CN104151411B

CN104151411B - Albumen and pharmaceutical composition for diagnosis of tuberculosis

Info

Publication number: CN104151411B
Application number: CN201310176083.5A
Authority: CN
Inventors: 刘立国; 金奇; 张笑冰; 张维佳
Original assignee: Institute of Pathogen Biology of CAMS
Current assignee: Institute of Pathogen Biology of CAMS
Priority date: 2013-05-14
Filing date: 2013-05-14
Publication date: 2018-03-20
Anticipated expiration: 2033-05-14
Also published as: CN104151411A

Abstract

The invention belongs to biochemistry and field of immunology, is related to a kind of albumen and pharmaceutical composition for diagnosis of tuberculosis.In particular it relates to a kind of albumen of separation, its amino acid sequence such as SEQ ID NO：1 or SEQ ID NO：Shown in 2.The albumen of the present invention can have good coincidence rate and response intensity as the tuberculosis immunodiagnosis molecular marker or vaccine candidate object as cellular level.

Description

Albumen and pharmaceutical composition for diagnosis of tuberculosis

Technical field

The invention belongs to biochemistry and field of immunology, is related to a kind of albumen and medicine group for diagnosis of tuberculosis Compound.

Background technology

Mycobacterium tuberculosis is to threaten human health one of maximum pathogen, and tuberculosis is adult in the world today In Infectious Diseases, to international public health form severe challenge.Tulase is more in latent shape after human body is infected State or persistent infection state, it can escape the defense reaction of host in host cells infected and amount reproduction simultaneously reaches with host A kind of delicate balance, the infected in life be whenever likely to fall ill.Generally, 10% the infected will send out Open up as active tuberculosis.The active tuberculosis patient of one untreated, 10-15 people can be infected within 1 year.

In the late five decades, control theory lungy and technology, clinical conditions are horizontal considerable enters with studying to have Step.Because the commonly used of effective chemotherapeutics (isoniazid, rifampin, pyrazine acyl ammonium, streptomysin, ethambutol etc.) and card are situated between Seedling is in worldwide popularization so that the incidence of disease lungy was once once reducing, so that people optimistically think, tuberculosis will It is the same with smallpox to be utterly destroyed by the mankind.But in the eighties mid-term, made an appraisal of the situation due to starry-eyed, reduce investment, contracting Subtract mechanism, loosen to treatment lungy and management, tuberculosis is again revivable in many countries, and the incidence of disease is in worldwide Inside go up again.WHO in 1993 announces that tuberculosis is in " global emergency state ", and appeals " swing into action and tuberculosis Crisis is waged a struggle ", this delivers such statement in WHO history and still belonged to the first time.Resistance and multi-drug resistance tuberculosis have also turned into Significant threat in current Tuberculosis control work.

There is positive rate is low, the cycle is long, complex operation, false positive rate height etc. are asked in conventional diagnosis of pulmonary tuberculosis technology Topic.So need a kind of quick, easy, sensitive, special diagnostic method.

Tuberculosis virus diagnostic method has at present：（One）Sputum inspection：Phlegm applies bacterium inspection and sputum examination for tubercle bacillus is simple and easy to do, Accuracy is higher, and tulase is found in phlegm, has suffered from tuberculosis with regard to that can make a definite diagnosis.General first go to a doctor will look into three sputum specimens, i.e. night Between phlegm, early morning phlegm and instant phlegm.Although it is diagnosis pulmonary tuberculosis " golden index " but diagnosis is low, cultivation cycle length.Sputum Culture, credible result degree are high, and can do tulase drug sensitive test, but take 6-8 weeks, using being restricted.（Two）Ⅹ ray is examined Look into：The ray examination of chest Ⅹ can be with early detection tuberculosis, and can determine the position of focus, property, scope, understands morbidity feelings Condition and the judgement for therapeutic effect, and carry out conveniently, patient is willing to accept.Chest CT can be found that less or hidden portion The lesion of position, the deficiency of general Ⅹ ray examination can be made up.But easily obscure with other PUD Ds, it is necessary to which medical practitioner is true Card.（Three）Pulmonary tuberculosis immunology diagnosis：1. conventional has purified protein derivative of tuberculin（PPD）Experiment, the experiment are positive It is to infect one of evidence of tulase, but false positive rate is high, easy mistaken diagnosis.2. in blood, in phlegm, antibody detection is positive Also contribute to diagnose, false positive rate also easily occur.3.BACTEC methods survey the metabolin of mycobacterium tuberculosis, and general two weeks separable Go out mycobacteria, but how much bacterium amount can influence the number of days of positive findings appearance.5. polymerase chain reaction（PCR）, advantage is sensitive Property up to 98%-100%, shortcoming be specificity it is poor.（Four）Other are checked：Auxiliary diagnosis can only be used as, it is impossible to as diagnosis according to According to.1. fiberoptic bronchoscopy, can directly observe or judge bronchus, Pulmonary lesion indirectly, and have biopsy, The functions such as lavation, video recording, shooting tracheal strips photo, it is particularly useful for diagnosis and differential diagnosis.2. thoracoscope and mediastinoscopy Look into, be used equally for observing thoracic cavity, enlarged lymph node in mediastinum, and can be taken off biopsy with sharp diagnosis and differential diagnosis.3. Ultrasonic examination, it is mainly used in the diagnosis and differential diagnosis of pleural effusion.

Mycobacterium tuberculosis（MTB）There are a kind of Early insulin secretion antigen target protein (early secretory in culture filtrate Antigenic target, ESAT-6), GenBank accession number is：NC_00962, gi：57117165, relative molecular mass is 6000, it can be identified in MTB infection early stages by the immune system of body, be important T cell antigen antigen.ESAT-6 sources Synthesis polypeptide be widely used in the detection of peripheral blood MTB specificity gamma interferon (IFN-γ) response situation, clinically use Whether there are one of MTB infection indexs [Ravn P.Munk ME.Andersen AB Prospective evaluation in diagnosis of a whole-blood test using Mycobacterium tuberculosis-specific antigens ESAT-6and CFP-10for diagnosis of active tuberculosis2005；Harbce M.Oetteinger T.Wiker HG Evidence for oceurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycabacterium bovis BCG1996；Zhang,M.,H.Wang,M.Liao,et al.(2010)."Diagnosis of latent tuberculosis infection in bacille Calmette-Guerin vaccinated subjects in China by interferon-gamma ELISpot assay."Int J Tuberc Lung Dis14(12):1556- 1563.]。

Nevertheless, new diagnosis of tuberculosis will be found or detect the mark of mycobacterium tuberculosis by still needing at present, and New checkout and diagnosis tuberculosis or the method and reagent of detection mycobacterium tuberculosis.

The content of the invention

The present inventor passes through in-depth study and performing creative labour, has obtained an albuminoid, the present inventor sends out in surprise Existing, albumen of the invention can be as the mark of diagnosis of tuberculosis or detection mycobacterium tuberculosis, and it has well Sensitivity and Specificity.Thus provide following inventions：

One aspect of the present invention is related to a kind of albumen of separation, its amino acid sequence such as SEQ ID NO：1（In the present invention Sometimes referred to as Rv0232）Or SEQ ID NO：2（Rv1031 is sometimes referred to as in the present invention）It is shown.The albumen is for tuberculosis Diagnosis or the albumen of tubercle bacillus detection.

Rv0232 amino acid sequence：150aa

KGSLFQYFADKRDLYAFIADIASQRVRSYMEDLIRELDPNR PFFEFLTDLLDGWVAYFAEHPRERALHAAATLEVDTDARISVRS VLHRHYLDVLRPLVRDAHARGDLRADSDTGALMSLLLLIFPHL ALAPYMRGLDPILGLDEPTPEQ（SEQ ID NO： 1）

Rv1031 amino acid sequence：189aa

MRRQLLPALTMLLVFTVITGIVYPLAVTGVGQLFFGDQAN GALLERDGQVIGSAHIGQQFTAAKYFHPRPSSAGDGYDAAASSG SNLGPTNEKLLAAVAERVTAYRKENNLPADTLVPVDAVTGSGS GLDPAISVVNAKLQAPRVAQARNISIRQVERLIEDHTDARGLGF LGERAVNVLRLNLALDRL（SEQ ID NO：2）

A kind of nucleotides of separation of another aspect of the present invention, it encodes the albumen of the present invention；Specifically, the nucleotides Sequence such as SEQ ID NO：3 or SEQ ID NO：Shown in 4.

Rv0232 nucleotide sequence（Reading frame）：450bp

AAGGGCAGCCTGTTCCAGTACTTCGCGGACAAGCGCGAC CTCTACGCGTTTATTGCCGACATCGCCAGCCAGCGAGTCCGC TCCTACATGGAGGACCTGATCCGCGAGCTGGACCCGAACCG GCCGTTCTTCGAATTCCTCACCGACCTGCTCGATGGCTGGGT CGCCTACTTCGCCGAGCATCCTCGGGAACGTGCGTTGCATGC TGCGGCGACCCTGGAGGTCGACACCGATGCCCGCATCAGCG TGCGCAGCGTCCTGCACCGCCACTACCTGGACGTGCTACGG CCGCTGGTGCGCGACGCGCACGCGCGGGGCGACCTGCGCGC AGATTCCGACACCGGTGCATTGATGTCGCTGCTGCTGCTGAT CTTTCCGCACCTGGCGCTGGCTCCATACATGCGTGGTTTGGA TCCGATCCTGGGCCTCGACGAGCCCACACCTGAGCAG（SEQ ID NO：3）

Rv1031 nucleotide sequence（Reading frame）：570bp

ATGCGTCGTCAATTACTGCCCGCGCTCACCATGCTGTTG GTGTTCACCGTCATCACCGGCATCGTCTACCCGCTTGCCGTG ACCGGCGTCGGGCAACTGTTCTTCGGTGACCAGGCGAACGG CGCGCTGCTCGAGCGGGACGGGCAGGTCATCGGCTCCGCCC ACATCGGCCAGCAGTTCACCGCCGCGAAGTACTTCCACCCGC GCCCCTCGTCGGCAGGCGACGGTTACGACGCTGCGGCGAGC TCGGGCTCCAACCTGGGACCGACGAACGAGAAGCTGCTGGC GGCCGTCGCTGAACGGGTCACCGCCTACCGCAAGGAAAACA ATCTGCCGGCCGATACGCTGGTTCCGGTCGACGCGGTTACC GGCTCGGGTTCCGGGCTGGACCCGGCCATATCGGTGGTCAA TGCCAAGCTGCAGGCACCGCGGGTGGCGCAGGCGCGCAATA TCTCGATAAGGCAGGTCGAGCGTCTGATCGAGGACCACACC GACGCGCGTGGTCTCGGCTTCCTGGGCGAGCGCGCGGTGAA CGTGCTCAGGCTGAACCTCGCATTGGATCGCCTCTGA（SEQ ID NO：4）

The nucleotide sequence of Rv0232 and Rv1031 reading frame can be by the corresponding plasmid or PCR in embodiment 1 or 2 Product is sequenced to obtain, and the amino acid sequence of coding can be further deduced according to reading frame nucleotide sequence.Certainly, above-mentioned reading The nucleotide sequence of code frame can also be obtained by artificial synthesized.The albumen can be suitable by the way that reading frame sequence is passed through Expression vector and host cell obtain.

Another aspect of the invention is related to a kind of recombinant vector, and it contains the nucleotides of the present invention.

Another aspect of the invention is related to a kind of recombinant host cell, and it contains the recombinant vector of the present invention.

Another aspect of the invention is related to a kind of primer pair, and it includes SEQ ID NO：7 and SEQ ID NO：Core shown in 8 Nucleotide sequence, and/or include SEQ ID NO：9 and SEQ ID NO：Nucleotide sequence shown in 10.Specifically, the primer pair Nucleotide sequence such as SEQ ID NO：7 and SEQ ID NO：Shown in 8, and/or such as SEQ ID NO：9 and SEQ ID NO：10 institutes Show.

Another aspect of the invention is related to a kind of antibody, and it can be with the albumen of the present invention（Rv0232 and/or Rv1031） Specific binding；Specifically, the antibody is monoclonal antibody or polyclonal antibody；Specifically, the antibody, which is connected with, to examine The label of survey.

The polyclonal antibody or monoclonal antibody are referred to the method that those skilled in the art know and prepared.

In the present invention, term " specific binding " has immunologic general sense, such as the combination between antigen-antibody.

Another aspect of the invention is related to a kind of antibody coupling matter, including antibody moiety and coupling moiety, wherein, it is described anti- Body portion is the antibody of the present invention, and the coupling moiety is selected from radionuclide, medicine, toxin, cell factor, enzyme, fluorescence One or more in element, carrier protein, lipid and biotin.

Another aspect of the invention is related to a kind of composition, and it contains the albumen of the present invention（Rv0232 and/or Rv1031）、 The present invention nucleotides, the present invention recombinant vector, the present invention recombinant host cell, the present invention primer pair, the present invention The antibody coupling matter of antibody or the present invention；Alternatively, the composition（Pharmaceutical composition）Also containing pharmaceutically acceptable Carrier or excipient；Specifically, the composition is vaccine combination, and the pharmaceutically acceptable carrier or excipient For vaccine carrier or excipient.

In the present invention, term " vaccine carrier or excipient " refers to the one or more selected from following material, including But it is not limited to：PH adjusting agent, surfactant, adjuvant, ionic strength reinforcing agent.Specifically, for example, pH adjusting agent is included but not It is limited to phosphate buffer, surfactant includes but is not limited to cation, anion or nonionic surface active agent.Its In, nonionic surface active agent includes but is not limited to:Tween-80.Adjuvant includes but is not limited to aluminium hydroxide, and Fu Shi is complete Adjuvant.Ionic strength reinforcing agent includes but is not limited to sodium chloride.

Another aspect of the invention is related to a kind of kit, its include the present invention antibody, the present invention antibody coupling matter, Or the primer pair of the present invention；Specifically, the kit is for diagnosis of tuberculosis particularly lung pulmonary tuberculosis or detection knot The kit of core bacillus.

Another aspect of the invention is related to a kind of model for screening medicine, and it includes the albumen of the present invention（Rv0232 and/or Rv1031）Or the nucleotides of the present invention；Specifically, the medicine is controlled for diagnosis and/or treatment and/or prevention and/or auxiliary Treat tuberculosis and be particularly phthisical medicine or the medicine for anti-mycobacterium tuberculosis.

For example, the albumen of the present invention whether can be detected by the medicine being screened（Rv0232 and/or Rv1031） Or the present invention nucleotides, the medicine to judge to be screened whether can act as diagnosing and/or treating and/or prevent and/or Auxiliary treatment tuberculosis is particularly phthisical medicine or the medicine for anti-mycobacterium tuberculosis.

Another aspect of the invention is related to the albumen of the present invention, and either of the invention nucleotides is being prepared or as screening Purposes in the model of medicine, specifically, the medicine are diagnosis and/or treatment and/or prevention and/or auxiliary treatment knot Core disease is particularly phthisical medicine or to detect the medicine of tubercle bacillus or anti-mycobacterium tuberculosis.

Another aspect of the invention is related to the albumen of the present invention（Rv0232 and/or Rv1031）, the present invention nucleotides, this The recombinant vector of invention, the recombinant host cell of the present invention, the primer pair of the present invention, the antibody of the present invention or the present invention it is anti- Body conjugate is particularly phthisical medicine in preparation diagnosis and/or treatment and/or prevention and/or auxiliary treatment tuberculosis（Bag Include but be not limited to vaccine）Or the purposes in the medicine of preparation detection tubercle bacillus or anti-mycobacterium tuberculosis.

Another aspect of the invention is related to a kind of method for detecting tubercle bacillus in vivo or in vitro, including the use of the present invention Antibody or the present invention primer pair the step of；Specifically, methods described is antigen detection method（Such as western blot Or ELISA）Or PCR methods.A kind of method for detecting tubercle bacillus in vivo or in vitro, it is ELISPOT methods.The sample of detection Originally include but is not limited to：The sputum of detected person, blood, tissue, lymphocyte, DNA etc..

Another aspect of the invention is related to a kind of diagnosis of tuberculosis and is particularly phthisical method, including the use of the present invention's The step of primer pair of antibody or the present invention；Specifically, methods described is antigen detection method（Such as western blot or Person ELISA）Or PCR methods.A kind of diagnosis of tuberculosis is particularly phthisical method, and it is ELISPOT methods.

The explanation of part term of the present invention is given below.

Expression vector

The invention further relates to carried comprising the recombination expression of nucleotide sequence of the present invention, promoter and transcription and translation termination signal Body.Above-mentioned various nucleic acid and regulating and controlling sequence can be joined together to Prepare restructuring expression vector, the carrier can include one Individual or multiple convenient restriction sites, so as to which the nucleic acid sequence of the albumen of the coding present invention or antibody is inserted or substituted in these sites Row.Or can be by the way that nucleotide sequence or nucleic acid construct comprising the sequence be inserted into appropriate expression vector to express this hair The bright nucleotide sequence.When preparing expression vector, coded sequence can be made to be located in carrier so as to appropriate expression regulation sequence It is operably connected.

Recombinant expression carrier can be any carrier for being convenient for recombinant DNA operation and express nucleic acid sequence（Such as matter Grain or virus）.The selection of carrier generally depends on the compatibility for the host cell that carrier will import with it.Carrier can be line Property or closed circular form plasmid.

Carrier can be autonomous replicating vector（It is present in extrachromosomal complete structure, can enters independently of chromosome Row replicates）, such as plasmid, extra-chromosomal element, minute chromosome or artificial chromosome.Carrier, which can include, ensures self-replacation Any mechanism.Or carrier be one when importing host cell, would be integrated into genome and the chromosome with being incorporated into The carrier replicated together.In addition, single carrier or plasmid can be applied, or overall include will import the whole of host cell gene group DNA two or more carriers or plasmid, or transposons.

It is preferred that carrier of the present invention contains one or more selected markers for being easy to select transformed cells.Selected marker is Such a gene, its product is assigned to the resistance of biocide or virus, the resistance of heavy metal, or assigns auxotroph Prototrophy etc..The dal genes of the example of bacterial selectable markers such as bacillus subtilis or bacillus licheniformis, or antibiotic is such as Ampicillin, kanamycins, the resistance marker of chloramphenicol or tetracycline.

It is preferred that carrier of the present invention, which includes, can make carrier stable integration into host cell gene group, or ensure that carrier exists In cell the element of autonomous replication is carried out independently of cellular genome.

For carrying out the situation of autonomous replication, carrier can also include replication orgin, enable carrier thin in target host Independently replicated in born of the same parents.Replication orgin, which can carry, makes it turn into the mutation of responsive to temperature type in host cell（See, for example, FEhrlich, 1978, NAS's journal 75：1433）.

The nucleotide sequence of the present invention of more than one copy can be inserted to host cell to improve the yield of the gene outcome. The copy number increase of the nucleotide sequence can by least one additional copies Insertion Into Host Cell genome by the sequence, Or an amplifiable selected marker is inserted together with the nucleotide sequence, it is thin by being cultivated in the presence of appropriate selectable agent Born of the same parents, pick out the selected marker containing amplification copy, so as to the cell containing additional copies nucleotide sequence.

For connecting above-mentioned each element to build the operation of recombinant expression carrier of the present invention be those skilled in the art Known（See, for example, Sambrook etc., molecular cloning experiment handbook, the second edition, CSH Press, cold spring Port, New York, 1989）.

Host cell

The invention further relates to include the albumen that can be used to the recombinant production present invention or the nucleotide sequence of the present invention of antibody （Polynucleotides）Recombinant host cell.Can be by the vector introduction host cell of the nucleotide sequence comprising the present invention, so that should Carrier is maintained with carrier format outside above-mentioned chromosomal integrant or the chromosome of self-replacation.Term " host cell " is covered Any offspring different from parental cell due to the mutation occurred during duplication.The selection of host cell is largely depended on Albumen or antibody-encoding genes and its source in the present invention.

Host cell can be prokaryotic or eukaryotic, such as bacterium or yeast cells.This area can be passed through Technology known to technical staff is by vector introduction host cell.

Preparation method

The invention further relates to the method for the albumen or antibody for being prepared by recombinant invention, this method includes：(a) suitable Under conditions of the albumen or antibody of the present invention is produced, the host cell containing nucleic acid construct is cultivated, the nucleic acid construct bag Containing the nucleotide sequence for encoding the albumen of the invention or antibody；Reclaim the peptide (b).

In preparation method of the present invention, caused by albumen or antibody of the means known in the art in the suitable present invention Cell is cultivated in nutrient medium.For example, the albumen or antibody expression of the present invention can allowed in suitable culture medium And/or under conditions of separation, pass through Shaking culture, laboratory or industrial fermentation tank middle and small scale or large scale fermentation（Including even It is continuous, in batches, batch charging or solid state fermentation）To cultivate cell.Including the suitable culture of carbon source and nitrogen source and inorganic salts In base, cultivated using step known in the art.Suitable culture medium can provide or be referred to disclosure by supplier Composition（For example, described in the catalogue of American type culture collection）To prepare.If the albumen or antibody of the present invention It is secreted into culture medium, then directly can be reclaimed from culture medium.If the albumen or antibody of the present invention are not secreted, Ke Yicong Reclaimed in cell lysate.

Albumen or antibody that can be of the invention caused by means known in the art reclaim.For example, routine can be passed through Operation（Include, but are not limited to centrifuge, filter, extract, be spray-dried, evaporate or precipitate）Reclaimed from culture medium.

Albumen or antibody of the invention of the present invention can be purified by various operations known in the art, these behaviour Work includes, but are not limited to chromatograph（For example, ion-exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing and big float Resistance layer is analysed）, HPLC, electrophoresis（For example, preparative isoelectric focusing）, differential solubility（Such as ammonium sulfate precipitation）、 SDS-PAGE Or extracting（For example, see, protein purification, J.C.Janson and Lars Ryden are compiled, VCH Publishers, New York, 1989）.

In the present invention, term " tubercle bacillus ", include but is not limited to, for example, mycobacterium tuberculosis；Term " tuberculosis point Branch bacillus " includes but is not limited to, Mycobacterium tuberculosis H37Rv.

The beneficial effect of invention

The albumen of the present invention can be used as tuberculosis（Such as pulmonary tuberculosis）Or tubercle bacillus diagnosis of cell mediated immunity molecule Mark, has good coincidence rate and response intensity.

Brief description of the drawings

Fig. 1：Target gene Rv0232 and Rv1031 electrophoresis result, the sample of each swimming lane are successively from left to right： Rv1031、DNA marker、Rv0232。

Fig. 2：Rv0232 and Rv1031 PAGE results after expression.The sample of each swimming lane is albumen successively from left to right marker、Rv1031、Rv2032。

Fig. 3：The PAGE results of Rv0232 and Rv1031 protein purifications.The sample of each swimming lane is albumen successively from left to right marker、Rv1031、Rv2032。

Fig. 4：Expressing protein primary dcreening operation result example.

Fig. 5：The ELISPOT identification partial results of Partial Protein.

Embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment.Those skilled in the art will manage Solution, the following examples are merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted specific in embodiment Technology or condition person, according to the technology or condition described by document in the art（Such as write with reference to J. Pehanorm Brookers etc., it is yellow What training hall etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press）Or carried out according to product description.Examination used Agent or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.

Embodiment 1：The clone of MTB H37Rv related gene and expression（Gateway methods）

The Gateway technologies of Invitrogen companies provide a kind of possibility for the restructuring and expression of albumen, and it is from PCR Product starts to create Gateway^TMEntry clones.This method is by merging attB sites to upstream and downstream primer, so It is incubated pcr amplification product and pDONR jointly afterwards^TM(including attP sites) and Gateway^TM BP Clonase^TMEnzymatic mixture. Then it is transformed into Escherichia coli, it will obtain the entry clones for including target gene, while target gene both sides have attL Recombination site.This entry clones can be with Gateway^TMPurpose carrier pDEST17 is recombinantly expressed, and rapidly and efficiently obtains mesh Albumen [Walhout AJ, Temple GF, Brasch MA.et al.GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes.Methods Enzymol,.2000,328:575-592；Rachael M.Goldstone,Nicole J.Moreland,Ghader Bashiri,et al.A new Gateway_vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis.Protein Expression and Purification57(2008)81–87；A high throughput screen to identify secreted and transmembrane proteins involved in Drosophila embryogenesis.PNAS1998;95;9973-9978.]。

This experiment using in MTB H37Rv genome as template, utilize Invitrogen companies provide Gateway skills Art, the protein of whole ORF codings is expressed, being obtained by western blot methods detection clinical blood has higher sensitivity Property specific antigen, solid material base is provided for clinical diagnosis, disease treatment, prevention and control.

First, experimental method

1. the acquisition of gene order

According to disclosed MTB H37Rv ORFs gene orders, primer sequence is designed with Primer designer, According to the design of primers scheme of Gateway systems, attB arms are added for primer sequence, primer is completed by Beijing Xin Qingke companies Synthesis.

It is that forward primer arm is：

GGGGACAAGTTTGTACAAAAAAGCAGGCTTC（SEQ ID NO：5）；

Reverse primer arm is：

GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA（SEQ ID NO：6）.

Completion design of primers 3535 is right altogether, and because length is limited, part primer is as shown in Table 1 below.

Table 1：Partially synthetic primer（Part without att arms）

Using H37Rv genomes as template（Institute is controlled from Beijing tuberculosis prophylaxis）, with the addition of the positive and negative of attB arms Different ORFs are expanded to primer, PCR system is as follows：

PCR reaction conditions are：98 DEG C of thermal startings（Preliminary examination temperature is 94 DEG C）, 97 DEG C denaturation 1min, 60 DEG C（58-65 DEG C） Anneal 1min, 72 DEG C of extension 3min, 30 circulations.Last 72 DEG C of incubations, 30min.

5 μ l pcr amplification products are taken to carry out electrophoresis in 0.8% Ago-Gel, with DNA Marker DL2,000 makees standard Molecular weight, electrophoresis result is observed under uviol lamp, and taken pictures.

After agarose gel electrophoresis, the blob of viscose containing purpose size DNA segment is cut out under uviol lamp, is often cut once, is used Ultrapure water blade, and wipe clean, the film cut is cut into pieces as far as possible, is put into Eppdorf pipes；Of short duration centrifugation, Volume is watched, adds the QG of 3 times of volumes, 50 DEG C of water-baths, 10min, until glue is completely dissolved（Solution is in yellow）, during water-bath, Eppendorf pipes 2-3 times are inverted every 3min, if segment size in below 400bp or more than 4000bp, in EP pipes Add the isopropanol of 1 times of volume, mix；Dissolved gum liquid is added in QIAquick spin column, 12000r/min, from Heart 1min discards liquid in collecting pipe；500 μ l sol solutionses QG, 12000r/ is added in QIAquick spin column Min, 1min is centrifuged, to remove unnecessary agar, liquid in collecting pipe is discarded, is added in QIAquick spin column 750 μ l washing lotions PE, static 3-5min, 12000r/min, centrifuge 1min；Waste liquid is discarded, 12000r/min, centrifuges 1min again； QIAquick spin column are put into the new numbering 1.5mLEppendorf that finishes writing to manage, in QIAquick spin Column centers add the DNA lysates that 50 μ l are warmed（EB solution）, static 5min, then 12000r/min centrifuge 1min, In Eppendorf pipes liquid repeated centrifugation once, to increase DNA elution amounts.

Agarose gel electrophoresis detects, quantitative, -20 DEG C of preservations, standby.

The BP reactions of 2.Gateway systems

Restructuring is to create Gateway from PCR primer^TMAnother method of entry clones.This method is to pass through merging Then attB sites are incubated pcr amplification product and pDONR jointly on upstream and downstream primer^TMCarrier (includes attP sites) And Gateway^TMBP Clonase^TMEnzymatic mixture.Then it is transformed into Escherichia coli, it will obtain comprising target gene Entry clones, while target gene both sides have attL recombination sites.This entry clones can be with any Gateway^TMPurpose Carrier is recombinated.

Reaction system sets positive control simultaneously, pEXP7-tet50ng/ μ l, takes 1 μ l, and remaining uses water polishing；

attB-PCR product(≥10ng/μl;Final amount15-150ng), 7 μ l；

pDONR^TM211,1 μ l；

TE buffer, pH8.0,8 μ l.

BP Clonase are taken out from low temperature refrigerator^TMII enzyme mix melt on ice, and whirlpool shakes secondary, each 2s, often Hole sample adds 2 μ lBP Clonase^TMII, shake secondary, gentle centrifugation；By BP Clonase^TMII enzyme mix to are fast Speed puts back to low temperature refrigerator；25 °C of overnight incubations；Add 1 μ l per hole 2 μ g/ μ l protein kinase Ks, 37 °C, 1h, terminating reaction.Will be even The reaction product connected, transformed competence colibacillus cell.

3. the conversion of recombinant plasmid

According to bibliography（J. Pehanorm Brooker, D.W. Russells edit Molecular Cloning:A Laboratory guides [M] third edition, [9]：1217-1265.）Perform, take out the competence bacteria prepared from low temperature refrigerator and add 3.5 μ l connections production thereto Thing, ice bath 30min after mixing.96 orifice plates are moved into 42 water-baths, stop 90s just（Never vibrate）；96 orifice plates are put immediately In ice bath, 5min；450 μ l LB fluid nutrient mediums are added into 96 orifice plates, in 37 constant temperature oscillations（2000r/min）Culture 60min.The bacterium of culture is collected, is applied on the LA flat boards with kalamycin resistance, 37 DEG C of overnight incubations；Picking monoclonal bacterium Fall inoculation, be dispersed in 50 μ l sterilizing ultra-pure waters, 99 DEG C of denaturation, be bacterium colony PCR, verify whether for positive colony, to separate simultaneously 25 μ l are seeded in containing in the orifice plate of kalamycin resistance 96（LB culture mediums）；37 DEG C of overnight incubations, prepare extraction plasmid.

The selection of bacterium colony PCR primer：According to pDONR221 plasmids, this bacterium colony is expanded since M13 bacteriophages, From：

M13forward primer：GTAAAACGACGGCCAGT（SEQ ID NO：39）；reverse primer: CAGGAAACAGCTATGAC（SEQ ID NO：40）；

Positive control：The μ l of H37Rv genomic DNAs 0.5,25 μ l systems, with water polishing；Negative control：It is not added with bacterium.

25 μ lPCR systems：

PCR reaction conditions are：98 DEG C of thermal startings, 97 DEG C of denaturation 1min, 60 DEG C of annealing 1min, 72 DEG C of extension 3min, 30 are followed Ring.Last 72 DEG C of incubations 30min.

0.8% agarose gel electrophoresis, identify positive colony bacterium colony（Size is correctly the positive）To treat lower step upgrading grain Establish Eetry clone.

4. plasmid extraction

The extracting method of 96 orifice plate plasmids, DNA after purification is obtained in 96V type bottom plates.

Qiagen kits extract high copy number plasmid pDONR221 and low-copy plasmid pDEST17（http// www.qiagen.com）

Extract plasmid buffer solution：Buffer I（Re-suspension liquid）Room temperature preservation, Buffer II（Lysate）Room temperature preservation, Buffer III（Neutralizer）Room temperature preservation, Buffer IV（Impurity removes liquid A）, 4 DEG C of preservations.Buffer V（Impurity removes liquid B）Room temperature preservation, Buffer VI（Impurity removes liquid C）, 4 DEG C of preservations, RNase A（20mg/mL）- 20 DEG C of preservations.

Required other reagents：TE（Tris-HCl10mM, EDTA1mM, pH8.0, ultra-pure water are prepared, and high pressure or filtering are gone out Bacterium）, isopropanol and 70% ethanol（Ultra-pure water is prepared with absolute ethyl alcohol）.

Kit can be made 20 times（<150mL bacterium solutions/time）Plasmid extraction purifies.Normal temperature transports.RNase A are in -20 DEG C of guarantors Deposit, add in Buffer I and mix when using first, put 4 DEG C of preservations.Buffer IV and Buffer VI put 4 DEG C of preservations, remaining Solution is stored in room temperature.The term of validity 6 months.Buffer II and Buffer V might have precipitation and produces.Please respectively in 37 DEG C of water-baths Dissolved with 50 DEG C of heating water baths, then mix and use.Buffer IV are mixed and can be used if any layering.

Prepare before plasmid extraction：RNase A are added into the final concentration of 100 μ g/mL of Buffer P1；If Buffer P2 have Precipitation can be heated to 37 DEG C of dissolvings；Precooling Buffer P3 to 4 DEG C

The extraction step of plasmid：

1）10mL QBT is added to QIAGEN-tip500, makes solution with gravity streamline, pre-equilibration it；

2）Picking monoclonal colony inoculation is into 4mL LB culture mediums, 300r/min overnight incubations；Press 1 within second day：500 put Greatly, the final concentration of 50 μ g/mL of ampicillin are added, cultivate 12-16h, 500mL bacterium solutions is collected, loads suitable centrifugal bottle In, 6,000g, 15min precipitation thalline, complete reject supernatant are centrifuged in 4 DEG C.

3) 125mL Buffer I are added, be fully suspended concussion bacterial sediment, is completely dispersed it, is deposited to without wadding block .125mL Buffer II are added, gently overturn centrifuge tube 6-8 times, room temperature places 5min, bacterium is cracked completely, solution It is transparent.

4) 125mL precooling Buffer III are added, overturn centrifuge tube immediately 6-8 times, are fully mixed, to White Flocculus Produce.30min is placed on ice.

5) above-mentioned lysate centrifuges 30min in 4 DEG C of 20000g, carefully suctions out supernatant, moves into new centrifuge tube.

6) 4 DEG C of 20000g centrifuge 15min again, carefully suction out supernatant, in the GIAGEN-tip balanced in advance.By gravity Trickle, plasmid enter resin；

7) 600mL Buffer QC washings are added；

8) 100mLBuffer QF, elution are added；

9）The isopropanol that 70mL room temperature is placed is added, mixing, 15,000g, 4 DEG C centrifuge 30min immediately；

10）70% ethanol placed with room temperature washs, 15,000g, 4 DEG C of centrifugation 10min；

11）10-20min is dried, adds 1mLTEe（pH8.0）；

12）Dissolve repeatedly once.

13）DNA is extracted to obtain in agarose electrophoresis identification.

5.LR reacts, and BP reactions produce entry clones

LR reactions are the entry clones in the site containing attL（Entry clone）Between the purpose carrier comprising attR sites Recombining reaction.LR reactions produce an expression cloning.Expression cloning include target gene and the special promoter of purpose carrier or Some elements of person.

Reaction system, while set positive control pENTR^TM-gus（Kalamycin resistance）

The μ l of entry clones (50-150ng) 7

Destionation vector (150ng/ μ l, pDEST17) 1 μ l

TE buffer, pH8.0 amount to 8 μ l

LR Clonase are taken out from low temperature refrigerator^TMII enzyme mix melt on ice, and whirlpool shakes secondary, each 2s,

2 μ lLR Clonase are added per hole sample^TMII enzyme mix, shake secondary, gentle centrifugation；

By LR Clonase^TMII enzyme mix put back to rapidly low temperature refrigerator；

25 °C of overnight incubations；

The μ l of 2 μ g/ μ l protein kinase Ks 1, terminating reaction are added per hole.

The reaction product that will be connected, transformed competence colibacillus cell.

Plasmid extraction is same as above, and positive findings, 25 μ l systems, the selection of primer are verified with expression plasmid PCR:attB Site is recovered,

Forward primer attB1：

GGGG-ACA AGT TTG TAC AAA AAA GCA GGC TTC（SEQ ID NO：5）

Reverse primer attB2：

GGGG-AC-CAC-TTT-GTC-CAA-GAA-AGC-TGG-GTC- CTA（SEQ ID NO：6）.

Positive control：The μ l of H37Rv genomic DNAs 0.5, with the μ l of water polishing 25.Negative control：Not plus bacterium.

25 μ lPCR systems：

PCR reaction conditions are：98 DEG C of thermal startings 97 are denatured 1min, and 60 DEG C of annealing 1min, 72 DEG C of extension 3min, 30 are followed Ring.Last 72 DEG C of incubations 30min.

0.8% agarose gel electrophoresis, identify positive colony bacterium colony（Size is correctly the positive）To treat lower step extraction matter Grain, establishes Expression clone；

6. expression cloning is transformed into expression bacterial strain

The expression plasmid for carrying target gene carries amicillin resistance, is transformed into expression bacterial strain BL21（DE3）With take Band chlorampenicol resistantIn Competent Cells, step of converting is same as above 2.5, picking Dan Ke Grand bacterium colony, it is inoculated into the LB liquid containing different resistances, 37 DEG C, 200r/min, overnight；

IPTG induced expressions

Take the μ l of 96-well-plate bacterium solutions 50 of overnight growth in 2.9.1（1:20）It is diluted to 1mL LB（Ampr）96- well-plate（2 plates）In, 37 DEG C of culture bacteriums, 300r/min, about 3-4h, to OD600=0.6.

IPTG induction steps：

Take 100mM IPTG to add in 96-well-plate, cultivated to final concentration 0.5mM. (200x dilutions), such as l mL Add 5 μ l in base.It is Amp resistance culture bases in 96-well-plate

One 37 DEG C of plate, 250r/min induction 5h, 37 DEG C of a plate, 250r/min overnight inductions, if plus IPTG it is negative right According to gus positive controls.Then 4 DEG C, 3950r/min centrifugation 5min, supernatant is abandoned, the rear processing for carrying out loading sample

Note：Three different inducing temperature 0.2mM, 0.5mM, 0.8mM can be set to.28 DEG C, 37 DEG C of temperature.

7. discontinuous sds polyacrylamide gel electrophoresis

Upper strata：Concentrate glue（staking or concertration gel）5% pH of buffer 6.8；

Lower floor：Separation gel（separation or resolving gel）12% pH of buffer 8.8.

Add after TEMED, mix rapidly and be poured in offset plate, pay attention to reserving space needed for perfusion spacer gel（Comb Tooth length again plus 0.5-1cm）.With watertight sealing (pressing and anti-oxidation).At room temperature（About 30-45min）Polymerization.

Water seal is discarded after polymerization, is blotted with filter paper.

Prepare spacer gel：

Comb is first inserted, then adds TEMED, spacer gel is irrigated on the separation gel of polymerization immediately after mixing, carefully keeps away Exempt to be mixed into bubble, gel is disposed vertically at room temperature.

After spacer gel polymerization completely, about 20min, comb is carefully removed from, gel slab is transferred on electrophoretic apparatus, prepared Loading.

The processing of loading sample（Can first it handle）：The sample 4000r/min induced, 10min is centrifuged, abandons supernatant, so Afterwards plus 50 μ l sterilizing ultra-pure water concussions mix, and then take out 20 μ l to 96-well plate, add the μ l of 2x sample buffers 20, even Mended with Marker100 DEG C of albumen denaturation 10min, the μ l of loading 20, emptying aperture with sample buffer, negative control is with the bacterium not induced The competent cell of culture simultaneously.

Loading：Every block of offset plate is assembled, sees whether leakage, with octal road pipettor or single sample-adding, will carefully be inserted Enter above sample well, slide into sample 15-20 μ l, prevent in syringe in bubble access aperture（Albumen Marker adds 20 μ l）.

Different protein combinations is distinguished with diverse location albumen Marker.

Electrophoresis：Electrophoretic buffer will be added in Vertial electrophorestic tank（Liquid level exceeds electrode circuit）.Connect with the mains and start electrophoresis. Start voltage 80V, after dyestuff enters separation gel（About 0.5h）, voltage increases to 100V, continues electrophoresis to bromophenol blue and reaches point From being cut off the electricity supply behind glue bottom（About 2h）.

Glue is taken, glass plate is pried open with spatula, different staining jars is put into by different combinations, dyes 1h, 1h after decolouring, and Compare albumen size corresponding to different genes and cut off the protein gel of correct size in 1.5mLEP pipes, -20 DEG C of holdings, to treat Subsequent analysis is used.

2nd, experimental result

96 orifice plate gene electrophoresis results after glue purification are cut, are obtained 3535 target gene, 3372 entry clones, 3245 Expression cloning, expressing protein 1916.

Wherein, Rv0232 and Rv1031 gene electrophoresis result is as shown in Figure 1.

Albumen Rv0232 and Rv1031 SDS-PAGE results are as shown in Figure 2.

96 orifice plate genes expand one and share 32 plates, and the size of each gene has different lists, because more picture money Material, therefore only list Rv0232 and Rv1031 electrophoretogram.

3rd, interpretation of result

With from http:The H37Rv genome ORFs that //www.ncbi.nlm.nih.gov/ is downloaded（ORFs）Sum And the distribution results of expressing protein wherein are compared, as shown in Table 2 below.

Table 2：The function distribution of expressing protein

From table 2, the albumen expressed in this experiment has good coverage rate to full-length genome.

Embodiment 2：Protein expression and purification

First, experimental method

Protein purification is the necessary condition for carrying out downstream experiment, can include after procaryotic cell expression it is thousands of not Same protein, some content very abundants, some only contain several copies.In order to study some protein, it is necessary to first will Protein side of being purified from other protein and non-proteinaceous molecule can remove false sun caused by the foreign protein of external source Property, the albumen of high-efficiency high-purity needs by high-throughout protein purification.The expressing protein of this experiment is to be marked containing 6His The prokaryotic expression of label, therefore carried out using the affinity of the adjacent histidine of His.Tag sequences and immobilization metal nickel ion pure Change.

1. strain is brought back to life and amplification

LB culture mediums needed for strain recovery and amplification are prepared, corresponding antibiotic is added after its cooling.Ampicillin stores up Deposit concentration 50mg/mL, the μ g/mL of concentration 50；Chloramphenicol stores concentration 50mg/mL, the μ g/mL of concentration 34）, by chapter 1 Identify that the strain of expression takes out from low temperature refrigerator, often pipe is inoculated with 10 μ l strains after defrosting.37 DEG C, 200r/min, overnight.Strain expands Increasing, expression and fungi preservation：Expand and respective concentration antibiotic is equally added in LB, 1：50 amplifications（Optionally 1：20）, about 2 hours Left and right is to OD600 to 0.6（Stay the LB of 1mL not added with antibiotic to do negative control, arbitrarily connect a bacterium and be used to measure OD values）.Strain During to 0.6, first respectively take 1mL bacterium solutions to do and do not induce control, remaining adds IPTG (1mL：50 μ l), according to 37 DEG C of former inductive condition or 28 DEG C, 200r/min, 12-16 hours are induced overnight.Remaining bacterium solution is used to preserve strain, each two pipe（Final glycerol concentration 15%）.

2mL is first taken in bacterium solution after induction, 4 DEG C, 6000r/min, 15min collect precipitation, are dissolved in 100 μ l pure water, 4 DEG C of remaining bacterium solution of control after inducing, 6000r/min, 15min collect bacterial sediment, stay 200 μ l supernatants, concentrate, and detection is It is no to belong to secreted protein.

Precipitation is weighed, and is dissolved with 5-10mLlysisbuffer/g.

Add 0.1mg/mLlysozyme, 1mMPMSF（Final concentration）, 4 DEG C of stirring 30min.

Ultrasonic degradation.10sec ultrasounds, 10sec intervals, common 10-15 minutes.Pay attention to operating on ice always.

Per one sample of ultrasound, thoroughly cleaning is popped one's head in.

4 DEG C, 12000r/min, 20min collect supernatant and precipitation respectively.

First take supernatant precipitation to do soluble identification before being purified, be distributed according to albumen and determine purification process.

Cracking supernatant can be directly used for purifying, and note：30 μ l supernatants are stayed to compare.Precipitation is dissolved again with cleaning solution, It washed once.

4 DEG C, 12000r/min, 20min collect precipitation, weigh, again with bindingbuffer（Urea containing 8M）Dissolving, 10-20mg/mL, room temperature act on 1 hour.

4 DEG C, 12000r/min, 20min collect denaturation supernatant, are directly used in purifying, stay 30 μ l to compare.

2. protein purification

Filler mixture is anticipated, ethanol sedimentation, with pure water rinsing once, buffer solution balance.

Supernatant and denaturation supernatant are cracked through 0.45um membrane filtrations, is combined with appropriate filler 1 hour（Crack supernatant It should be consistently placed on ice）.

Collect efflux.

Washed 1-2 times with bindingbuffer, collect cleaning solution respectively.

With 1mL various concentrations elution destination proteins, eluent is collected respectively.

SDS-PAGE is analyzed.Wherein, albumen Rv0232 and Rv1031 SDS-PAGE results are as shown in Figure 3.

BCA kit protein quantifications.

BCA kit protein quantification quantitative comparisons are adapted to multiple protein quantifications, based on 96 orifice plates, by kit program Operation.

2nd, experimental result

1600 albumen are obtained in purifying, for the experiment in embodiment 3.

Remaining 316 albumen does not obtain in purge process, is not limited to the limitation of theory, and reason is probably：Purifying Process presses inclusion body purification, if fusion protein may be lost；Large scale purification is not carried out again to these albumen again Purifying；It is probably that expression quantity is very low, small size purifying cannot.

Embodiment 3：ELISPOT is analyzed

ELISpot（ELISPOT）Technology is that the optimal skill of biological cell immune level is detected in the world today Art.It collects many advantages, such as high sensitivity, high confidence level, high flux, individual cell level, feature detect and are inexpensive in one Body, educational circles is at home and abroad immunized and obtains a wide range of applications, turns into one of main flow immunology detection technology.ELISPOT detections are former Reason is as follows：

A. the bottom of detection hole is coated on the monoclonal antibody of anti-gamma interferon.

B. the lymphocyte of sample to be detected is separated.

C. cell to be measured is put into detection hole and cultivated about 20 hours, while add antigen（Stimulant）.In the training period, Resisting the T lymphocytes of original idiosyncrasy will be activated, and start to secrete specific cell factor（Such as gamma interferon）, this A little cell factors are captured by the monoclonal antibody at plate bottom simultaneously；It is then not stimulated to the responseless cell of antigen, also do not secrete Specific cell factor（Such as gamma interferon）.

D. emigrated cells, plate bottom leave the potential image of cell factor.

E. this potential image can become real image-speckle by way of enzyme-linked colour developing.

F. each spot represents a T lymphocyte specific for having reaction to specific antigen.The number of spot is more The identification state of few cellular immunity for just reflecting sample：Blurring illustrates that Immune discrimination state is good, and spot is saved your breath bright immune knowledge There is immune tolerance in other state difference.

The immune response of tuberculosis infection is based on cellular immunity, and as a part for immune response, T cell is by tuberculosis Antigenic stimulus sensitization, form the effector T cell of activation, including CD4 and CD8, be individually separated from whole blood, in vitro by The stimulation of antigentic specificity is simultaneously counted.Therefore diagnosis lungy can be diagnosed with adopting said method.

Two individually have specific antigen ESAT-6 and CFP10, and use in conjunction can improve detection sensitivity. ELISPOT detections are highly sensitive, and before the cell factor diffusion dilution of cell secretion, it can capture cell peripheral immediately Secreted cell factor.Therefore this experiment detects positive patient to ESAT-6 with the detection platform of clinical patient（Clinic is examined Disconnected standard is：ESAT-6 is positive, and phlegm applies the positive or culture positive or PCR is positive）For research object, its PBMC is extracted, is pressed Screening of the ELISPOT detection methods screening available for diagnosis of cell mediated immunity molecule diagnostic marker.

First, experimental method

Kit using Shenzhen up to section for offer（Human IFN-γ precoated Elispot Kit, article No.： DKW22-1000-500）Tested.

Periphery lymphocyte is separated, and makes cell number be 2x10⁶Every milliliter, 100 μ l cell suspensions are added per hole, and (serum-free is trained Support base diluting cells)（Shenzhen is DKW34-EU0100 up to section）, be separately added into stimulant, if blank control, positive control and The positive control, negative control etc. of expression is tested, covers plate, puts cell in 37 DEG C, CO₂Incubator, suitable time 15-20 are small When（It not shake or translate during this period flat board）；Wherein：

Positive control is ESAT-6（The μ g/ml of final concentration 5）And the ESAT-6 of this experiment expression and purification（8μg/ml）, simultaneously If negative control（System buffer solution）And blank control（Refinement born of the same parents are not added with stimulant）, using the albumen of expression and purification as to be checked Survey albumen（8μg/ml）.If the positive control ESAT-6 assays of kit are the positive, the ESAT- of this experiment expression and purification 6 controls are similarly positive, and it is also the positive to treat Reichl's test, then this treats that Reichl's test can be used as positive reaction antigen.Concentration Remarks：The albumen of expression and purification only purifies by HIS labels, is not purified further, therefore the egg really to work White amount is by glue map analysis, is calculated in the ratio shared by actual expressing protein after purification total protein concentration.

This experiment uses phytolectin PHA as positive charge condition, as long as it has with regard to spottiness in cell hole, because It is that non-specific stimulation cell produces some factors and can show spotting out（The μ g/ml of final concentration 5）.

Culture plate is flicked on sink and empties liquid, and is patted dry on blotting paper；

Add PBSs of the 100 μ l containing 0.1%Tween20 per hole, 4 DEG C are placed 2 minutes；

Liquid is discarded, repeatedly with the PBS board-washings 5 times containing 0.1%Tween20, and is thoroughly patted dry；

Each block of plate, dilution 100 μ l detection antibody（The anti-human INF gamma antibodies of biotin labeling, i.e., 2 is anti-）It is reagent There is provided, can be combined with coated monoclonal antibody in box；

Into PBSs of the 10ml containing 1%BSA, 100 μ l are added per hole, closing culture plate is put in 37 DEG C 1 hour；

Empty culture plate and washed 5 times with the PBS containing 0.1%Tween20, patted dry；

Each block of plate, dilution 10 μ l Streptavidins-alkaline phosphatase multienzyme complex is into PBSs of the 10ml containing 1%BSA, per hole Add 100 μ l, closing culture plate is put in 37 DEG C 1 hour；

Empty, washed 5 times, inhaled repeatedly with blotting paper to remove remaining liquid all to the greatest extent with the PBS containing 0.1%Tween20.

Add the 100 standby chromophoric solutions of μ l per hole, allow reaction to carry out 5-20 minutes in room temperature.Visually visible fleck is formed.

With distillation washing three times.

Dry per hole, count spot.Spot may become obvious after being stayed overnight at 4 DEG C, place culture plate in room temperature, avoid Direct illumination.

2nd, experimental result

The identification of 1600 albumen has been carried out altogether, and part of test results is as shown in Figure 4.

Primary dcreening operation has been carried out to 120 clinical patients, has obtained 61 positive proteins.Wherein, decision method is：Readings is more than 40 Individual point is determined as the positive, and standard positive hole is positive and expressing protein records statistical result, and calculation expression albumen and mark to be positive The coincidence rate of quasi- positive protein.

Secondary screening is carried out to reacting 10 stronger albumen in this 61 positive proteins, secondary screening method is identical with primary dcreening operation.

Secondary screening as shown in figure 5, patient's number of secondary screening 10, normal healthy controls 4, has obtained 2 and has been used now Positive criteria the reactive protein albumen Rv0232 and Rv1031 that meet, coincidence rate is respectively 70% and 60%, and computational methods are tables The ratio that the protein positive number reached accounts in the ESAT-6 number positives in kit.

Table 3：The ELISPOT results of two albumen of checking high level reaction

It is computed, Rv0232 and Rv1031 and ESAT-6 response intensity are respectively 56% and 46%（Expressing protein reflecting point Number and the ESAT-6 of expression react the ratio between points）, for the two albumen with lower than ESAT-6 coincidence rate, response intensity is also slightly weak, but Immunodiagnosis molecular marker and vaccine candidate object as cellular level have important application value.

Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to disclosed all teachings, those details can be carried out with various modifications and replacement, these change in the guarantor of the present invention Within the scope of shield.The four corner of the present invention is provided by appended claims and its any equivalent.

Claims

1. purposes of the albumen in medicine for diagnosing tuberculosis is prepared, wherein, the amino acid sequence such as SEQ ID of the albumen NO：Shown in 1.

2. purposes according to claim 1, wherein, the tuberculosis is pulmonary tuberculosis.

3. purposes of the nucleotides in medicine for diagnosing tuberculosis is prepared, wherein, the nucleotide coding amino acid sequence such as SEQ ID NO：Albumen shown in 1.

4. purposes according to claim 2, wherein, the sequence such as SEQ ID NO of the nucleotides：Shown in 3.

5. the purposes according to claim 3 or 4, wherein, the tuberculosis is pulmonary tuberculosis.