CN102584962A - Preparation method and application of mycobacterium tuberculosis Rv3117 recombinant protein - Google Patents
Preparation method and application of mycobacterium tuberculosis Rv3117 recombinant protein Download PDFInfo
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- CN102584962A CN102584962A CN201210070593XA CN201210070593A CN102584962A CN 102584962 A CN102584962 A CN 102584962A CN 201210070593X A CN201210070593X A CN 201210070593XA CN 201210070593 A CN201210070593 A CN 201210070593A CN 102584962 A CN102584962 A CN 102584962A
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Abstract
The invention relates to a preparation method and an application of a mycobacterium tuberculosis Rv3117 recombinant protein, effectively solving the problems of low diagnostic detection rate, high expense, complex operation and poor effect of a diagnostic reagent in tuberculosis. The preparation method of the Rv3117 recombinant protein comprises the following steps: according to an Rv3117 gene sequence, designing a target gene primer; taking a mycobacterium tuberculosis strain genome DNA as a template for PCR (polymerase chain reaction) amplification, and purifying to obtain a purified Rv3117 gene sequence; carrying out double enzyme digestion to obtain enzyme-digested products; inserting the enzyme-digested products into expression plasmids; cloning to create recombinant plasmids; transforming the created recombinant plasmids into host cells matched with the expression plasmids for carrying out protein expression; inducing the expressed protein Rv3117; separating and purifying the induced expression product to obtain the Rv3117 recombinant protein. The Rv3117 recombinant protein is high-sensitivity in tuberculosis detection, is a high-activity B cell target antigen, is used for the preparation of a tuberculosis detection kit, is high in sensitivity and is safe and reliable.
Description
Technical field
The present invention relates to biomedicine field, particularly a kind of preparation method and application thereof of mycobacterium tuberculosis Rv3117 recombinant protein.
Background technology
Diagnose mycobacterium tuberculosis (MTB) to infect fast and accurately, significant for control lungy.MTB diagnosis of infection method has a lot, and clinical method the most commonly used still relies on traditional phlegm smear bacteriology checking, but recall rate is low; MTB cultivates " gold standard " that can make a definite diagnosis as white plaque, but its incubation time is oversize, in general 4-6 week, is difficult to meet clinical needs; Though shortened incubation time, expense is higher for the full-automatic culture systems of mycobacterium (Bactec MGIT 960), is difficult in the short period of time popularize; X line and CT examination only provide the diagnosis of iconography possibility; Polymerase chain reaction (PCR) technology and other gene diagnosis method remain at complicated operation as clinical diagnosis, and be higher to technology and personnel specialty competency profiling, and still can not be used for the cloudy quick diagnosis lungy of bacterium.At present, diagnosis of tuberculosis widespread use skin test detection reagent---mycobacterium tuberculosis purified protein derivative (PPD), yet there is the cross reaction with environment mycobacterium and BCG vaccine strain in PPD, so diagnostic value is on the low side, thus improvement and innovate imperative.
Summary of the invention
To above-mentioned situation; For overcoming the prior art defective, the present invention's purpose just provides a kind of preparation method and application thereof of mycobacterium tuberculosis Rv3117 recombinant protein, and it is low effectively to solve diagnosis recall rate lungy; Expense is high, the problem of complicated operation and diagnostic reagent weak effect.
The technical scheme that the present invention solves, the preparation method of this mycobacterium tuberculosis Rv3117 recombinant protein, realized by following steps:
(1), (the Rv3117 gene order NCBI number of landing is BX842582.1 according to the Rv3117 gene order; Albumen number is: CAB08374.1) design the target gene primer;
(2), be template with the mycobacterium tuberculosis strain gene group DNA, pcr amplification, the PCR product, after the PCR product is purified Rv3117 isogeneity sequence, double digestion, enzyme cut product;
(3) enzyme being cut product is inserted in the expression plasmid (expression plasmid is claimed carrier again) with enzyme and cuts the used corresponding restriction enzyme site of the restriction restriction endonuclease place of Rv3117 isogeneity sequence; Adopt ordinary method clone construction recombination plasmid, among the gene order that order-checking shows recombinant plasmid and the GenBank (genomic library) in the full genome of H37Rv tuberculosis corresponding gene order (the Rv3117 gene NCBI number of landing is BX842582.1; Albumen number is: CAB08374.1) in full accord;
(4) recombinant plasmid transformed that makes up is gone into to carry out protein expression in the host cell that is complementary with expression plasmid, get expressing protein Rv3117;
(5) abduction delivering albumen Rv3117 gets the abduction delivering product;
(6) separation and purification abduction delivering product; Obtain Rv3117 recombinant protein (SEQ ID NO:1); The gene order of Rv3117 recombinant protein is compared with the CAE55554.1 protein sequence in the American National biotechnology information center (NCBI), has added expression plasmid correlated series and purification tag, as; When using the pET32a expression plasmid, recombinant protein has added correlated series and the one section His Tag purification tag on the pET32a at the back;
Rv3117 recombinant protein of the present invention can be separately as detecting diagnostic reagent lungy;
The invention provides a kind of detection test kit lungy of the Rv3117 of containing recombinant protein, this test kit is based on the test kit of antigen antibody reaction principle preparation, and the result is positive like antigen antibody reaction; Think that then this tester suffers from white plaque; Antigen antibody reaction comprises and is not limited only to agglutination reaction, or precipitin reaction, or immunofluorescence technique; Or the reaction of E garland, or the ELISA reaction etc.;
It is a kind of through including the reagent box for tuberculate diagnosis of Rv3117 recombinant protein with detection specificity antibody that the present invention also provides; The above-mentioned Rv3117 of the containing recombinant protein of the present invention or contain in the detection test kit lungy of specific antibody of Rv3117 recombinant protein also comprises:
(1) encapsulates diluent: the carbonate buffer solution of pH9.6 concentration 0.05M (commercially available molecular biology reagent commonly used);
(2) PBST lavation buffer solution (commercially available molecular biology reagent commonly used);
(3) sample diluent: the PBST (commercially available molecular biology reagent commonly used) that contains 1%BSA;
(4) resist through two of mark: the goat-anti people IgM of the goat anti-human igg of horseradish peroxidase-labeled or horseradish peroxidase-labeled;
(5) positive reference substance and negative control article;
(6) substrate solution: substrate buffer solution A: sodium acetate 2.4g, Hydrocerol A 0.28g adds successively, is dissolved in 88ml ultrapure water (Millipore, US, as follows), and stirring and dissolving in magnetic stirring apparatus adds 53 μ l 30%H
2O
2(superoxol), the magnetic stirrer dissolving; Adding 0.03g TMB in the substrate buffer solution B:80ml ultrapure water (3,3 ', 5,5 '-TMB) and 0.32g EDTA-Na2 (EDTA Disodium, commercially available molecular biology reagent commonly used), and then add 8ml glycerine, stir dissolving;
(7) stop buffer: 2M sulfuric acid;
(8) 96 hole enzyme plates (Costar, US);
Described expression plasmid is a kind of among pET21a, pET32a, pET28b, pET30a or the pET32a etc.; Described host cell is prokaryotic cell prokaryocyte or eukaryotic cell etc., like BL21 (DE3) bacterial strain or BL21 (DE3) the physS bacterial strain of intestinal bacteria (E.coli); Described mycobacterium tuberculosis strain gene group DNA is mycobacterium tuberculosis H
37The Rv genomic dna; Described Rv3117 gene order comprises one or more codons among the BX842582.1 is encoded that the degenerate codon of same amino acid replaces the back and the sequence that produces; Because the degeneracy of codon, so be low to moderate about 70% the degenerate sequence identical aminoacid sequence of also encoding with the BX842582.1 nucleotide sequence homology; Described abduction delivering albumen Rv3117 can use sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) inductor that expressing protein Rv3117 is induced; The method of described separation and purification abduction delivering product is methods such as affinity chromatography or ion exchange chromatography.
Rv3117 recombinant protein of the present invention is in the preparation process; Can select related vector known in the art for use, be connected in expression regulation sequence with encoding Rv3117 nucleotide sequence operability of the present invention then, thereby form recombinant plasmid; Rv3117 recombinant protein of the present invention susceptibility aspect the detection pulmonary tuberculosis is high; Be the B cell target antigen that activity is strong, be used for the preparation of white plaque detection kit, susceptibility is high and safe and reliable.
Description of drawings
Fig. 1 is Rv3117 Recombinant Protein Expression purifying figure of the present invention.
Fig. 2 is the SDS-PAGE figure of Rv3117 recombinant protein of the present invention after being further purified and concentrating.
Embodiment
Elaborate below in conjunction with the accompanying drawing specific embodiments of the invention.
1), the structure of recombinant plasmid pET32a-Rv3117
(1) target gene design of primers
Rv3117-F (upstream primer): 5 '-CATA
CCATGGCACGCTGCGAT-3 ';
Rv3117-R (downstream primer): 5 '-CTT
CTCGAGTCAGCTTCCCAA-3 ';
Restriction enzyme site is respectively Nco I, Xho I;
(2) pcr amplification of target gene, clone and sequencing
With mycobacterium tuberculosis H
37The Rv genomic dna is a template, and Rv3117-F is that upstream primer and Rv3117-R are downstream primer, uses the Taq enzyme, through pcr amplification, gets the PCR product, the reaction conditions of pcr amplification: 94 ℃ of preparatory sex change 5min; (94 ℃ of sex change 30s; 58 ℃ of annealing 30s; 72 ℃ of extension 40s) 35 circulations; 72 ℃ are extended 5min; 4 ℃ of preservations; Separate the PCR product with 1% agarose gel electrophoresis then; (Invitrogen US) reclaims, and gets Rv3117 isogeneity sequence to reclaim test kit with DNA again; Rv3117 isogeneity sequence is cut with restriction restriction endonuclease Xho I enzyme with restriction restriction endonuclease Nco I; Be inserted in the Nco I restriction enzyme site and Xho I restriction enzyme site of pET32a expression plasmid, clone construction recombination plasmid pET32a-Rv3117, order-checking shows that the full genome corresponding gene sequences of H37Rv tuberculosis is in full accord among gene order and the GeneBank of recombinant plasmid pET32a-Rv3117;
2) abduction delivering of recombinant plasmid pET32a-Rv3117 and purifying
Get 100ul BL21 (DE3) physS competent cell (Tiangen, China) pack into eppdorf pipe is put on ice, after liquid in pipe melted in 3-5 minute; 0.5ul checked order recombinant plasmid pET32a-Rv3117 correct and be transformed into carry out protein expression in the competent cell, place 45min on ice, in 42 ℃ of water-baths, thermal shock 90s; Leave standstill 3min on ice, expressing protein Rv3117, what in expressing protein Rv3117, add 500ul does not contain antibiotic LB substratum, 37 ℃ of shaking tables; 220rmp cultivates 45-60min, gets 100ul then and is coated with containing on the solid LB substratum plate of kantlex, after the 18-25 ℃ of drying; Be inverted in 37 ℃ of incubators and cultivated 10-12 hour, picking is cloned, and puts into the LB liquid nutrient medium of the resistance that contains the 50ug/ml kantlex; 220rmp cultivates the OD value to 0.4-0.7 for 37 ℃, and adding final concentration is the IPTG inductor of 10mM; Induce 3h for 37 ℃, collect the thalline after inducing, press 10ml lavation buffer solution I (KCl:300mM; KH2PO4:50mM; Imidazoles: 5mM, as follows) the resuspended back ultrasonication of ratio of every gram thalline, mixed solution; Get the centrifugal 30min of mixed solution 10000g, collection contains the target protein supernatant of (target protein promptly refers to the Rv3117 recombinant protein), and (Bio-Rad) carries out affinity purification with the IMAC affinity chromatographic column; Promptly first with 2CV deionized water rinsing 2ml/min, 3min, the lavation buffer solution I balance affinity column of 5CV; Get appearance on the supernatant that 6CV comprises target protein again, wash 2ml/min successively with 6CV lavation buffer solution I; 3min, 6CV lavation buffer solution II (KCl:300mM; KH2PO4:50mM; Imidazoles: 10mM) flushing, 2ml/min, 3min, the lavation buffer solution III (KCl:300mM of 10CV; KH2PO4:50mM; Imidazoles: 250mM) wash-out 2ml/min, 5min collects elutriant, and the target protein that gets purifying is (as shown in Figure 1; The 1st, molecular weight of albumen standard, the 2nd, inductive BL21-pET32a:Rv3117 bacterial strain not, the 3rd, the BL21-PET32a:Rv3117 bacterial strain after inducing, the 4th, broken supernatant; The 5th, broken deposition, the 6th, go up all article, the 7th, lavation buffer solution I washing sample, the 8th, lavation buffer solution 2 washing sample before the affinity purification; The 9th, the elutriant elution samples), with the 20mM Tris-HCl damping fluid dialysis of pH 8.0, get target protein liquid again; With 10kDa ultrafiltration pipe the target protein liquid after dialysing is concentrated, get spissated target protein, measure the concentration of spissated target protein with the BCA method; The result shows, SDS-PAGE electrophoretic analysis target protein purity be 95% (as shown in Figure 2, the 1st, the molecular weight of albumen standard; The 2nd, the target protein (5 μ g) after purifying concentrates, the 3rd, the target protein (2.5 μ g) after purifying concentrates), promptly get Rv3117 recombinant protein (SEQ ID NO:1).
Contain the application of Rv3117 recombinant protein as detection of antigens test kit lungy: assessment Rv3117 recombinant protein is as detecting the reaction effect of antigen to IgG in PPD-normal healthy controls, PPD+ normal healthy controls serum and the tuberculosis patient serum; Utilize indirect enzyme-linked immunosorbent assay to detect the IgG reaction of anti-Rv3117 in the different serum specimens: concrete steps are following: contain in the detection test kit lungy of Rv3117 recombinant protein, also comprise:
(1) encapsulates diluent: the carbonate buffer solution of pH9.6 concentration 0.05M (commercially available molecular biology reagent commonly used);
(2) PBST lavation buffer solution (commercially available molecular biology reagent commonly used);
(3) sample diluent: the PBST (commercially available molecular biology reagent commonly used) that contains 1%BSA;
(4) resist through two of mark: the goat-anti people IgM of the goat anti-human igg of horseradish peroxidase-labeled or horseradish peroxidase-labeled;
(5) positive reference substance and negative control article;
(6) substrate solution: substrate buffer solution A: sodium acetate 2.4g, Hydrocerol A 0.28g adds successively, is dissolved in 88ml ultrapure water (Millipore, US, as follows), and stirring and dissolving in magnetic stirring apparatus adds 53 μ l 30%H
2O
2(superoxol), the magnetic stirrer dissolving; Adding 0.03g TMB in the substrate buffer solution B:80ml ultrapure water (3,3 ', 5,5 '-TMB) and 0.32g EDTA-Na2 (EDTA Disodium, commercially available molecular biology reagent commonly used), and then add 8ml glycerine, stir dissolving;
(7) stop buffer: 2M sulfuric acid;
(8) 96 hole enzyme plates (Costar, US);
96 hole enzyme reaction plates encapsulated 16 hours with the antigen Rv3117 (claiming the Rv3117 recombinant protein again) of 2 μ g/ml, discarded coating buffer, and PBST lavation buffer solution washing 5 times was washed 2 minutes at every turn; Dry, every hole adds the PBST sample diluent that 300 μ l contain 1%BSA, and 37 ℃ of incubation 2h dry; Every hole adds the to be checked serum of 100 μ l with the PBST sample diluted that contains 1%BSA, and the PBST sample diluent of 1%BSA and the volume ratio of serum to be checked are 1: 50,37 ℃ of incubation 0.5h; Discard serum, PBST lavation buffer solution washing 5 times dries; Every hole add 100 μ l with contain 1%BSA PBST sample diluted horseradish peroxidase-labeled the goat anti-human igg antibody (Jackson, USA), 37 ℃ of incubation 0.5h; Discard liquid, PBST lavation buffer solution washing 5 times dries; Every hole adds 100 μ l substrate solution mixtures, and the substrate solution mixture is to be mixed with at 1: 1 with volume ratio with substrate buffer solution A and substrate buffer solution B, 37 ℃ of lucifuge incubation 10min; Every hole adds 50 μ l 2M sulfuric acid stop buffer termination reactions, detects the OD value behind the 450nm, adds the judgement criteria of 3 times of standard variances as Cutoff with the average OD value of normal healthy controls person's serum (comprising PPD-normal healthy controls, PPD+ normal healthy controls serum) sample; When the OD of serum specimen to be checked value during more than or equal to judgement criteria, promptly be judged as the positive, on the contrary negative.
The result shows that as the proteantigen of single detection, the Rv3117 recombinant protein of the present invention's preparation has higher susceptibility and specificity aspect diagnosis of tuberculosis, through TE, obtained good effect, and is as shown in table 1:
Table 1Rv3117 recombinant protein is respectively to normal healthy controls serum and tuberculosis patient serum IgG reaction result
From the above: the positive rate of IgG is 25% in tuberculosis patient serum; Compare with the PPD+ normal healthy controls with PPD-, its detection specificity is respectively 100% and 91.7%, and total specificity is 96.9%; Therefore; Rv3117 recombinant protein of the present invention can also can be united with existing known diagnostic antigen, thereby improve the susceptibility that detects tuberculosis patient separately as diagnostic reagent.
Claims (5)
1. a mycobacterium tuberculosis Rv3117 recombinant protein is characterized in that, the sequence of Rv3117 recombinant protein is SEQ ID NO:1.
2. the preparation method of a mycobacterium tuberculosis Rv3117 recombinant protein is characterized in that, is realized by following steps:
(1), designs the target gene primer according to the Rv3117 gene order;
(2), be template with the mycobacterium tuberculosis strain gene group DNA, pcr amplification, the PCR product, after the PCR product is purified Rv3117 isogeneity sequence, double digestion, enzyme cut product;
(3) enzyme being cut product is inserted in the expression plasmid with enzyme and cuts the used corresponding restriction enzyme site of the restriction restriction endonuclease place of Rv3117 isogeneity sequence; Adopt ordinary method clone construction recombination plasmid, among the gene order that order-checking shows recombinant plasmid and the GenBank in the full genome of H37Rv tuberculosis corresponding gene order Rv3117 gene in full accord;
(4) recombinant plasmid transformed that makes up is gone into to carry out protein expression in the host cell that is complementary with expression plasmid, get expressing protein Rv3117;
(5) abduction delivering albumen Rv3117 gets the abduction delivering product;
(6) separation and purification abduction delivering product; Obtain Rv3117 recombinant protein SEQ ID NO:1; The gene order of Rv3117 recombinant protein is compared with the CAE55554.1 protein sequence on the NCBI; Added expression plasmid correlated series and purification tag, when promptly using the pET32a expression plasmid, recombinant protein has added correlated series and the one section His Tag purification tag on the pET32a at the back;
Described expression plasmid is a kind of among pET21a, pET32a, pET28b, pET30a or the pET32a; Described host cell is colibacillary BL21 DE3 bacterial strain or BL21 DE3 physS bacterial strain; Described mycobacterium tuberculosis strain gene group DNA is mycobacterium tuberculosis H
37The Rv genomic dna; Described Rv3117 gene order comprises one or more codons among the BX842582.1 is encoded that the degenerate codon of same amino acid replaces the back and the sequence that produces; Because the degeneracy of codon, so be low to moderate about 70% the degenerate sequence identical aminoacid sequence of also encoding with the BX842582.1 nucleotide sequence homology.
3. the preparation method of mycobacterium tuberculosis Rv3117 recombinant protein according to claim 1 and 2 is characterized in that, is realized by following steps:
1), the structure of recombinant plasmid pET32a-Rv3117
(1) target gene design of primers
Rv3117-F：5’-CATA
CCATGGCACGCTGCGAT?-3’;
Rv3117-R：5’-?CTT
CTCGAGTCAGCTTCCCAA?-3’;
Restriction enzyme site is respectively
Nco I,
Xho I
(2) pcr amplification of target gene, clone and sequencing
With mycobacterium tuberculosis H
37The Rv genomic dna is a template, and Rv3117-F is that upstream primer and Rv3117-R are downstream primer, uses the Taq enzyme, through pcr amplification, gets the PCR product, the reaction conditions of pcr amplification: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s; 58 ℃ of annealing 30s; 72 ℃ are extended 40s, 35 circulations; 72 ℃ are extended 5min; 4 ℃ of preservations separate the PCR product with 1% agarose gel electrophoresis then, reclaim test kit with DNA again and reclaim, and get Rv3117 isogeneity sequence, and Rv3117 isogeneity sequence is used the restriction restriction endonuclease
NcoI and restriction restriction endonuclease
XhoThe I enzyme is cut, and is inserted into the pET32a expression plasmid
NcoThe I restriction enzyme site with
XhoIn the I restriction enzyme site, clone construction recombination plasmid pET32a-Rv3117, order-checking shows that the full genome corresponding gene sequences of H37Rv tuberculosis is in full accord among gene order and the GeneBank of recombinant plasmid pET32a-Rv3117;
2) abduction delivering of recombinant plasmid pET32a-Rv3117 and purifying
Get pack into eppdorf pipe of 100ul BL21 DE3 physS competent cell, put on ice, after liquid in pipe melted in 3-5 minute, 0.5ul checked order recombinant plasmid pET32a-Rv3117 correct and be transformed into carry out protein expression in the competent cell; Place 45min on ice, in 42 ℃ of water-baths, thermal shock 90s leaves standstill 3min on ice; Expressing protein Rv3117, what in expressing protein Rv3117, add 500ul does not contain antibiotic LB substratum, 37 ℃ of shaking tables, 220rmp; Cultivate 45-60min, get 100ul then and be coated with containing on the solid LB substratum plate of kantlex, after the 18-25 ℃ of drying, be inverted in 37 ℃ of incubators and cultivated 10-12 hour; Picking is cloned, and puts into the LB liquid nutrient medium of the resistance that contains the 50ug/ml kantlex, and 220rmp cultivates the OD values to 0.4-0.7 for 37 ℃; Adding final concentration is the IPTG inductor of 10mM, induces 3h for 37 ℃, collects the thalline after inducing, in the resuspended back ultrasonication of the ratio of the every gram thalline of 10ml lavation buffer solution I; Get mixed solution, get the centrifugal 30min of mixed solution 10000g, collect the supernatant that contains target protein, Bio-Rad carries out affinity purification with the IMAC affinity chromatographic column; Promptly earlier with 2CV deionized water rinsing 2ml/min, 3min, the lavation buffer solution I balance affinity column of 5CV is got on the supernatant that 6CV comprises target protein kind again; Wash 2ml/min, 3min, the flushing of 6CV lavation buffer solution II successively with 6CV lavation buffer solution I; 2ml/min, 3min, the lavation buffer solution III wash-out 2ml/min of 10CV, 5min; Collect elutriant, get the target protein of purifying, dialyse with the 20mM Tris-HCl damping fluid of pH 8.0 again; Get target protein liquid, the target protein liquid after dialysing is concentrated, get spissated target protein with 10 kDa ultrafiltration pipes; Measure the concentration of spissated target protein with the BCA method, SDS-PAGE electrophoretic analysis target protein purity is 95%, promptly gets Rv3117 recombinant protein SEQ ID NO:1; Described lavation buffer solution I is mixed by KCl:300mM, KH2PO4:50mM, imidazoles: 5mM and forms; Described lavation buffer solution II is mixed by KCl:300mM, KH2PO4:50mM, imidazoles: 10mM and is formed; Described lavation buffer solution III is mixed by KCl:300mM, KH2PO4:50mM, imidazoles: 250mM and is formed.
4. claim 1 or the 2 or 3 described mycobacterium tuberculosis Rv3117 recombinant proteins application in detecting diagnostic reagent lungy.
5. claim 1 or 2 or 3 described mycobacterium tuberculosis Rv3117 recombinant proteins or the application of specific antibody in detecting test kit lungy that contain the Rv3117 recombinant protein, described test kit also includes:
(1) encapsulates diluent: the carbonate buffer solution of pH9.6 concentration 0.05M;
(2) PBST lavation buffer solution;
(3) sample diluent: the PBST that contains 1%BSA;
(4) resist through two of mark: the goat-anti people IgM of the goat anti-human igg of horseradish peroxidase-labeled or horseradish peroxidase-labeled;
(5) positive reference substance and negative control article;
(6) substrate solution: substrate buffer solution A: sodium acetate 2.4g, Hydrocerol A 0.28g adds successively, is dissolved in the 88ml ultrapure water, and stirring and dissolving in magnetic stirring apparatus adds 53 μ l 30%H
2O
2, the magnetic stirrer dissolving; Add 0.03g TMB and 0.32g EDTA-Na2 in the substrate buffer solution B:80ml ultrapure water, and then add 8ml glycerine, stir dissolving;
(7) stop buffer: 2M sulfuric acid;
(8) 96 hole enzyme plates.
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| CN115184603A (en) * | 2022-06-30 | 2022-10-14 | 首都医科大学附属北京胸科医院 | Application of EspC protein in preparation of mycobacterium tuberculosis separation or enrichment product |
| CN115197306A (en) * | 2022-07-29 | 2022-10-18 | 天津鸿宇泰生物科技有限公司 | Induction medium and method for carrying out induced expression on MPT64 protein by using same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115184603A (en) * | 2022-06-30 | 2022-10-14 | 首都医科大学附属北京胸科医院 | Application of EspC protein in preparation of mycobacterium tuberculosis separation or enrichment product |
| CN115184603B (en) * | 2022-06-30 | 2024-02-06 | 首都医科大学附属北京胸科医院 | Application of EspC protein in preparation of mycobacterium tuberculosis separation or enrichment product |
| CN115197306A (en) * | 2022-07-29 | 2022-10-18 | 天津鸿宇泰生物科技有限公司 | Induction medium and method for carrying out induced expression on MPT64 protein by using same |
| CN115197306B (en) * | 2022-07-29 | 2023-08-29 | 天津鸿宇泰生物科技有限公司 | Induction medium and method for carrying out induction expression on MPT64 protein by using induction medium |
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Application publication date: 20120718 |