CN1696289A - Protein of tuberculosis in use for diagnosing tuberculosis - Google Patents
Protein of tuberculosis in use for diagnosing tuberculosis Download PDFInfo
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- CN1696289A CN1696289A CN 200410044568 CN200410044568A CN1696289A CN 1696289 A CN1696289 A CN 1696289A CN 200410044568 CN200410044568 CN 200410044568 CN 200410044568 A CN200410044568 A CN 200410044568A CN 1696289 A CN1696289 A CN 1696289A
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Abstract
A tuberculosis mycobacterium protein used for diagnosing tuberculosis and epidemiologic survey and monitor of tuberculosis, the polynucleotide for coding it, the hybrid plasmid and procaryotic host cell containing said polynucleotide, and the process for preparing said protein are disclosed.
Description
Technical field
The present invention relates to a kind of Mycobacterium tuberculosis albumen that utilizes genetically engineered preparation, and this albumen is in diagnosis of tuberculosis, tuberculosis epidemiology investigation and Application in Monitoring with it is as the application of antigen composition in vaccine.
Background technology
Mycobacterium tuberculosis is pathogenic bacterium lungy, and about three million peoples in the annual whole world suffer from the tuberculosis that tubercule bacillus causes.China is that 22 tuberculosis height are born one of country in the world, and patient's number occupies the second place of the world, is only second to India.In recent years, number of the infected lungy is in rising trend again in China.
Tuberculosis is a kind of respiratory infectious disease, therefore, to this disease early stage, accurately diagnosis is a key of effectively controlling this disease wide-scale distribution.
In the twenties in 20th century, the foreign study person the thickening filtration thing of the tubercule bacillus cracked soluble product of growing in the liquid nutrient medium has been made allergen for skin test (ot, OT).Seiber in 1934 sets up ammonium sulfate salting-out process and has prepared PPD.Nineteen eighty-two, China began to develop PPD, its basic preparation method is: the logical substratum of will reviving is cultivated 8-10 week culture for 37 ℃, 100 ℃ of 60min deactivation after-filtration are removed thalline, precipitate with the final concentration 2% Tricholroacetic Acid method of saltouing then, the purifying protein derivative, this protein derivatives is PPD[kingdom and controls, middle national defence consumptive disease magazine, 1991].OT and PPD all can cause the DTH reaction of mycobacterium sensitization body, are widely used in the tuberculosis tentative diagnosis, tuberculosis epidemiology investigation and monitoring.But, since among OT and the PPD except that the albumen derivative, also comprise the tubercule bacillus meta-bolites, some compositions in thalline polysaccharide, nucleic acid, lipid material and the substratum etc. usually cause non-specific cross-reaction and other side reactions.U.S. Konstantin points out that in report PPD antigen and nearly all mycobacterium have common antigen, exists cross reaction widely [Konstantin L, et al., Infection and Immunity, 1998]; Huang Jian report, in 1184 crowds' epidemiology survey, the skin abnormality reaction appears in injection site, as blister appears, downright bad to account for 1.7%[yellow strong, middle national defence consumptive disease magazine, 1994]; Reports such as Liu Yuandong, blister wherein appears in PPD skin test strong positive 266 people, downright bad number is 132 people, accounts for 49.6%[Liu Yuan east etc., middle national defence consumptive disease magazine, 2000]; Among report such as Wu Lanzhen 89 routine tuberculosis diseases (pulmonary tuberculosis, the outer tuberculosis of the lung) patient, blister appears in the PPD skin test, and necrosis and reaction range surpass the above person of 20mm and account for 34.7%[Wu Lan treasure etc., middle national defence consumptive disease magazine, 1991].In addition, also have the only a few patient systemic anaphylaxis reaction behind the PPD skin test, to occur, as allergy necrotizing vasculitis, anaphylactoid purpura, lymphangitis, anaphylactic shock etc.
In view of PPD exists above-mentioned toxic side effects and poor specificity problem, in recent years, that the researchist can replace in searching, more special, many good tries have been carried out in safer allergen aspect, in numerous researchs, Mycobacterium tuberculosis 38KDa albumen receives much attention.
Mycobacterium tuberculosis 38KDa albumen is the main immunogen of Mycobacterium tuberculosis, has stronger immunocompetence.National scholar's joint studies such as Britain such as Wilkinson, Denmark, Italy, India, Germany, in report in 1997, do the test of cavy DTH allergen with 38KDa albumen, its effect is better than 17KDa, 19KDa, 24KDa and 32KDa albumen, and its effectiveness is similar substantially to PPD.This 38KDa albumen brings out by mycobacterium sensitized guinea pig in Mycobacterium tuberculosis, BCG (bacille Calmette-Guerin vaccine) and the born of the same parents and produces DTH, and do not take place or the only extremely slight reaction [Wilkinson et al, Journal of Clinical Microbiology 1997] of generation with bird mycobacterium, mycobacterium kansasii, scrofula mycobacterium.Although this 38KDa albumen has allergen characteristic preferably, the problem that exists is that the synthetic 38KDa albumen of Mycobacterium tuberculosis is few, only accounts for the thousandth of total protein.Because it is low and need cultivate and operate poisonous bacterial classification in a large number to obtain the 38KDa protein yield by Mycobacterium tuberculosis, there is the danger that infects the people, therefore, the research worker has dropped into a large amount of experimental studies, produces reorganization 38KDa albumen by genetic engineering technique fermentation intestinal bacteria.At present, though produce 38KDa albumen its output is increased greatly by genetically engineered, for example, M.Singh etc. are by recombination bacillus coli K-12 bacterial strain CAG629[pMS9-2] prepared reorganization Ag38 albumen, the Recombinant Protein Expression amount has reached 10% of total cell protein, but such expression amount can not satisfy the needs of clinical diagnosis of tuberculosis and tuberculosis epidemiology investigation far away, has influenced the practical application of this albumen in the tuberculosis field.
At present the utmost point need find a kind of can mass production, have good specificity again, and a safer Mycobacterium tuberculosis allergen, overcome people and thirst for the existing problem of diagnosis of tuberculosis and screening aspect that solves for a long time.
The present invention is by big quantity research, found a kind of Nucleotide of particular sequence, with importing prokaryotic host cell behind the nucleic acid construct recombinant expression vector with this nucleotide sequence, as expression in escherichia coli, the expression of recombinant proteins amount is multiplied, reaches and account for 36% of total bacterioprotein.And the albumen after the expression confirms that by animal experiment and clinical trial it has good allergen to render a service and specificity highly, and side reaction is minimum.
Summary of the invention
The invention summary
The invention discloses the proteic nucleotide sequence shown in sequence 2 of a kind of Mycobacterium tuberculosis of encoding, utilize this nucleotide sequence to prepare the proteic method of Mycobacterium tuberculosis, and by the Mycobacterium tuberculosis albumen of this method preparation.
Mycobacterium tuberculosis albumen of the present invention is by the expression of nucleic acid of nucleotide sequence shown in the sequence 2.Expressed proteic aminoacid sequence is shown in sequence 3.
The nucleotide sequence of the present invention shown in sequence 2 can insert in the carrier that is adapted at expressing in the prokaryotic host cell and be built into expression vector, thereby expresses in prokaryotic host cell.Preferred procaryotic cell expression carrier is PET9d, PET22b (+) or PET15b plasmid; Preferred prokaryotic host cell is intestinal bacteria, more preferably BL21 (DE3) or HMS174 (DE3).
The present invention utilizes nucleotide sequence shown in the sequence 2 to prepare Mycobacterium tuberculosis albumen by gene engineering method and can realize as follows:
1) obtains to have the polynucleotide sequence shown in the sequence 2;
2) this polynucleotide sequence is imported plasmid, preferred PET9d, PET22b (+) or PET15b plasmid;
3) this plasmid is imported prokaryotic host cell, preferred, e. coli host cell is more preferably among BL21 (DE3) or the HMS174 (DE3);
4) under the condition that helps described polynucleotide sequence expression, cultivate described host cell; With
5) recombinant protein that recovery, purifying and renaturation are expressed.
The invention still further relates to and comprise proteic composition of Mycobacterium tuberculosis of the present invention and test kit.Described composition can be used as diagnosis of tuberculosis and tuberculosis epidemiology investigation and monitoring reagent, is used for investigation, screening and the monitoring of clinical diagnosis lungy and tuberculosis epidemiology.
Described albumen or composition also can be prepared into reagent box for tuberculate diagnosis or tuberculosis epidemiology investigation screening reagent box, are advantageously used in investigation, screening and the monitoring of clinical diagnosis lungy and tuberculosis epidemiology.
The present invention relates to investigation of a kind of new diagnosis of tuberculosis method and tuberculosis epidemiology and monitoring method, this method comprises that utilization comprises proteic reagent of Mycobacterium tuberculosis of the present invention or test kit, by tuerculoderma, or serological test comes the diagnosis of tuberculosis patient, or filters out the tuberculosis suspected patient.Therefore, the invention still further relates to the reagent or the test kit that comprise the proteic tuberculosis skin diagnosis of Mycobacterium tuberculosis of the present invention or epidemiology survey and monitoring, and the reagent or the test kit that comprise the proteic tuberculosis serological diagnosis of Mycobacterium tuberculosis of the present invention or epidemiology survey and monitoring.
Mycobacterium tuberculosis albumen of the present invention confirms to have good immunogen by animal and clinical trial and renders a service, and have high specificity, the little characteristics of side effect as the tubercule bacillus allergen.Therefore, it may occur to persons skilled in the art that Mycobacterium tuberculosis albumen of the present invention can be used to prepare the Mycobacterium tuberculosis vaccine, as subunit vaccine etc.Equally, the nucleotide sequence shown in sequence 2 disclosed in this invention also can be used to prepare dna vaccination, in order to prevention tuberculosis.Therefore, the invention still further relates to the Mycobacterium tuberculosis vaccine that comprises Mycobacterium tuberculosis albumen of the present invention or nucleic acid.
Detailed Description Of The Invention
The invention discloses the proteic nucleotide sequence shown in sequence 2 of a kind of Mycobacterium tuberculosis of encoding, utilize this nucleotide sequence to prepare the proteic method of Mycobacterium tuberculosis, the Mycobacterium tuberculosis albumen that comprises aminoacid sequence shown in the sequence 3 by this method preparation, and comprise this proteic composition and test kit, their application in diagnosis of tuberculosis and tuberculosis epidemiology investigation and monitoring are also disclosed simultaneously.In addition, Mycobacterium tuberculosis albumen of the present invention and nucleic acid of the present invention also can be used for the preparation of Mycobacterium tuberculosis vaccine.
One embodiment of the invention relate to the proteic nucleotide sequence shown in sequence 2 of a kind of Mycobacterium tuberculosis of encoding.Base be numbered conventional 5 ' to 3 ' (initiation codon of translation chain is to the termination codon order) in proper order.
Nucleotide sequence shown in the sequence 2 can be by the method preparation of this area routine, preferably according to the Pab gene order, nucleotide sequence shown in sequence 1 (being numbered conventional 5 ' to 3 ' in proper order of base), the synthetic one couple of PCR primers of design, the primer of sequence increases shown in the preferred sequence 4 and 5, and amplified production is carried out following sudden change: make its disappearance 5 ' end 1-72 bit base 1.; 2. 76-78 bit base TCG is sported TCT; 3. making termination codon is TAA.
The change of polynucleotide sequence structure influences for example secondary structure of nucleic acid higher structure, thereby influences the expression level of this nucleic acid in host cell.The present invention finds that have as mentioned above the nucleic acid molecule of structure can improve the expression productive rate greatly with suitable carrier and host cell expression the time.
One embodiment of the invention relate to the nucleotide sequence of application shown in sequence 2 and prepare the proteic method of Mycobacterium tuberculosis.According to conventional methods, can with contain code book invention Mycobacterium tuberculosis proteic shown in sequence 2 cDNA or the DNA nucleic acid molecule of nucleotide sequence be connected in the expression vector, then by the ordinary method transformant.Conversion is meant as the outer composition of karyomit(e) or DNA is imported biological so that this DNA can duplicate through chromosomal integration.According to used host cell, use the standard technique that is suitable for this cell to transform.Cohen, institute of NAS newspaper, 69:2110 (1972) and Mandel etc., the molecular biology magazine, the calcium processing of the described employing calcium chloride of 53:154 (1970) generally is used for protokaryon or other contains the cell of a large amount of cell walls obstacles.
Usually, preferred prokaryotic organism are used for the initial clone of dna sequence dna and are used for vector construction of the present invention.For example, intestinal bacteria, genus bacillus such as subtilis and such as the various bacteriums of other bacterium of enterobacteriaceaes such as Salmonella typhimurium or serratia marcescens and Rhodopseudomonas.
In general, contain with the plasmid vector of matched replicon of prokaryotic host cell and regulating and controlling sequence and can both be used in combination with described host.But the proteic dna sequence dna of optimized encoding the present invention is inserted into PET9d, PET22b (+) or PET15b (+) carrier forms recombinant expression vector.The heterozygosis plasmid that code book is invented proteic nucleic acid and PET9d, PET22b (+) or PET15b (+) formation has the stability of height, helps the proteic expression of the object of the invention.
The promotor of Mycobacterium tuberculosis can not be discerned by the e. coli rna polysaccharase, the T7 phage rna polymerase can only be discerned the T7 phage promoter, so select T7 phage promoter should select the to encode host bacterium of T7 phage rna polymerase could realize expression.Contain (DE3) lysogen in theory, and the host bacterium that does not produce the T7 phage rna polymerase before expressing protein all can be selected, for example, HMS174 (DE3), JM109 (DE3), BL21 (DE3) all can efficiently express the object of the invention albumen, but preferred HMS174 (DE3) and BL21 (DE3), these two kinds of proteic output of host cell expression the object of the invention are higher, and its fast growth, help enhancing productivity.
In a preferred embodiment of the invention, as shown in Figure 1, preparation contains the expression construct of the nucleic acid molecule and the PET9d plasmid of polynucleotide sequence shown in the sequence 2, and this construct is transformed BL21 (DE3), after 4-5 hour, collects thalline through the IPTG inducing culture.The target protein expression amount is through the quantitative assay of gel densitometric scan, and the result accounts for the proteic 36%-40% of whole cell, and the gained target protein detects through mass spectroscopy, and molecular weight is 35.8KDa.
Can adopt the conventional resulting albumen of purification process purifying, and make its renaturation.Known purification process comprises, for example the absorption and desorption, ultracentrifugation, gel-filtration, affinity chromatography or the specificity purification process that carry out of ion-exchanger.Albumen of the present invention behind the purifying can be with comprising, for example, dithiothreitol (DTT) or 3-mercaptoethanol are in interior conventional reduction agent renaturation.
One embodiment of the invention relate to the Mycobacterium tuberculosis albumen with method for preparing, purifying, renaturation, and this albumen has aminoacid sequence shown in the sequence 3.
One embodiment of the invention relate to and comprise proteic composition of Mycobacterium tuberculosis of the present invention and test kit.Described composition or test kit can be prepared into diagnosis of tuberculosis and tuberculosis epidemiology investigation and monitoring reagent or kit form.
One embodiment of the invention relate to and comprise the proteic diagnosis of tuberculosis of Mycobacterium tuberculosis of the present invention and tuberculosis epidemiology investigation and monitoring reagent.In this product, albumen of the present invention can dry powdered form exist, and is diluted to desired concn before use; Also can certain density solution form exist.Comprising the investigation of proteic diagnosis of tuberculosis of Mycobacterium tuberculosis of the present invention and tuberculosis epidemiology can comprise a container and be positioned at label or package insert this vessel surface or relevant with this container with monitoring reagent.Proper container has bottle, bottle, syringe etc.Container can be made by various materials such as glass or plastics.This container can include and detect the proteic composition of the present invention lungy, and has aseptic access port (for example this container can be the bottle that has stopper that can be by the intradermal injection needle penetration).Described label or described using method and the description of use that comprises the proteic composition of Mycobacterium tuberculosis of the present invention of package insert explanation.In addition, this product also can further comprise second container, wherein can contain pharmaceutically useful buffer reagent, for example the sterilized water of injection (BWFI), phosphate-buffered salt, Ringer ' solution and glucose solution.Also can comprise other material that satisfies user's needs, as other damping fluid, thinner, filter, syringe needle and syringe.
Another embodiment of the present invention relates to the test kit that contains the proteic diagnosis of tuberculosis of Mycobacterium tuberculosis of the present invention and epidemiology survey, screening and monitoring, be used for clinically easily and fast and diagnosis of tuberculosis accurately, and, more apparent its advantage of this test kit in carrying out large-scale tuberculosis epidemiology investigation and monitoring.
" test kit " described herein is meant and utilizes albumen of the present invention to finish that tuberculosis detects and reagent set that assembly is made.This test kit is used for diagnosis of tuberculosis, or is used for epidemiological survey lungy and monitoring.In this test kit, comprise a container, this container contains Mycobacterium tuberculosis albumen of the present invention.Mycobacterium tuberculosis albumen of the present invention can be any easily, suitable packaging means packs.For example, if it is a freeze-dried preparation, the ampoule or the phial of band elastic plug can be used as container usually, prepare certain density protein solution of the present invention so that can inject liquid through elastic plug.The ampoule most convenient of ampoule of nonelastic removable lid (as sealed glass) or band elastic plug is used for the proteic injectable forms of Mycobacterium tuberculosis of the present invention.Test kit of the present invention also can comprise other a plurality of containers, wherein can contain to detect used standard substance antibody, the enzyme of antibody or process mark, substrate or damping fluid etc. respectively.In this test kit, comprise that also label and package insert are in order to provide the operation instruction of test kit.Also can comprise other material that meets user's needs, as microtiter plate or the like.
At the test kit that is used for the tuberculosis serological auxiliary detection, Mycobacterium tuberculosis albumen of the present invention also can be through mark.Specifically can use marks such as enzyme, fluorescent substance, luminophore, radioactive substance, metallo-chelate.Preferred mark enzyme for example, peroxidase, alkaline phosphatase, beta-D-galactosidase, malate dehydrogenase (malic acid dehydrogenase), staphylococcal nuclease, δ-5-steroid isomerase, α-Gan Youlinsuantuoqingmei, Triosephosphate isomerase, horseradish peroxidase, asparaginase, glucose oxidase, rnase, urease, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase, and acetylcholinesterase etc.Preferred fluorescent substance has fluorescein isothiocyanate, phycobiliprotein, rhodamine, phycoerythrin, Phycocyanins, C-, allophycocyanin, and Phthalyldicarboxaldehyde.Preferred luminophore has different luminol,3-aminophthalic acid cyclic hydrazide, lucigenin, luminol,3-aminophthalic acid cyclic hydrazide, aromatic series acridinium ester, imidazoles, acridinium salt and modification type ester thereof, luciferin, luciferase, and aequorin.Preferred radioactive substance has
125I,
127I,
131I,
14C,
3H,
32P,
35S etc.Preferred metallics has Radioactive colloidal gold etc.
With above-mentioned marker bonded method be known.Specifically can use direct and indirect labelling method.Direct labelling method commonly used is, with albumen of the present invention and marker by a kind of linking agent with chemical covalent bonds.Linking agent has N, N '-adjacent phenylene dimaleimide, 4-(N-maleimide aminomethyl) cyclohexylenedinitrilotetraacetic acid N-succinimide ester, 6-maleimide lpsilon N-succinimide ester, 4,4 '-dithio pyridine, and other known linking agent.The example of indirect labelling method has, albumen of the present invention is combined with lower molecular weight haptens such as vitamin H, dinitrobenzene, pyridoxal or fluorescamine, and with albumen of the present invention with haptenic in conjunction with the counterpart indirect labelling.The recognition ligand that avidin and streptavidin can be used as vitamin H uses.
When marker was enzyme, its substrate and developer can be used for measuring its activity.When using peroxidase, with H
2O
2As substrate solution, and with 2,2 '-azine-two-[3-ethyl benzo thiazole phenanthroline sulfonic acid] ammonium salt (ABTS), 5-aminosalicylic acid, O-Phenylene Diamine, the amino antipyrine or 3 of 4-, 3 ', 5,5 '-tetramethyl benzidines etc. are as developer.When using alkaline phosphatase, available ortho-nitrophenyl phosphoric acid, p-nitrophenyl phosphoric acid etc. are as substrate.When using beta-D-galactosidase, can use fluorescein-two-(β-D-gala pyranoside), 4-methyl umbrella shape base (umbelliferyl)-β-D-gala pyranoside etc. as substrate.
One embodiment of the invention relate to using and comprise the proteic reagent of Mycobacterium tuberculosis of the present invention or test kit to individuality, for example tuberculosis suspected patient, the epidemic-stricken area crowd individuality that maybe might infect tubercule bacillus detects, with auxiliary diagnosis lungy or filter out suspicious patient.
In one embodiment, the present invention relates to a kind of new diagnosis of tuberculosis or epidemiology survey monitoring method, described diagnosis or epidemiology survey and monitoring comprise proteic reagent of Mycobacterium tuberculosis of the present invention or test kit by application, adopt conventional serological method to realize, for example, can use the tuberculosis mycobacteria antibodies that comprises in proteic reagent of Mycobacterium tuberculosis of the present invention or the body fluid such as test kit detection serum, cerebrospinal fluid.The method that detects the tuberculosis mycobacteria antibodies has indirect test, as Mycobacterium tuberculosis proteantigen of the present invention is adsorbed in solid phase carrier, add serum to be checked then, exist if any corresponding antibodies, then on carrier, form antigen-antibody complex with antigen, the washing back adds enzyme mark anti-antibody or colloid gold label anti-antibody, the colour developing observations.Described indirect test method is ELISA method, spot enzyme immunity percolation method, spot gold-marking immunity percolation process etc. for example.Perhaps, serology detects can use sandwich assay, for example, makes sample to be checked and the albumen test of the present invention that is bonded to the activated protein of the present invention and the mark of insoluble carrier, detects the antibody amount in the sandwich complex of this reaction generation.Perhaps also available competition experiments is carried out the detection of tuberculosis mycobacteria antibodies, for example, make tuberculosis mycobacteria antibodies and the tuberculosis mycobacteria antibodies in the sample and the protein competition reaction of the present invention of mark, according to determining the amount of the tuberculosis mycobacteria antibodies in the sample with the amount of the tuberculosis mycobacteria antibodies of the mark of albumen test of the present invention.Tiring of tuberculosis mycobacteria antibodies is higher than contrast 1-4 doubly in the sample, and preferred 2-3 times, more preferably 2 times, clinically tuberculosis there is the auxiliary diagnosis meaning, aspect the tuberculosis epidemiology investigation meaning of monitoring is being arranged.
Any biological sample as body fluid such as blood plasma, serum, blood, urine, tissue juice or cerebrospinal fluid, as long as they contain the tuberculosis mycobacteria antibodies, with regard to available albumen of the present invention, comprises this proteic composition or test kit and detects.
In one embodiment, the present invention relates to a kind of new diagnosis of tuberculosis or epidemiology survey monitoring method, described diagnosis or epidemiology survey and monitoring comprise proteic reagent of Mycobacterium tuberculosis of the present invention or test kit by application, adopt for example tuberculosis intradermal vaccination tuerculoderma commonly used to carry out auxiliary diagnosis lungy, or carry out epidemiology survey lungy and monitoring.For example, albumen of the present invention can for example pass through, available Motouw method, and sterilization 1ml disposable syringe is drawn 0.1ml Mycobacterium tuberculosis albumen of the present invention in the 1/3 place intradermal injection of left forearm palmar, and seeing has skin mound proof to inject successfully.Measured horizontal stroke, the vertical footpath (mm) of injection site blush or subcutaneous scleroma in back 24 hours in injection.Average horizontal stroke, the vertical diameter 〉=5mm of injection site blush (or scleroma) are positive, and mean diameter＜5mm or reactionless (with 0 expression) are negative.In epidemiology survey lungy, behind the healthy population intradermal vaccination tuerculoderma reagent, to the skin test reactions positive particularly the healthy population of strong positive be the main crowd that tuberculosis prophylaxis need be monitored.
Detect and skin detection is used in testing comprises proteic all reagent of Mycobacterium tuberculosis of the present invention or test kit all comprises within the scope of the invention at above-mentioned tuberculosis serological.
Description of drawings
Fig. 1 shows the recombinant plasmid cleavage map.The linear plasmid DNA of the segmental goal gene of the as seen about 1.1kb of expression plasmid double digestion and about 4.5kb; The single endonuclease digestion expression plasmid only sees and is about the 5.6kb fragment that the dna sequence dna of proteins encoded contains the proteic gene of pre-expression in restriction enzyme site and carrier successful connection accurately in the recombinant plasmid, and the foreign gene that inserts size is consistent with theory.
Fig. 2-A-Fig. 2-B is dna sequence dna figure.This figure show dna sequence meets fully with design, and the mutational site sequence is correct, and is in full accord with genomic dna sequence with the genome complementation part.
Fig. 3 shows recombinant bacterial strain SDS-polyacrylamide gel electrophoresis figure.This figure shows engineering bacteria express recombinant protein under the IPTG inductive condition, and engineering bacteria is induced express recombinant protein hardly without IPTG.
Fig. 4 shows engineering bacterium expression expression of recombinant proteins amount.IPTG induces the scanning of engineering bacteria whole cell proteins gel electrophoresis to show that target protein accounts for 36% of total tropina, and peak 14 is the object of the invention recombinant proteins among the figure.
Fig. 5 is the purification result of recombinant protein of the present invention.Solubilization of inclusion bodies liquid obtains the higher recombinant protein of purity through twice anion column chromatography.During chromatography purification, the albumen applied sample amount is 10 μ g.Purifying is a band after the SDS-polyacrylamide gel electrophoresis is identified.
Fig. 6 is the proteic amino acid composition analysis figure of the present invention, and framework is accurate when inserting the gene coded protein in the carrier, and frameshit does not take place, and amino acid is formed with theoretical consistent, shows that Mycobacterium tuberculosis protein nucleic acid molecule of the present invention obtains to express in intestinal bacteria.
Fig. 7 is N end sequencer map.The N terminal sequence shows that the methionine(Met) of ATG coding is fallen N terminal amino acid sequence entirely accurate by the enzyme enzymolysis in the intestinal bacteria; Framework accurately is not shifted when further showing gene coded protein.
Specific embodiments
Hereinafter, reference example of the present invention is specifically addressed, but these embodiment are not construed as limiting the invention.
Embodiment 1: the proteic preparation of Mycobacterium tuberculosis of the present invention
According to the Pab gene order, by round pcr the Pab gene to be suddenlyd change, author designed has been synthesized one couple of PCR primers.Long 37 bases of upstream primer a1, shown in sequence 4,5 ' end has designed protection base and NCOI restriction enzyme site (containing codon ATG), long 34 bases of downstream primer a2, shown in sequence 5,5 ' end design BamHI restriction enzyme site and termination codon TAA.With Mycobacterium tuberculosis H37Rv genomic dna is template, archaeal dna polymerase round pcr by high-fidelity obtains code book and invents proteic polynucleotide (wherein 5 ' the end base sequence is ATGGGCTCT-(sequence 1) or ATGGGCTCA-(sequence 2), and wherein the most last base is suddenlyd change by former bases G.The pcr amplification product of this primer is through 1.2% agarose gel electrophoresis, and ultraviolet detection presents the specific amplified band, is about 1050bp, adheres to specification.The coding triplet TCG that the PCR product has removed N end coding 24 amino acid whose coding triplets (72 bases), original encoding the 26th amino acids sports TCT, termination codon is TAA.
Polynucleotide are cut with the BamHI enzyme through NCOI with expression vector after separate through 1.0% agarose gel electrophoresis, and cutting contains the agar block of DNA band, and press DNA fast purifying test kit specification sheets recovery DNA.Polynucleotide that reclaim and PET9d plasmid expression vector are at T
4Connect into reorganization heterozygosis plasmid under the dna ligase effect, reorganization heterozygosis plasmid transformed competence colibacillus e. coli jm109, by selecting the screening of recombination bacillus coli bacterium colony and identifying the intestinal bacteria bacillus JM109 that contains reorganization heterozygosis plasmid, require the single endonuclease digestion and the double digestion collection of illustrative plates of reorganization heterozygosis plasmid to meet Fig. 1.The goal gene that inserts in the reorganization heterozygosis plasmid automatically checks order through DNA and proves that sequence and design meet fully.The correct reorganization heterozygosis plasmid of target gene sequences transforms expressive host bacterium BL21 (DE3), when this recombination bacillus coli is cultured to optical density(OD) and is 0.6-0.8, adds 0.4mmol/L IPTG inducing culture, induces 4-5 hour, collects thalline.Under this inducing culture condition, the target protein expression amount accounts for the proteic 36%-40% of whole cell through the gel densitometric scan.Albumen of the present invention has been realized efficiently expressing by the change of nucleotide sequence and the selection of carrier and host cell.
Embodiment 2: the proteic purifying of Mycobacterium tuberculosis of the present invention, renaturation and evaluation
Thalline adds lysis buffer (the 1g bacterium adds the 3ml lysis buffer) suspension thalline.The ultrasonication bacterium, power 200W, ultrasonic 20 seconds, gap 20 seconds, ultrasonic 80 times altogether; Centrifugal 15 minutes of 4 ℃, 12000rpm are abandoned supernatant, and precipitation is washed 2-3 time with containing 1%Triton-X100 respectively.20mmol/LTris.Cl/8M urea (pH8.0) dissolving inclusion body, 4 ℃, the centrifugal 15min of 12000rpm collect supernatant.
The solubilization of inclusion bodies supernatant liquor is splined on the DEAE-52 anion-exchange column of handling well.Last sample finishes, with the abundant unconjugated albumen of flush away of 20mmol/L Tris.Cl/8 M urea (pH8.0), 0-0.3MNaCl gradient elution then, collect the solution of the corresponding collection tube of protein SDS-PAGE electrophoresis detection elution peak simultaneously, after the collection tube that contains target protein merges dialysis desalting, after the QFF anion column, 0-0.5M NaCl gradient elution, SDS-PAGE detects elution peak, contains the more and purer collection tube of target protein and merges, dialysis desalination renaturation.Renaturation process: damping fluid is 20mmol/L Tris.Cl/6M urea (pH8.0), 4 ℃ of dialysed overnight; Damping fluid is 20mmol/L Tris.Cl/3 M urea (pH8.0), 4 ℃ of dialysed overnight; Damping fluid be 20mmol/L Tris.Cl (pH8.0), 4 ℃ the dialysis 2 days.All contain the 5mM mercaptoethanol in all liquid.By preliminary reduction damping fluid urea concentration, thereby slowly remove the urea in the albumen and reach renaturation.
Whole purifying work is to finish on LKB chromatographic system instrument.
Purifying protein is through the SDS-polyacrylamide gel electrophoresis, and the gel densitometric scan shows this purity of protein near about 96%, and the HPLC purity testing shows that purity is also near 96%.
Mass spectrometric determination recombinant protein molecular weight shows that molecular weight of albumen of the present invention is 35.8KDa.
The analysis of N terminal amino acid sequence is finished by Peking University, and 15 amino acid of this protein N terminal are glycine (G)-Serine (S)-Methionin (K)-proline(Pro) (P)-proline(Pro) (P)-Serine (S)-glycine (G)-Serine (S)-proline(Pro) (P)-L-glutamic acid (E)-Threonine (T)-glycine (G)-L-Ala (A)-glycine (G)-L-Ala (A) successively.N end result proves that initial amino acid is correct, the coded amino acid coding triplet is correct.
Isoelectric point determination is 5.64.
Amino acid is formed and is conformed to peptide sequence.
Peptide quality fingerprinting atlas analysis shows that polypeptide Methionin of the present invention and arginine position in peptide sequence are accurately, thereby the indirect proof polypeptide structure is accurately.The methionine(Met) of this proteic codon ATG coding of encoding is not on the permanent staff in the albumen framework of sign indicating number.The proteic amino acid of the present invention is formed and is shown that this albumen is a kind of new Mycobacterium tuberculosis albumen.
Embodiment 3: Mycobacterium tuberculosis albumen of the present invention is used for the former cavy test of skin allergic reaction
Cavy DTH test: 100 300-350g cavys are divided into two groups at random, and 80 cavys are in the tubercule bacillus H37Rv of subcutaneous injection deactivation 0.2ml, and 4 weeks are carried out the DTH test in the injection back.Control group cavy injection 0.2ml physiological saline.All guinea pig back unhairings, in back, vertebra both sides corresponding site difference intradermal injection 0.1ml albumen of the present invention and 0.1ml PPD (requiring the injection site skin mound to occur is as the criterion), injected back 24 hours and detected in 48 hours the blush or horizontal, the vertical diameter (mm) of scleroma of injection site, horizontal, vertical mean diameter 〉=5mm is positive for injection site blush (or scleroma), mean diameter＜5mm or reactionless (with 0 expression) are negative, the results are shown in Table 1.
Table 1 albumen of the present invention is used for the cavy DTH test-results of tubercule bacillus sensitization
| Grouping | Reagent | The experimental guinea pig number of elements | Positive reaction number (%) | Average scleroma reaction (mm) |
| Control group | The PPD contrast | ????20 | ????0(0) | |
| Albumen of the present invention | ????20 | ????0(0) | ||
| The sensitization group | The PPD contrast | ????80 | ????80 | ????9.95±1.5 |
| Albumen of the present invention | ????80 | ????80 | ????9.23±1.9 |
The protein induced DTH of the present invention on average hardens and PPD ratio is 0.93, and the result shows that recombinant protein induces sensitized guinea pig to produce the DTH suitable with PPD and react.
The test of cavy DTH specificity: different mycobacterium sensitized guinea pigs, carry out the DTH test then.The result is as shown in table 2.
Table 2 Mycobacterium tuberculosis albumen of the present invention skin test allergen immunologic opsonin detected result
| Allergen | Detection time (h) | The local blush of the cavy of different sensitinogen sensitization (scleroma) reaction | |||||||||
| Tuberculosis | The Kansas | Bacille Calmette-Guerin vaccine | Accidental | Scrofula | |||||||
| Number positive | Diameter (mm) * | Number positive | Diameter (mm) | Number positive | Diameter (mm) | Number positive | Diameter (mm) | Number positive | Diameter (mm) | ||
| Human-like-PPD | ??24 | ????5/5 | ??/ | ????5/5 | ????/ | ????5/5 | ??/ | ????5/5 | ??/ | ????5/5 | ????/ |
| ??48 | ????5/5 | ??10.9/54.5 | ????5/5 | ????9.0/45.0 | ????5/5 | ??8.6/43.0 | ????4/5 | ??6.1/30.5 | ????5/5 | ????9.2/46.0 | |
| Albumen of the present invention | ??24 | ????5/5 | ??/ | ????2/5 | ????/ | ????3/5 | ??/ | ????0/5 | ??/ | ????1/5 | ????/ |
| ??48 | ????5/5 | ??10.7/53.5 | ????0/5 | ????0 | ????2/5 | ??2.4/12.0 | ????0/5 | ??0 | ????1/5 | ????1.3/6.5 | |
Annotate: diameter: mean value/accumulated value; /: do not detect
Table 2 result shows that albumen of the present invention and human-like PPD and Mycobacterium tuberculosis sensitized guinea pig all are positive, and albumen of the present invention and mycobacterium kansasii and accidental mycobacterium sensitized guinea pig no cross reaction, is lower than human-like PPD with bacille Calmette-Guerin vaccine sensitized guinea pig and scrofula mycobacterium sensitized guinea pig positive reaction rate.
More than test shows that the recombinant protein of the present invention of escherichia coli expression has the good allergen property renderd a service, and the DTH reaction of inducing the mycobacterium tuberculosis sensitized guinea pig to produce is suitable with PPD, but specificity is far above PPD.
Embodiment 4: Mycobacterium tuberculosis albumen of the present invention is used for the former clinical trial of skin allergic reaction
Clinical trial: selected lunger is essential through clinical definite.Consubstantiality both arms Motouw method, the 1ml disposable syringe of promptly sterilizing is drawn 0.1ml PPD in the 1/3 place intradermal injection of right forearm palmar, and seeing has skin mound proof to inject successfully.Other gets the 1ml disposable syringe, draws 0.1ml albumen of the present invention in the 1/3 place intradermal injection of left forearm palmar, and seeing has skin mound proof to inject successfully.In injection back 24 hours and 48 hours double blinding sentence read result, horizontal, vertical mean diameter 〉=5mm is positive for injection site blush (or scleroma), and mean diameter＜5mm or reactionless (with 0 expression) are negative.510 routine pulmonary tuberculosis patient tuerculoderma results are as shown in table 3.
Table 3 pulmonary tuberculosis patient skin test reactions result
| Observing time (hour) | Positive reaction rate (%) | Reaction is footpath (mm) X ± SD all | Positive reaction is footpath (mm) X ± SD all | Untoward reaction (%) | |
| Albumen of the present invention | ????24 | ?73.3(374/510) | ??7.31±4.58 | ????9.63±2.94 | ????0 |
| ????48 | ?68.2(347/510) | ??5.24±4.06 | ????7.64±2.42 | ????0 | |
| ????PPD | ????24 | ?76.3(389/510) | ??8.16±5.59 | ????10.66±3.79 | ????0.2(1/510) |
| ????48 | ?78.2(399/510) | ??9.74±6.23 | ????12.40±4.09 | ????5.9(30/510) |
Table 3 shows that albumen tuerculoderma result of the present invention observed with 24-48 hour, 24 hours albumen tuerculoderma positive reaction rates of the present invention and 48 hours suitable (X of PPD tuerculoderma positive reaction rate
2=3.07, P＞0.05).
It is suitable with PPD that above-mentioned evidence, albumen of the present invention are used for tuberculosis skin test diagnostic reaction positive rate, can meet clinical needs, and do not see any side reaction.On the contrary, detect with PPD, bubble or lymphangitis appear in 5.9% pulmonary tuberculosis patient PPD tuerculoderma.
Embodiment 5: Mycobacterium tuberculosis albumen of the present invention is used for serum diagnosis
The ELISA method detects 291 parts of serum: the every hole of albumen of the present invention bag is by 0.2-1 μ g, and PPD wraps by 0.2-1 μ g, and control wells adds coating buffer.This tests used coating buffer is 2.94g/L NaHCO
3With 1.50g/L Na
2CO3.Bag is discarded liquid by more than 2 hours, washes plate 3 times with PBST then, each 3 minutes.Sample diluting liquid (PBST that contains 0.1%BSA) 1: 100 dilution serum sample to be checked, every hole adds 100 μ l, 37 ℃ 1 hour, ditto wash plate 3 times.Dilute two anti-rabbit anti-human iggs of HRP mark on request with sample diluting liquid (PBST that contains 0.1%BSA), every hole adds 100 μ l, 37 ℃ 1 hour, ditto wash plate 3 times, every hole adds 100 μ 1 colour developing liquid, and (citric acid and biphosphate are received damping fluid dissolving DAB, face with before adding H
2O
2) colour developing, add 2 termination reactions of 2M sulfuric acid, measure each OD of 490nm wavelength place, hole on the microplate reader.Positive greater than 2 times of contrast OD values.29l part serum ELISA detected result sees Table 4.
Table 4 recombinant protein detects the result of serum with the ELISA method
| The serum source | Umber | Susceptibility (%) | Specificity (%) | ||
| ??TB-PPD | Albumen of the present invention | ??TB-PPD | Albumen of the present invention | ||
| Pulmonary tuberculosis patient | |||||
| The smear feminine gender | ??78 | ??54(69.2) | ??52(66.7) | ??--- | ??--- |
| The smear positive | ????30 | ????30(100.0) | ????29(96.7) | ????--- | ????--- |
| The healthy blood donor | ????105 | ????6(5.7) | ????3(2.9) | ????94.3 | ????97.1 |
| BCG (Bacille Calmette-Guerin) vaccination | |||||
| Sun commentaries on classics person | ????78 | ????26(32.1) | ????11(14.0) | ????67.9 | ????86.0 |
To show that albumen of the present invention is used for the serum of tuberculosis patients detection sensitivity suitable with PPD for the result in the table, but specificity is higher than PPD, influenced by the BCG inoculation and be starkly lower than PPD, this country to BCG plan inoculation adopts tuberculosis serological diagnosis significant.
Embodiment 5: Mycobacterium tuberculosis albumen of the present invention is used for the cerebrospinal fluid diagnosis
Spot enzyme immunity percolation method (DIEFA) rapid detection CSF antibody: get suitable concn the dry rear enclosed of albumen 0.1ml point film of the present invention, film, add CSF, wash film, add ELIAS secondary antibody, wash film, colour developing.Occur the black colorant spot on every film and be judged to the positive.Entire operation was finished in 15 minutes.130 parts of cerebrospinal fluid samples detect with DIEFA, the results are shown in Table 5.
Table 5 Mycobacterium tuberculosis albumen of the present invention is used for the result of cerebrospinal fluid diagnosis
| Number of samples | The sample type | Enzyme mark percolation process detects IgG antibody | ||
| Positive anti-property number | The positive reaction rate | Specificity | ||
| 100 parts | Tuberculosis patient cerebrospinal fluid | ????55 | ????55％ | |
| 32 parts | Non-tuberculosis patient cerebrospinal fluid | ????1 | ??97％ | |
Table 5 shows that albumen tuberculosis patient cerebrospinal fluid of the present invention detects positive rate and reaches 55%, and specificity reaches 97%.Set up the tuberculosis antibody in the rapid detection cerebrospinal fluid, can be the tuberculous meningitis quick diagnosis supplementary means is provided.
It is higher that table 4 and 5 results show that recombinant protein is used for antibody test susceptibility, and have higher specificity.
Sequence table
＜110〉village, beautiful brightness
What, elegant cloud
Open, little firm
＜120〉be used for the Mycobacterium tuberculosis albumen of diagnosis of tuberculosis
<130>PLMD4562N
<160>5
<170>PatentIn?version?3.1
<210>1
<211>1125
<212>DNA
＜213〉Mycobacterium tuberculosis H37Rv (Mycobacterium tuberculosis H37Rv)
<400>1
gtgaaaattc?gtttgcatac?gctgttggcc?gtgttgaccg?ctgcgccgct?gctgctagca??????60
gcggcgggct?gtggctcgaa?accaccgagc?ggttcgcctg?aaacgggcgc?cggcgccggt?????120
actgtcgcga?ctacccccgc?gtcgtcgccg?gtgacgttgg?cggagaccgg?tagcacgctg?????180
ctctacccgc?tgttcaacct?gtggggtccg?gcctttcacg?agaggtatcc?gaacgtcacg?????240
atcaccgctc?agggcaccgg?ttctggtgcc?gggatcgcgc?aggccgccgc?cgggacggtc?????300
aacattgggg?cctccgacgc?ctatctgtcg?gaaggtgata?tggccgcgca?caaggggctg?????360
atgaacatcg?cgctagccat?ctccgctcag?caggtcaact?acaacctgcc?cggagtgagc?????420
gagcacctca?agctgaacgg?aaaagtcctg?gcggccatgt?accagggcac?catcaaaacc?????480
tgggacgacc?cgcagatcgc?tgcgctcaac?cccggcgtga?acctgcccgg?caccgcggta?????540
gttccgctgc?accgctccga?cgggtccggt?gacaccttct?tgttcaccca?gtacctgtcc?????600
aagcaagatc?ccgagggctg?gggcaagtcg?cccggcttcg?gcaccaccgt?cgacttcccg????660
gcggtgccgg?gtgcgctggg?tgagaacggc?aacggcggca?tggtgaccgg?ttgcgccgag????720
acaccgggct?gcgtggccta?tatcggcatc?agcttcctcg?accaggccag?tcaacgggga????780
ctcggcgagg?cccaactagg?caatagctct?ggcaatttct?tgttgcccga?cgcgcaaagc????840
attcaggccg?cggcggctgg?cttcgcatcg?aaaaccccgg?cgaaccaggc?gatttcgatg????900
atcgacgggc?ccgccccgga?cggctacccg?atcatcaact?acgagtacgc?catcgtcaac????960
aaccggcaaa?aggacgccgc?caccgcgcag?accttgcagg?catttctgca?ctgggcgatc???1020
accgacggca?acaaggcctc?gttcctcgac?caggttcatt?tccagccgct?gccgcccgcg???1080
gtggtgaagt?tgtctgacgc?gttgatcgcg?acgatttcca?gctag???????????????????1125
<210>2
<211>1056
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: the sudden change of sequence 1
<400>2
atgggctcta?aaccaccgag?cggttcgcct?gaaacgggcg?ccggcgccgg?tactgtcgcg?????60
actacccccg?cgtcgtcgcc?ggtgacgttg?gcggagaccg?gtagcacgct?gctctacccg????120
ctgttcaacc?tgtggggtcc?ggcctttcac?gagaggtatc?cgaacgtcac?gatcaccgct????180
cagggcaccg?gttctggtgc?cgggatcgcg?caggccgccg?ccgggacggt?caacattggg????240
gcctccgacg?cctatctgtc?ggaaggtgat?atggccgcgc?acaaggggct?gatgaacatc????300
gcgctagcca?tctccgctca?gcaggtcaac?tacaacctgc?ccggagtgag?cgagcacctc????360
aagctgaacg?gaaaagtcct?ggcggccatg?taccagggca?ccatcaaaac?ctgggacgac????420
ccgcagatcg?ctgcgctcaa?ccccggcgtg?aacctgcccg?gcaccgcggt?agttccgctg????480
caccgctccg?acgggtccgg?tgacaccttc?ttgttcaccc?agtacctgtc?caagcaagat????540
cccgagggct?ggggcaagtc?gcccggcttc?ggcaccaccg?tcgacttccc?ggcggtgccg????600
ggtgcgctgg?gtgagaacgg?caacggcggc?atggtgaccg?gttgcgccga?gacaccgggc????660
tgcgtggcct?atatcggcat?cagcttcctc?gaccaggcca?gtcaacgggg?actcggcgag????720
gcccaactag?gcaatagctc?tggcaatttc?ttgttgcccg?acgcgcaaag?cattcaggcc????780
gcggcggctg?gcttcgcatc?gaaaaccccg?gcgaaccagg?cgatttcgat?gatcgacggg????840
ccggccccgg?acggctaccc?gatcatcaac?tacgagtacg?ccatcgtcaa?caaccggcaa????900
aaggacgccg?ccaccgcgca?gaccttgcag?gcatttctgc?actgggcgat?caccgacggc????960
aacaaggcct?cgttcctcga?ccaggttcat?ttccagccgc?tgccgcccgc?ggtggtgaag???1020
ttgtctgacg?cgttgatcgc?gacgatttcc?agctaa?????????????????????????????1056
<210>3
<211>350
<212>PRT
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: reorganization
<400>3
Gly?Ser?Lys?Pro?Pro?Ser?Gly?Ser?Pro?Glu?Thr?Gly?Ala?Gly?Ala?Gly
1???????????????5???????????????????10??????????????????15
Thr?Val?Ala?Thr?Thr?Pro?Ala?Ser?Ser?Pro?Val?Thr?Leu?Ala?Glu?Thr
20??????????????????25??????????????????30
Gly?Ser?Thr?Leu?Leu?Tyr?Pro?Leu?Phe?Asn?Leu?Trp?Gly?Pro?Ala?Phe
35??????????????????40??????????????????45
His?Glu?Arg?Tyr?Pro?Asn?Val?Thr?Ile?Thr?Ala?Gln?Gly?Thr?Gly?Ser
50??????????????????55??????????????????60
Gly?Ala?Gly?Ile?Ala?Gln?Ala?Ala?Ala?Gly?Thr?Val?Asn?Ile?Gly?Ala
65??????????????????70??????????????????75??????????????????80
Ser?Asp?Ala?Tyr?Leu?Ser?Glu?Gly?Asp?Met?Ala?Ala?His?Lys?Gly?Leu
85??????????????????90??????????????????95
Met?Asn?Ile?Ala?Leu?Ala?Ile?Ser?Ala?Gln?Gln?Val?Asn?Tyr?Asn?Leu
100?????????????????105?????????????????110
Pro?Gly?Val?Ser?Glu?His?Leu?Lys?Leu?Asn?Gly?Lys?Val?Leu?Ala?Ala
115?????????????????120?????????????????125
Met?Tyr?Gln?Gly?Thr?Ile?Lys?Thr?Trp?Asp?Asp?Pro?Gln?Ile?Ala?Ala
130?????????????????135?????????????????140
Leu?Asn?Pro?Gly?Val?Asn?Leu?Pro?Gly?Thr?Ala?Val?Val?Pro?Leu?His
145?????????????????150?????????????????155?????????????????160
Arg?Ser?Asp?Gly?Ser?Gly?Asp?Thr?Phe?Leu?Phe?Thr?Gln?Tyr?Leu?Ser
165?????????????????170?????????????????175
Lys?Gln?Asp?Pro?Glu?Gly?Trp?Gly?Lys?Ser?Pro?Gly?Phe?Gly?Thr?Thr
180?????????????????185?????????????????190
Val?Asp?Phe?Pro?Ala?Val?Pro?Gly?Ala?Leu?Gly?Glu?Asn?Gly?Asn?Gly
195?????????????????200?????????????????205
Gly?Met?Val?Thr?Gly?Cys?Ala?Glu?Thr?Pro?Gly?Cys?Val?Ala?Tyr?Ile
210?????????????????215?????????????????220
Gly?Ile?Ser?Phe?Leu?Asp?Gln?Ala?Ser?Gln?Arg?Gly?Leu?Gly?Glu?Ala
225?????????????????230?????????????????235?????????????????240
Gln?Leu?Gly?Asn?Ser?Ser?Gly?Asn?Phe?Leu?Leu?Pro?Asp?Ala?Gln?Ser
245?????????????????250?????????????????255
Ile?Gln?Ala?Ala?Ala?Ala?Gly?Phe?Ala?Ser?Lys?Thr?Pro?Ala?Asn?Gln
260?????????????????265?????????????????270
Ala?Ile?Ser?Met?Ile?Asp?Gly?Pro?Ala?Pro?Asp?Gly?Tyr?Pro?Ile?Ile
275?????????????????280?????????????????285
Asn?Tyr?Glu?Tyr?Ala?Ile?Val?Asn?Asn?Arg?Gln?Lys?Asp?Ala?Ala?Thr
290?????????????????295?????????????????300
Ala?Gln?Thr?Leu?Gln?Ala?Phe?Leu?His?Trp?Ala?Ile?Thr?Asp?Gly?Asn
305?????????????????310?????????????????315?????????????????320
Lys?Ala?Ser?Phe?Leu?Asp?Gln?Val?His?Phe?Gln?Pro?Leu?Pro?Pro?Ala
325?????????????????330?????????????????335
Val?Val?Lys?Leu?Ser?Asp?Ala?Leu?Ile?Ala?Thr?Ile?Ser?Ser
340?????????????????345?????????????????350
<210>4
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: upstream primer
<400>4
cgcatgccat?gggctctaaa?ccaccgagcg?gttcgcc???????????????????????????????37
18
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: downstream primer
<400>5
cgggatccaa?tgctggaaat?cgtcgcgatc?aacg??????????????????????????????????34
Claims (10)
1. nucleic acid, it has the nucleotide sequence shown in sequence 2.
2. proteic genetically engineered recombination and preparation of Mycobacterium tuberculosis, it is characterized in that application rights requires 1 nucleic acid construct expression vector, and in the prokaryotic host cell that this carrier was fit to, express the Mycobacterium tuberculosis albumen of the described nucleic acid encoding of claim 1.
3. the method for claim 2, wherein said expression vector is PET9d, PET22b (+) or PET15b plasmid.
4. the method for claim 2, wherein said host cell is e. coli bl21 (DE3) or HMS174 (DE3).
5. according to the Mycobacterium tuberculosis albumen of each described method preparation in the claim 2 to 4.
6. the proteic composition of Mycobacterium tuberculosis that comprises claim 5.
7. the described composition of claim 6, it is a diagnostic reagent of tuberculosis.
8. the described composition of claim 6, it is a tuberculosis epidemiology investigation and monitoring reagent.
9. the proteic test kit of Mycobacterium tuberculosis that comprises claim 5.
10. the test kit of claim 9, it is diagnosis of tuberculosis and tuberculosis epidemiology investigation and monitoring test kit.
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|---|---|---|---|
| CNB200410044568XA CN100393876C (en) | 2004-05-11 | 2004-05-11 | Protein of tuberculosis in use for diagnosing tuberculosis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200410044568XA CN100393876C (en) | 2004-05-11 | 2004-05-11 | Protein of tuberculosis in use for diagnosing tuberculosis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102713629A (en) * | 2009-11-20 | 2012-10-03 | 俄勒冈健康科学大学 | Methods for detecting a mycobacterium tuberculosis infection |
| CN105181837A (en) * | 2015-08-28 | 2015-12-23 | 程永娟 | Mycobacterium tuberculosis detection method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5714593A (en) * | 1995-02-01 | 1998-02-03 | Institut Pasteur | DNA from mycobacterium tuberculosis which codes for a 45/47 kilodalton protein |
| AU2003224313A1 (en) * | 2002-04-27 | 2003-11-17 | The Secretary Of State For Environment, Food And Rural Affairs | Mycobacterial antigens and uses thereof |
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2004
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102713629A (en) * | 2009-11-20 | 2012-10-03 | 俄勒冈健康科学大学 | Methods for detecting a mycobacterium tuberculosis infection |
| CN105181837A (en) * | 2015-08-28 | 2015-12-23 | 程永娟 | Mycobacterium tuberculosis detection method |
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Assignee: Guangdong Hansen Biological Pharmaceutical Co., Ltd. Assignor: The No. 309 Hospital of PLA Contract record no.: 2011990000438 Denomination of invention: Protein of tuberculosis in use for diagnosing tuberculosis Granted publication date: 20080611 License type: Exclusive License Open date: 20051116 Record date: 20110608 |