulus thing of test kit, and wherein Rv2645 group has Variant statistical meaning.

Fig. 3 is that ELISA method detects corresponding antibodies total IgG in 24 routine patients TB and 24 routine Healthy Peoples of Rv2645, Rv1768, Rv1773 and IgM level.A figure is the detection of IgG antibody, and wherein Rv2645 and Rv1768 is p=0.0236 and p=0.0292, has significant difference p<0.05, and the p=0.229 of Rv1773.B figure is the detection of IgM antibody, and wherein Rv2645 and Rv1768 is p=0.0229 and p=0.0394, has significant difference p<0.05, and the p=0.4789 of Rv1773.

Fig. 4 is Rv2645 for the detection of 104 routine tuberculosis patients and 104 routine Healthy Human Serum IgG, IgM, IgG2 and IgG4.Wherein A is the scatter diagram of the ELSIA detection of Rv2645, and B is its corresponding ROC curve.Each antibody p<0.0001 that Rv2645 detects, the AUC value of its ROC curve is respectively 0.7865,0.7760,0.6781 and 0.8205.

Fig. 5 is CE for the detection of 104 routine tuberculosis patients and 104 routine Healthy Human Serum IgG, IgM, IgG2 and IgG4.Wherein A is the scatter diagram of the ELSIA detection of CE, and B is its corresponding ROC curve.The p value that CE detects is <0.0001, <0.001, >0.05, <0.001 respectively.The AUC value of its ROC curve is respectively 0.6936,0.6516,0.5419 and 0.6475.

Fig. 6 is that the rabbit polyclonal antibody of preparation Rv2645 detects for 2 routine non-tuberculosis people and 2 routine tuberculosis patient lung tissue sections, and makes positive control (visual field is 100 times of amplifications) with CE.Wherein to be CE rabbit polyclonal antibody and Rv2645 rabbit polyclonal antibody for the lung tissue of 1-2 non-tuberculosis people cut into slices A-D detects as negative control.E-F is the detection that CE rabbit polyclonal antibody and Rv2645 rabbit polyclonal antibody are cut into slices for 1-2 tuberculosis patient lung tissue.Wherein place shown in arrow is positive brown region tubercle (should be brown color, be darker regions in non-coloured picture).

Fig. 7 is the typical mono-clonal bacterium colony figure blocked after BCG electricity turns insertion pMV361-Rv2645 recombinant shuttle plasmid on that resistance plate screen plate.A is the colonial morphology figure of rBCG-Rv2645 after electricity turns, B is the figure aspect graph that goes down to posterity after rBCG-Rv2645 electricity turns, and C is partial enlargement aspect graph.

Fig. 8 is PCR and the WB qualification figure of rBCG-Rv2645.A is PCR proof diagram, and wherein 1,3 swimming lanes are respectively negative and positive contrast, and 2 is experimental group, and the clip size of Rv2645 is 432bp.B is Western-blot proof diagram, and wherein 2,3 swimming lanes are yin and yang attribute contrast, and 1 is experimental group.Result shows, and rBCG-Rv2645 has positive band.C-E utilizes biotin labeled Rv2645 gene probe to carry out Southern blot checking to the rBCG-Rv2645 genome extracted.

Fig. 9 is that recombiant vaccine rBCG-Rv2645 immune mouse attacks the lungs (A) after poison and spleen (B) enumeration.Result shows, and rBCG-Rv2645 group and BCG and PBS have significant difference.

Figure 10 is the lungs HE colored graph after attacking poison.Wherein PBS group, alveolar structure is imperfect, and the tubercle of place shown in arrow is larger; BCG comparatively PBS group is greatly improved, and rBCG-Rv2645 is the most complete, and tubercle is rare.Wherein ABC figure is 40 times of amplifications, and DEF figure is 100 times of mirror figure below amplified.

Figure 11 is the spleen HE colored graph after attacking poison.Wherein PBS group, the lymph tubercle edge blurry inflammation of place shown in arrow is heavier, and BCG comparatively PBS group is greatly improved, rBCG-Rv2645 group lymph tubercle neat in edge boundary clear.Wherein ABC figure is 40 times of amplifications, and DEF figure is 100 times of mirror figure below amplified.

Embodiment

Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.

The structure of each prokaryotic expression carrier pET28a-RDs in embodiment 1RD10-14 district

The design of 1.1 each RD gene primers, builds required concrete primer in table 1:

Table 1. each RD gene primer sequence

Wherein, setting-out part be restriction enzyme site.

The amplification of 1.2 each RD genes and recovery

With M.tb bacterial strain H37Rv (ATCC 25618) genome for template, use the primer PCR in table to increase each RD gene, response procedures is different according to each primer annealing temperature.For the Rv2645 gene that increases, PCR response procedures is: 95 DEG C of 3min; 94 DEG C of 20s, 58 DEG C of 20s, 72 DEG C of 20s, 30 circulations; 72 DEG C of 3min; PCR primer is detected by the agarose gel electrophoresis of 1%, and gel purification kit (Axygen) carries out reclaiming (according to the operation of reagent kit product specification sheets).

The PCR primer of 1.3 each RD genes and the enzyme of carrier pET28a are cut

For Rv2645 gene, it is as follows that PCR primer enzyme cuts system:

PCR primer 10 μ L, ddH ₂o 32 μ L, 10 × Buffer E 5 μ L, BamH I 1 μ L, Hind III 1 μ L, BSA 1 μ L, total system 50 μ L.

It is as follows that vector plasmid pET28a enzyme cuts system: pET28a 4 μ L, ddH ₂o 20 μ L, 10 × Buffer E 3 μ L, BamH I 1 μ L, Hind III 1 μ L, BSA 1 μ L, total system 30 μ L.

Enzyme is cut system and is reclaimed by Axygen gel purification kit specification sheets after 37 DEG C of water-bath enzymes cut 1-2h.

The connection of 1.4 digestion products

Large fragment of cutting PCR primer endonuclease bamhi and carrier enzyme runs agarose gel electrophoresis after reclaiming, and carries out the connection of next step endonuclease bamhi after judging respective concentration.As follows for its system of Rv2645 gene: Rv2645 enzyme cuts rear fragment amount: vector plasmid pET28a enzyme cuts rear amount ≈ 3:1, both supposing, concentration is suitable, then ligase enzyme (High ligase, Toyobo) system is that example in 15 μ L is as follows: Rv2645 7.5 μ L, pET28a 2.5 μ L, High ligase (containing buffer) 5 μ L, 16 DEG C of water-bath 30min; DH5 α competence is transformed after water-bath connects.

1.5 connect products conversion

Get connection product 5-10 μ L joins in competence DH5 α in Bechtop, mixture ice bath 30min; 42 DEG C of heat shock 90s; Add LB 800 μ L, in temperature control shaking table 37 DEG C, 200rpm, 40min; Centrifugal supernatant of abandoning retains the resuspended bacterium liquid of 100 μ L; Coating card that resistance (50 μ g/mL) LB solid board.

Extraction and the enzyme of 1.6 recombinant plasmids cut qualification

From LB solid culture plate, picking mono-clonal is in containing in LB liquid nutrient medium (kantlex (kana) concentration is 5 μ g/mL) 37 DEG C, 200rpm, 16h; Extract plasmid by the operation of Axygen plasmid extraction kit specification sheets after collection bacterium and do double digestion qualification by following system: plasmid 15 μ L, ddH ₂o 9 μ L, 10 × Buffer E 3 μ L, BamH I 1 μ L, Hind III 1 μ L, BSA 1 μ L, total system 30 μ L.37 DEG C of water-bath enzymes cut 2-4h, and enzyme runs glue qualification after cutting and terminating.The plasmid correct to qualification result send company to check order, determine further, wherein 31 construction of recombinant plasmid are determined successfully, to be respectively Rv1255c, Rv1256c, Rv1257c, Rv1766, Rv1767, Rv1768, Rv1769, Rv1770, Rv1771, Rv1773, Rv2072c, Rv2073c, Rv2075c, Rv2645, Rv2646, Rv2647, Rv2648, Rv2649, Rv2650, Rv2651c, Rv2652c, Rv2653c, Rv2654c, Rv2655c, Rv2658c, Rv2659c, Rv3425, Rv3426, Rv3427c, Rv3428c, Rv3429.

The Expression and purification of the target protein in embodiment 2 recombinant plasmid

The expression of 2.1RD albumen

For Rv2645, get pET28a-Rv2645 recombinant plasmid 1 μ L, add in escherichia coli expression engineering bacteria BL21 competence, ice bath 15min, 42 DEG C of heat shock 90s, ice bath 5min again, adds the LB liquid nutrient medium shaking culture 40min of 800 μ L, gets 100 μ L and is coated with that resistance LB solid board (the same) of card.After cultivation overnight, picking positive colony to forward in 5mL LB liquid nutrient medium (kana concentration is 5 μ g/mL) 37 DEG C to, and 200rpm, 16h do little shaking; Then get the little bacterium liquid shaken of 2mL to turn in 2 × YT (Tryptones yeast extract medium) liquid nutrient medium 250mL and shake greatly, when OD600 place absorbance about 0.6, add inductor IPTG, final concentration is 0.8mM.In temperature control shaking table 25 DEG C, 200rpm, 20h; After inducing culture terminates the 1mL bacterium cracking before getting 1mL collection bacterium and inducing, whether SDS-PAGE (polyacrylamide gel electrophoresis) comparative observation has the protein expression of object size.

The purifying of 2.2RD albumen

If have expression after induction, by the BL21 collection bacterium after shaking greatly, resuspended to 30mL with PBS, with the broken bacterium of high-pressure homogeneous instrument, collect that broken bacterium liquid is centrifugal discards bacterium slag, collect albumen supernatant, in the middle part of albumen supernatant, add Ni-NTA about 200 μ L vibrate in frozen water after in conjunction with 1-2h and collect sepharose 4B, centrifugal condition is 4 DEG C, 2500rpm, 5min.Utilize the imidazoles of different concns gradient such as 10mM, 20mM, 40mM, 80mM, 100mM, 200mM, 250mM to do gradient elution, wash away that be not combined with Ni-NTA or non-specific foreign protein with its combination thus obtain the target protein Rv2645 of purifying.The albumen of each concentration gradient wash-out is done SDS-PAGE qualification, leave and take the good and eluted protein that output is high of purity.Identified by SDS-PAGE after the protein expression and purification in RD10-14 district, the higher albumen of 22 purity, by successful expression and purifying, is respectively Rv1255c, Rv1257c, Rv1768, Rv1771, Rv1773, Rv2072c, Rv2073c, Rv2075c, Rv2645, Rv2646, Rv2647, Rv2649, Rv2650, Rv2651c, Rv2653c, Rv2655c, Rv2658c, Rv2659c, Rv3425, Rv3427c, Rv3428c, Rv3429.

The each RD albumen of embodiment 3 stimulates the ability of IFN-γ release to measure

3.1ELISPOT detects pre-immune mouse IFN-γ emission levels

Each RD albumen Bradford method of expression and purification is carried out protein quantification.The BALB/c mouse (concrete mouse immunization protocol as shown in Figure 1) that pre-for deactivation H37Rv immunity is crossed is put to death and gets spleen, obtain Mouse spleen cells with after the broken red corpuscle of AKT (erythrocyte cracked liquid).

Undertaken by the pre-coated test kit of Mouse IFN-gamma ELISPOT (reaching section is) specification sheets operation.Every hole paving 5 × 10 ⁵individual splenocyte, every hole add stimulator RD albumen 5 μ g, cultivate 48h in cell culture incubator.After experiment terminates, 96 orifice plate Song Yuda sections are that company reads spot number.

3.2ELISA detects the IFN-γ emission levels of human PBMC

Medical rescue center, Wuhan City obtains tuberculosis patient whole blood, is separated PBMC.Undertaken by Human IFN-gamma ELISA kit (pre-coated subtract footwork) (reach section for) specification sheets operation.Every hole paving 5 × 10 ⁵individual PBMC, every hole add in stimulator RD albumen 5 μ g, cell culture incubator cultivates 48h.Wash after lysing cell once, incubate primary antibodie, two anti-, colour developing stops, microplate reader detects in OD450 place.

After RD10-14 district albumen stimulates pre-immune mouse and human PBMC ELISPOT and ELISA method detect its produce IFN-γ level the results are shown in Figure 2.A figure detects its IFN-γ emission levels by ELISPOT method after each RD purifying protein stimulates the Mouse spleen cells 48h of the pre-immunity of deactivation H37Rv, and wherein shown in arrow, place is the RD albumen of emission levels front three, is respectively Rv2645, Rv1768 and Rv1773.B figure is the real figure of part spot in A result.C figure is that after Rv2645, Rv1768 and Rv1773 albumen stimulates tuberculosis patient and Healthy People PBMC, ELISA method detects its IFN-γ emission levels, and wherein Rv2645 has Variant statistical meaning.

The detection of the corresponding antibodies of RD albumen in tuberculosis patient and Healthy People of embodiment 4 high expression level IFN-γ

4.1ELISA method detects the corresponding antibodies of RD albumen in tuberculosis patient and Healthy People of high expression level IFN-γ

Evaluate in embodiment 3 high expression level INF-γ RD albumen Rv1768, Rv1773, Rv2645, these three kinds of RD albumen are wrapped separately and after 96 hole microwell plates, are detected Healthy People 24 example, total IgG, IgM in tuberculosis patient 24 example.Experiment flow is roughly as follows:

(1) 96 orifice plate envelope antigen: RD albumen coating buffer dissolved dilution, its final concentration is 100 μ g/mL, every hole 100 μ L.4 DEG C, wrap and spent the night.

The BSA of (2) 2% closes: wrap every hole after being washed twice by plate PBS and add the BSA of 2%, every hole 200 μ L, 37 DEG C, 1h.Close PBS after terminating and wash 2 times, pat dry micropore, elisa plate 4 DEG C preservation is stand-by.

(3) sample incubation: after serum sample PBS does 200 times of dilutions, every hole adds 100 μ L, 37 DEG C, 1h.Wash plate 6 times with PBST after end, pat dry.

(4) antibody is hatched: IgG, IgM of adding band HRP mark, antibody by specification dilutes.Every hole 100 μ L, 37 DEG C, 1h.Wash plate 6 times with PBST after end, pat dry.

(5) substrate colour developing is added: after tmb substrate A and B being done equal-volume mixing, every hole adds mixed substrates liquid 100 μ L, 37 DEG C, 1-5min; Wait for and become blue.

(6) stop: the H2SO4 solution 50 μ L adding stop buffer 2M after adding substrate color stability.

(7) detect: after termination turns yellow, elisa plate is placed in microplate reader and reads OD value, determined wavelength is 450nm.

What ELISA method detected corresponding antibodies total IgG in 24 routine patients TB and 24 routine Healthy Peoples of Rv2645, Rv1768, Rv1773 and IgM level the results are shown in Figure 3.A figure is the detection of IgG antibody, and wherein Rv2645 and Rv1768 is p=0.0236 and p=0.0292, has significant difference p<0.05, and the p=0.229 of Rv1773.B figure is the detection of IgM antibody, and wherein Rv2645 and Rv1768 is p=0.0229 and p=0.0394, has significant difference p<0.05, and the p=0.4789 of Rv1773.

The ELISA method of 4.2Rv2645 is used for the detection of tuberculosis patient serum and Healthy Human Serum corresponding antibodies in large sample (tuberculosis patient and each 104 examples of Healthy People)

Detection method is the same, adds CE albumen in contrast, and another two Testing index IgG2 and the IgG4 of many detections.Go out each group of P value according to result SPSS computed in software, evaluate at cellular immunization and the highest RD albumen of humoral immunity level, the results are shown in Figure 4-5 and table 2,3.Find in this screening experiment that this ability of Rv2645 albumen deriving from RD13 district is the highest.Organizing and the cut off value of Healthy People group, specific degree and sensitivity by calculating patient, finding that Rv2645 albumen is used for serodiagnosis lungy and has higher value.

Fig. 4 is Rv2645 for the detection of 104 routine tuberculosis patients and 104 routine Healthy Human Serum IgG, IgM, IgG2 and IgG4.Wherein A is the scatter diagram of the ELSIA detection of Rv2645, and B is its corresponding ROC curve.Each antibody p<0.0001 that Rv2645 detects, the AUC value of its ROC curve is respectively 0.7865,0.7760,0.6781 and 0.8205.

Fig. 5 is CE for the detection of 104 routine tuberculosis patients and 104 routine Healthy Human Serum IgG, IgM, IgG2 and IgG4.Wherein A is the scatter diagram of the ELSIA detection of CE, and B is its corresponding ROC curve.The p value that CE detects is <0.0001, <0.001, >0.05, <0.001 respectively.The AUC value of its ROC curve is respectively 0.6936,0.6516,0.5419 and 0.6475.

Table 2. compares Rv2645 and CE albumen for cut off value, specific degree and the Sensitirity va1ue in tuberculosis serological antibody diagnosis

"+" represents the number of cases that two diagnostic methods are judged as the tuberculosis positive, and "-" represents the number of cases that each two diagnostic methods are judged as tuberculosis feminine gender.IgG, IgM, IgG2, IgG4 sensitivity of Rv2645 is 74.04%, 77.88%, 48.08% and 80.77%; Specific degree is 72.12%, 71.15%, 86.54% and 82.69%; The accuracy rate of its diagnosis is 73.08%, 74.52%, 63.71% and 81.73%.IgG, IgM, IgG4 sensitivity of CE is 50.96%, 34.62% and 48.08%; Specific degree is 88.46%, 91.36% and 72.12%; The accuracy rate of its diagnosis is 69.71%, 62.98% and 60.09%.The IgG2 of CE is no difference of science of statistics in the differential diagnosis of tuberculosis and Healthy People, does not therefore include calculating in.

Table 3.Rv2645, CE are used for tuberculosis patient serology antibody diagnosis and conform to the result of T-Spot clinically the comparison of rate

"+" represents the number of cases that two diagnostic methods are judged as the tuberculosis positive, and "-" represents the number of cases that each two diagnostic methods are judged as tuberculosis feminine gender.Contrast the rate that conforms to that clinical T-spot result can compare two kinds of diagnostic methods, the rate that conforms to of Rv2645 IgG, IgM, IgG2 and IgG4 and T-spot diagnosis is 59.3%, 56%, 40% and 72%.CE IgG, IgM, IgG2, IgG4 and clinical T-Spot diagnose the rate that conforms to be respectively 35.48%, 40% and 70%.Find that Rv2645 albumen all has significant difference for serodiagnosis lungy is the same with reference protein CE by comparing.It should be noted that the IgG4 sensitivity of Rv2645 is 80.77%, specific degree reaches 82.69%, ROC area under curve AUC is 0.8205, find that the IgG4 of Rv2645 and CE is used for the conform to rate of clinical diagnosis higher than other indexs simultaneously, secondly the rate 72% that conforms to of Rv2645 is a little more than 70% of CE albumen, can be used as the potential index of Diagnosis of Tuberculosis.Wherein CE albumen detects not statistically significant at the antibody horizontal of IgG2 and does not include calculating in.

The rabbit polyclonal antibody of embodiment 5Rv2645 is used for the application of immunohistochemical methods

5.1 great expression purifying Rv2645 albumen are used for the preparation of new zealand rabbit polyclonal antibody

Concrete steps are as follows:

(1) preparation of antigen: Rv2645 mixes with Freund's incomplete adjuvant adjuvant equal-volume, with the syringe pull emulsification that two is hose connection, treats that Protein adjuvants mixture is that water-in-oil shape can be used.

(2) immunity: multi-point injection, in rabbit backbone both sides 4-6 point, in 100 μ g/ time/week, adds up to 4-5 time.

(3) bioactivity: taking preimmune serum as contrast, is experimental group at the serum of getting for 3,4,5 weeks of immunity.With Rv2645 bag by micropore, immunity before and Post-immunisation serum add micropore as primary antibodie as doubling dilution, the goat anti-rabbit igg of HRP mark resists as two, and ELISA method detects, and when the absorbancy of Post-immunisation serum is 2 times of preimmune serum, corresponding extension rate is and tires.

(4) blood sampling obtains polyclonal antibody, and when reaching ideal value when tiring, after Rabbit Heart being got blood, separation of serum is preserved.The antibody titer obtained is through surveying up to 1:102400.

5.2Rv2645 the application of polyclonal antibody clinical immunization group

The Rv2645 rabbit polyclonal antibody of acquisition is done immunohistochemical methods (biotech firm of Google) together with obtain clinically 2 routine non-tuberculosis people and 2 routine tuberculosis patient lung tissue (Haihe River Hospital, Tianjin City) tissue slicies.Using the rabbit polyclonal antibody of CE albumen as positive control, find that Rv2645 polyclonal antibody effect is suitable with CE group, in immunohistochemical methods section, the tubercle at brown color place can be revealed (in figure arrow indication dark parts).ImmunohistochemistryResults Results for tuberculosis patient lung tissue section is shown in Fig. 6.Wherein A, C are the detection of CE rabbit polyclonal antibody for two different non-tuberculosis people lung tissue sections, as negative control; B, D are the detection that Rv2645 rabbit polyclonal antibody is cut into slices to two non-tuberculosis people lung tissue, as negative control.E, G are the detection of CE rabbit polyclonal antibody to two different tuberculosis patient lung tissue sections, and F, H are the detection of Rv2645 rabbit polyclonal antibody for two different tuberculosis patient lung tissue sections.Result show, Rv2645 rabbit polyclonal antibody can detect corresponding antigens in tuberculosis patient lung tissue, in non-tuberculosis people then without.

Embodiment 6 improves recombinant BCG: the preparation of rBCG-Rv2645

The structure of 6.1pMV361-Rv2645 shuttle vectors

Specific implementation method is similar to the pET28a-Rv2645 that embodiment 1 builds.Wherein Rv2645 primer according to carrier pMV361 do design as follows:

Upstream primer: 5'-ATT gAATTCaTGACCACCACGC-3',

Downstream primer: 5'-AAT aAGCTTcCGCCGGTGTTCGC-3';

Underscore part is EcoR I and Hind III restriction enzyme site; Build and the same pET28a-Rv2645 of authentication method.The shuttle plasmid of restructuring is identified successfully through double digestion, and after order-checking, sequence is correct.

The preparation of 6.2rBCG-Rv2645

First pMV361-Rv2645 electricity will be gone to BCG, it is as follows that electricity turns scheme:

(1) after the competent preparation of BCG: BCG is inoculated in 7H9 liquid nutrient medium in 100rpm, 37 DEG C be cultured to OD600 value about 0.6 time, wash 2 times with the high glycerine pressed through and resuspended packing preserve.

(2) plasmid competence mixing: get BCG competence one on ice, gets pMV361-Rv2645 and is about after 1-2 μ L to add in competence mixing and is transferred in aseptic electric revolving cup (Bio-Rad), ice bath 30min.

(3) electricity turns: arrange electroporation (Bio-Rad) parameter, it is 2500V that electricity turns parameter, 1000 Ω, 25 μ F.

(4) cultivate: after electricity has turned, competence is joined in the 7H9 substratum of 5mL immediately.37 DEG C, 100rpm overnight incubation.

(5) coated plate: electricity turns the bacterium liquid after cultivating and gets 200 μ L and coat 7H10 and contain in the solid medium of that microbiotic of card (final concentration is 25 μ g/mL).

(6) choose mono-clonal and PCR qualification: picking 7H10 cultured on solid medium about 15 days with Rv2645 primer be verified as positive mono-clonal bacterium colony in 5mL containing the 7H9 liquid nutrient medium of that microbiotic of card (5 μ g/mL) in cultivate 15 days and turn to shake greatly.

(7) get after the positive bacterium liquid of PCR qualification shakes greatly and get 1mL collection bacterium, ultrasonic split bacterium after the rabbit polyclonal antibody of centrifuging and taking supernatant Rv2645 be primary antibodie, resist as two with goat anti-rabbit igg, do Western-blot checking.Be verified as positive to be rBCG-Rv2645 to protect bacterium for subsequent use simultaneously.

(8) rBCG-Rv2645 and BCG genome is extracted, carry out after genome enzyme cuts with Sau3AI enzyme, utilize that the gene probe of Rv2645 biotin labeling (5 ' end mark) 5 '-CCG GCG ATT CAC TAC ACG GAA CCG CCC GTG TTG GGG-3 ' of 289-324 position core conservative region (in the sequence of Rv2645) is primary antibodie, HRP-anti-vitamin H is two to resist, be Southern blot.Wherein BCG group is negative control, if rBCG-Rv2645 enzyme cut after genome can mix out band BCG without; could judge that Rv2645 gene successfully inserts in BCG genome.

In the resistant panel of the bacterium colony after electricity turns, bacterium colony figure as shown in Figure 7; What corresponding PCR checking and western-blot verified the results are shown in Figure 8 (A is PCR proof diagram, and B is western-blot proof diagram, and C-E is that Southern blot verifies series of drawing).Verify that rBCG-Rv2645 recombinates successfully by resistance screening, PCR, Western-blot checking and Sothern blot.

The detection of tuberculosis provide protection after embodiment 7 recombinant bacillus Calmette-Guerin vaccine rBCG-Rv22645 immune mouse

The pre-immunity of 7.1BALB/c mouse

Mouse often organizes 6, and viable bacteria immunity once.Its respective packets and immunizing dose as shown in table 4:

The grouping of table 4.BALB/c mouse and immunization route

7.2 attack poison and detection

Immunity carries out H37Rv aerosol infection by the dosage of 100CFU after spending 2 weeks.Observe 30 days, change such as record Mouse Weight, mood etc.Finally put to death and dissect mouse, get mouse lungs, spleen tissue, the enumeration of 7H10 solid medium is coated with after the quality organization homogenate such as part, remainder tissue fixedly does pathological analysis, comprise HE dyeing, acid-fast stain (the non-coloured picture of acid-fast stain result can not demonstrate result, does not put into result accompanying drawing) etc.

As shown in Figure 9, rBCG-Rv2645 group effectively can reduce tuberculosis infection to the enumeration result of lungs and spleen.The colony number of mouse lungs, spleen, demonstrates notable difference (p<0.05) after attacking malicious 6 weeks; The acid-fast stain of mouse lungs also show trend (coloured picture is not put into) identical with it, minimum number in the rBCG-Rv2645 group tubercule bacillus visual field.

The HE staining procedure of mouse lungs and spleen is as follows: the lungs that first 4% paraformaldehyde is fixed by (1) and spleen give conventional paraffin embedding, then make the section of 4 μm; (2) section is put plate to hot water, then be attached on slide glass, be positioned in 45 DEG C of thermostat containers and dry; (3) dewaxing dyeing: section is placed in dimethylbenzene and dewaxes, then section is placed in successively dehydrated alcohol, 95% ethanol, 80% ethanol, be finally placed in distilled water; (4) section is put the 5-10 minute that to dye to hematoxylin solution; The hydrochloride alcohol differentiation 30s of (5) 1%, running water; (6) anti-blue 1 minute of weak ammonia liquor solution; (7) running water 15 minutes; Yihong aqueous solution of (8) 0.5% redyes 7 minutes; (9) tap water 3 minutes; Place 2 minutes in (10) 95% ethanol I, place 2 minutes in 95% ethanol II, dehydrated alcohol I places 2 minutes, places 2 minutes in dehydrated alcohol II; (11) place 5 minutes in dimethylbenzene I, place 5 minutes in II in dimethylbenzene; (12) basis of microscopic observation after cover glass mounting.

The observations of lungs and spleen as shown in Figure 10 and Figure 11, and is used alone compared with BCG groups of immunized mice and PBS contrast, rBCG-Rv2645 group can the pathological change of lungs obviously comparatively light, fusion of pulmonary alveoli is less, and alveolar septum is more complete.In spleen, rBCG-Rv2645 group lymph tubercle edge is comparatively clear, and inflammation is lighter.

Claims (9)

1. mycobacterium tuberculosis Rv2645 albumen is as the application of mycobacterium tuberculosis dominant antigen, it is characterized in that: the aminoacid sequence of described Rv2645 albumen is as shown in SEQ ID NO.1.

2. the preparation method of mycobacterium tuberculosis Rv2645 albumen, is characterized in that comprising the steps:

(1) with the genome of mycobacterium tuberculosis for template, use following primer amplification Rv2645 gene;

Upstream primer: 5'-ATTGGATCCATGACCACCACGC-3',

Downstream primer: 5'-AATAAGCTTCCGCCGGTGTTCGC-3';

(2) Rv2645 gene is passed through restriction enzyme bamh I He hind III is building up on prokaryotic expression carrier pET28a and obtains recombinant plasmid pET28a-Rv2645;

(3) again pET28a-Rv2645 is transformed in e. coli bl21, obtains Rv2645 albumen by IPTG induction and Ni-NTA affinity purification.

3. the application of mycobacterium tuberculosis Rv2645 albumen in the Novel diagnosis reagent of preparation tuberculosis T-SPOT.

4. a Novel diagnosis reagent of tuberculosis T-SPOT, is characterized in that: go intracellular toxin to obtain in the Rv2645 albumen prepared by method according to claim 2.

5. the application of mycobacterium tuberculosis Rv2645 albumen in the Novel diagnosis reagent of the special IgG4 antibody of preparation tuberculosis.

6.Rv2645 albumen is as the application of dominant antigen in the novel recombinant BCG vaccine of tuberculosis or protein vaccine of mycobacterium tuberculosis.

7. a tuberculosis protein vaccine, is characterized in that: be the Rv2645 albumen prepared by method according to claim 2.

8. the novel recombinant BCG vaccine of tuberculosis, is characterized in that: for expressing the recombinant BCG of mycobacterium tuberculosis Rv2645 albumen.

9. the novel recombinant BCG vaccine of tuberculosis according to claim 8, is characterized in that the method by comprising following steps prepares:

(1) with the genome of mycobacterium tuberculosis for template, use following primer amplification Rv2645 gene;

Upstream primer: 5'-ATTGAATTCATGACCACCACGC-3',

Downstream primer: 5'-AATAAGCTTCCGCCGGTGTTCGC-3';

(2) Rv2645 gene is passed through restriction enzyme ecor I He hind III is building up in shuttle plasmid pMV361 and obtains recombinant plasmid pMV361-Rv2645;

(3) pMV361-Rv2645 electricity is forwarded in BCG to obtain expressing Rv2645 albumen recombinant BCG again.