CN103063843A - Specific marker of mycobacterium tuberculosis and application thereof - Google Patents
Specific marker of mycobacterium tuberculosis and application thereof Download PDFInfo
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- CN103063843A CN103063843A CN2011103201297A CN201110320129A CN103063843A CN 103063843 A CN103063843 A CN 103063843A CN 2011103201297 A CN2011103201297 A CN 2011103201297A CN 201110320129 A CN201110320129 A CN 201110320129A CN 103063843 A CN103063843 A CN 103063843A
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Abstract
The invention discloses a specific marker (Rv3119 recombinant protein) of mycobacterium tuberculosis and discloses an application of the Rv3119 recombinant protein in preparation a tuberculosis diagnostic reagent and a tuberculosis diagnostic kit. The Rv3119 specific antigen provided by the invention has high sensitivity in detecting phthisis, is a B cell target antigen with relatively strong activity, can be applied in preparation of rapid detection kit for the tuberculosis, and is safe and reliable.
Description
Technical field
The invention belongs to biomedicine field, more particularly, relate to a kind of diagnostic reagent of tuberculosis and its preparation method and application.
Background technology
Diagnosis of tuberculosis mycobacterium (MTB) infects fast and accurately, and is significant for control lungy.The diagnostic method that MTB infects has a lot, and at present clinical method the most commonly used still relies on traditional phlegm smear bacteriology checking, but recall rate is low; MTB cultivates " goldstandard " that can make a definite diagnosis as tuberculosis, but its incubation time is oversize, in general 4-6 week, is difficult to meet clinical needs; Though the Bactec technology has shortened incubation time, expense is higher, is difficult in the short time popularize; X-ray and CT examination only provide the diagnosis of iconography possibility; Polymerase chain reaction (PCR) technology and other gene diagnosis method remain difficulty 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.And the detection of plasma that MTB infects diagnosis with its intrinsic easy and simple to handle, quick, with the naked eye judged result, good stability, be convenient to promote, need not the advantage such as special exact instrument, be that most probable satisfies the diagnosis of tuberculosis technology that economically less developed region and tuberculosis district occurred frequently are needed badly under the new situation.
Summary of the invention
First purpose of the present invention is to provide a kind of Much's bacillus Specific marker.
Second purpose of the present invention is to provide the application of Rv3119 recombinant protein in preparation Diagnosis of Tuberculosis reagent.
The 3rd purpose of the present invention is to provide the application of Rv3119 recombinant protein in preparation Diagnosis of Tuberculosis kit.
The 4th purpose of the present invention is to provide a kind of detection kit lungy.
For realizing above purpose, the present invention discloses following technical scheme: one aspect of the present invention provides a kind of new Much's bacillus Specific marker Rv3119 recombinant protein.
The invention provides a kind of new recombinant plasmid, be about to the Rv3119 gene order and insert in the commercial expression plasmid sequence.The Rv3119 gene NCBI number of logging in is BX842582.1; The gene name is called moaE1; Albumen number is: CAE55554.1.
" Rv3119 gene order " of the present invention also comprises the sequence that one or more codons among the BX842582.1 are encoded and produce after the degenerate codon of same amino acid replaces.Because the degeneracy of codon, so be low to moderate approximately 70% the degenerate sequence identical amino acid sequence of also encoding with the BX842582.1 nucleotide sequence homology.This term also is included under the tight condition of moderate, more preferably under highly tight condition with the nucleotide sequence of the nucleotide sequence hybridization of BX842582.1.This term also comprises the nucleotide sequence homology at least 70% with BX842582.1, more preferably at least 90% nucleotide sequence.
Recombinant plasmid of the present invention, the multiple clone site place that usually the Rv3119 gene is inserted expression plasmid obtains.
Recombinant plasmid of the present invention can be selected various carrier known in the art in preparation process, the nucleotide sequence of the present invention of then will encoding is connected in expression regulation sequence operably, thereby forms protein expression vector.
Adoptable carrier comprises and not only for pET28a, pET28b, pET30a and pET32a among the present invention.In one embodiment of the present of invention, used plasmid is pET28b.
Another aspect of the present invention provides a kind of Much's bacillus recombinant protein of being correlated with, and this albumen changes the recombinant expression plasmid that inserts the Rv3119 gene over to host cell and obtains.
Compare with the CAE55554.1 protein sequence on the NCBI as the Rv3119 recombinant protein that detects reagent, added carrier correlated series and purification tag.For example, when using the pET28b plasmid, the protein sequence back has added correlated series and the one section His Tag purification tag on the pET28b.
Used host cell should be complementary with expression plasmid.Can use prokaryotic or eukaryotic etc., for example Escherichia coli (E. coli) commonly used.The host cell that is used for transforming can be BL21(DE3) or BL21 plysS(DE3) bacterial strain etc.Used in one embodiment of the present of invention is exactly BL21 plysS(DE3) bacterial strain.
Another aspect of the present invention provides the preparation method of above-mentioned Rv3119 recombinant protein, may further comprise the steps:
(1) Much's bacillus H
37The preparation of Rv genomic DNA;
(2) pcr amplification of Rv3119 gene;
(3) the Rv3119 gene expression plasmid makes up and identifies;
(4) expression plasmid that step (3) is made up is transformed into host cell;
(5) induction expression protein Rv3119;
(6) separation and purification abduction delivering product obtains the Rv3119 recombinant protein.
Among the above-mentioned preparation method, various experiment parameters are routinely operation selection all, is decided by concrete experiment condition.
The present invention uses conventional method well known by persons skilled in the art to prepare H
37The Rv genomic DNA; The Rv3119 gene order can be based on H
37The Rv genome is dna profiling, obtains with the pcr amplification method; Related nucleotide sequences disclosed according to the present invention, especially open reading frame sequence designs primer; The used expression plasmid of recombinant protein can be pET28a, pET28b, pET30a and pET32a; Expressing the used host cell of Rv3119 albumen is colibacillary BL21(DE3) bacterial strain or BL21 plysS(DE3) bacterial strain; Abduction delivering Rv3119 albumen can adopt several different methods, as using IPTG; Separation and purification restructuring Rv3119 albumen also can adopt several different methods, such as affinity chromatography and sieve chromatography etc.
In one embodiment of the present of invention, recombinant protein Rv3119 obtains as follows: the design primer (F:5 '-CATGCCATGGCCAATGTGGTAG-3 '; R:5 '-CTTCTCGAGTGGTCTATCGCCGAC-3 ').With H
37Rv pnca gene group DNA is template, pcr amplification Rv3119 gene, and the purified rear double digestion of the PCR product of this gene, restriction enzyme site is respectively Nco I, Xho I, and the clone makes up plasmid.Order-checking shows H among the sequence inserted and the GeneBank
37(the Rv3119 gene NCBI number of logging in is BX842582.1 to the corresponding gene order of the full genome of Rv tuberculosis; Albumen number is: CAE55554.1) in full accord.The recombinant plasmid transformed that checking is good is carried out protein expression to host cell.
In one embodiment of the present of invention, restructuring Rv3119 albumen can obtain as follows: will identify that good recombinant plasmid joins in the competent escherichia coli cell, place 45min on ice, thermal shock is 90 seconds in 42 ℃ of water-baths, ice bath 3min, the LB nutrient culture media that does not contain antibiotic preheating of adding 500ul.37 ℃ of shaking tables, 220rmp cultivates 45-60min.Get a certain amount of solid LB nutrient culture media plate containing kanamycins and be coated with, be inverted in overnight incubation in 37 ℃ of incubators after the drying at room temperature.Picking is cloned, and puts into the LB fluid nutrient medium of the resistance that contains the 50ug/ml kanamycins, and 220rmp cultivates about OD to 0.6 for 37 ℃, adds IPTG, and 37 ℃ of 3h collect the thalline after IPTG induces, resuspended rear ultrasonication.Destination protein is contained in the supernatant, and the centrifugal 30min of 10000g collects supernatant.Carry out affinity purification (2CV deionized water rinsing with the IMAC affinity chromatographic column, 5CV contains the washing Buffer I balance of 5mM imidazoles, 6CV contains destination protein supernatant loading, 6CV contains the washing Buffer I flushing of 5mM imidazoles, 6CV contains washing Buffer 2 flushings of 10mM imidazoles, 10CV contains the Elution Buffer wash-out of 500mM imidazoles), collected the destination protein of post purifying, use 20mM Tris-HCl, the solution dialysis of pH 8.0,10 kDa super filter tubes concentrate the destination protein liquid after dialysing, with the concentration of destination protein behind the BCA method mensuration purifying.
Another aspect of the present invention provides the application of Rv3119 recombinant protein in preparation Diagnosis of Tuberculosis reagent.
Another aspect of the present invention provides the application of Rv3119 recombinant protein in preparation Diagnosis of Tuberculosis kit.
Rv3119 is the member of molybdenum cofactor biosynthesis protein E, is positioned at the RD5 district.Recombinant protein of the present invention is used for the preparation of diagnosis kit, can carry out immune response with restructuring Rv3119, also can carry out PCR to the blood samples of patients extract with the Rv3119 gene primer, thereby reach testing goal.
The invention provides a kind of detection kit lungy, it contains recombinant protein Rv3119.
Kit of the present invention is mainly based on the antigen-antibody reaction principle, comprises and is not limited only to adopt agglutinating reaction, precipitation reaction, immunofluorescence technique, the reaction of E garland, ELISA, the immune responses such as colloidal gold chromatographic and chemiluminescence etc.Be positive such as reaction result, think that then this tester suffers from tuberculosis.
The present invention also provides a kind of diagnosis kit, and it contains the specific antibody of above-mentioned recombinant protein Rv3119.With person's sample to be detected, comprise and be not limited only to organize, the albumen in the body fluid separates, if any with the identical albumen of the specific antibody of recombinant protein Rv3119, judge that namely this tester suffers from tuberculosis.
In the kit of the present invention, except recombinant protein Rv3119 or its specific antibody are arranged, also have for detection of reagent and vessel various commonly used, comprise various damping fluids, dilution, stop buffer etc.In one embodiment of the invention, the tuberculosis detection kit comprises following reagent and article:
(1) the coated damping fluid (pH9.6) of the carbonate of coated dilution: 0.05M;
(2) lavation buffer solution: PBST;
(3) sample dilution: the PBST that contains 1%BSA;
(4) resist through two of mark: the goat anti-human igg of mark or IgM;
(5) positive reference substance and negative control product;
(6) substrate solution: substrate buffer solution A: sodium acetate 2.4g, citric acid 0.28g is dissolved in the 88ml ultrapure water, and the abundant stirring and dissolving of magnetic stirring apparatus adds 53 μ l 30%H at last
2O
2, the abundant stirring and dissolving of magnetic stirring apparatus and getting; Add 0.03g TMB and 0.32g EDTA-Na2 in the substrate buffer solution B:80ml ultrapure water, and then add 8ml glycerine, magnetic agitation makes its abundant stirring and dissolving and gets;
(7) stop buffer: 2M sulfuric acid;
(8) 96 hole enzyme reaction plates.
The invention has the advantages that: at present, diagnosis of tuberculosis widespread use skin test detects reagent---Much's bacillus 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.The invention provides a kind of new Much's bacillus Specific marker Rv3119 recombinant protein, Rv3119 specific antigen susceptibility aspect the detection pulmonary tuberculosis is higher, the B cell target antigen that activity is stronger, be used for the preparation of diagnosis kit, have higher specificity and susceptibility, and safe and reliable.
Description of drawings
Fig. 1 is the expression and purification figure of recombinant protein Rv3119,
Wherein, the 1st, the molecular weight of albumen standard, the 2nd, the BL21-pET28b:Rv3119 bacterial strain of not inducing, the 3rd, the BL21-pET28b:Rv3119 bacterial strain after inducing, the 4th, broken supernatant, the 5th, broken precipitation, the 6th, the front upper all product of affinity purification, the 7th, Washing Buffer I washing sample, the 8th, Washing Buffer II washing sample, the 9th, Elution Buffer elution samples.
Fig. 2 is the SDS-PAGE figure of recombinant protein Rv3119 after being further purified and concentrating,
Wherein, the 1st, molecular weight of albumen standard, the 2nd, the destination protein (5 μ g) after purifying is concentrated, the 3rd, the destination protein (2.5 μ g) after purifying is concentrated.
Fig. 3 be recombinant protein Rv3119 and 38kDa antigen respectively to PPD-normal healthy controls, PPD+ normal healthy controls serum and tuberculosis patient serum IgG reaction result, case load is 200.
Embodiment
Below in conjunction with accompanying drawing the present invention is elaborated, the effect of embodiment only is to explain and non-limiting the present invention.
The structure of embodiment 1, recombinant plasmid pET28b-Rv3119
(1) target gene design of primers
Rv3119-F：5’- CATGCCATGGCCAATGTGGTAG -3’;
Rv3119-R：5’-CTTCTCGAGTGGTCTATCGCCGAC-3’；
Restriction enzyme site is respectively Nco I, Xho I.
(2) pcr amplification of target gene, clone and sequence
With Much's bacillus H
37The Rv genomic DNA is for touching plate, and Rv3119-F and Rv3119-R are primer, uses the Taq enzyme, by the PCR Rv3119 protein gene that directly increases.PCR reaction conditions: 94 ℃ of denaturation 5min; (94 ℃, 30s; 58 ℃, 30s; 72 ℃, 40s) 35 circulations; 72 ℃ are extended 5min; 4 ℃ of preservations.Separate the purpose fragment with 1% agarose gel electrophoresis after reaction finishes, reclaim kit (Invitrogen) recovery with DNA afterwards.Cut with Nco I and Xho I enzyme, the clone is built in pET28b plasmid Nco I and the Xho I restriction enzyme site.Order-checking shows H among the sequence inserted and the GeneBank
37(the Rv3119 gene NCBI number of logging in is BX842582.1 to the full genome corresponding gene sequences of Rv tuberculosis; Albumen number is: CAE55554.1) in full accord.
The abduction delivering of embodiment 2, recombinant protein Rv3119 and purifying
100ul BL21(DE3 will be housed) the Eppdorf pipe of physS competent cell puts on ice immediately from-80 ℃ of refrigerators.After waiting liquid in pipe to melt after 3-5 minute, the restructuring pET28b-Rv3119 plasmid that 0.5ul checks order correct is put into competent cell, place 45min on ice, in 42 ℃ of water-baths, thermal shock 90s leaves standstill 3min on ice, the LB nutrient culture media that does not contain antibiotic preheating that adds 500ul, 37 ℃ of shaking tables, 220rmp cultivates 45-60min.Get a certain amount of solid LB nutrient culture media plate containing kanamycins and be coated with, be inverted in overnight incubation in 37 ℃ of incubators after the drying at room temperature.The picking clone, put into the LB fluid nutrient medium of the resistance that contains the 50ug/ml kanamycins, 220rmp, cultivate about OD to 0.6 for 37 ℃, adding final concentration is 10mM IPTG, 37 ℃ of 3h collect the thalline after IPTG induces, in the resuspended rear ultrasonication of ratio of the wet bacterium of the every gram of 10ml Washing Buffer I.Destination protein is contained in the supernatant as a result.The centrifugal 30min of 10000g collects supernatant.Carry out affinity purification [2 times column volume (CV) deionized water rinsing with IMAC affinity chromatographic column (Bio-Rad), 5CV contains the washing Buffer I balance of 5mM imidazoles, 6CV contains destination protein supernatant loading, 6CV contains the washing Buffer I flushing of 5mM imidazoles, 6CV contains washing Buffer 2 flushings of 10mM imidazoles, 10CV contains the Elution Buffer wash-out of 500mM imidazoles], collected the destination protein (Fig. 1) of post purifying, use 20mM Tris-HCl, the solution dialysis of pH 8.0,10 kDa super filter tubes concentrate the destination protein liquid after dialysing, with the concentration of destination protein behind the BCA method mensuration purifying, SDS-PAGE electrophoretic analysis destination protein purity (Fig. 2).
Utilize indirect enzyme-linked immunosorbent assay to detect the IgG reaction of anti-Rv3119 in the different serum specimens.Concrete steps are: 96 orifice plates are with antigen Rv3119(5 μ g/ml) or 38kDa antigen (5ug/ml) is coated spends the night, and discards coating buffer, and PBST washing 5 times dries.Every hole adds the PBST that 300 μ l contain 1%BSA, 37 ℃ of incubation 2h.Dry, every hole adds the serum to be checked (1:50) that 100 μ l dilute with the PBST that contains 1%BSA, 37 ℃ of incubation 0.5h.) discarding serum, PBST washing 5 times dries.Every hole adds the goat anti-human igg antibody that 100 μ l use the horseradish peroxidase-labeled of the PBST dilution that contains 1%BSA, 37 ℃ of incubation 0.5h.Discard liquid, PBST washing 5 times dries.Every hole adds the substrate solution (A:B=1:1) of the fresh configuration of 100 μ l, 37 ℃ of lucifuge incubation 10min.Every hole adds 50 μ l 2M sulfuric acid cessation reactions, and 450nm detects the OD value.Average OD value with normal healthy controls person's serum specimen adds 3 times of standard variances as the criterion of Cutoff value.If the OD value of serum specimen namely is judged as the positive more than or equal to this standard, otherwise negative.
The result shows: compare with 38kDa antigen, restructuring Rv3119 proteantigen has higher susceptibility and specificity aspect diagnosis of tuberculosis.The positive rate of its IgG in tuberculosis patient serum is 33.1%, compares with the PPD+ normal healthy controls with PPD-, and its detection specificity is respectively 100% and 95.8%, and total specificity is 98.4%(Fig. 3).Aspect the specificity that detects tuberculosis patient, recombinant protein Rv3119 is quite high.Restructuring Rv3119 albumen of the present invention can separately as diagnostic reagent, also can be united with existing known diagnostic antigen, thereby improve the susceptibility that detects tuberculosis patient.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
SEQUENCE LISTING
＜110〉Medical College, Shanghai Communication Univ.
＜120〉a kind of Much's bacillus Specific marker and application thereof
<130> /
<140> 201110320129.7
<141> 2011-10-20
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> M. tuberculosis
<400> 1
catgccatgg ccaatgtggt ag 22
<210> 2
<211> 24
<212> DNA
<213> M. tuberculosis
<400> 2
cttctcgagt ggtctatcgc cgac 24
Claims (7)
1.Rv3119 recombinant protein is as the application in the Much's bacillus Specific marker.
2.Rv3119 the application of recombinant protein in preparation Diagnosis of Tuberculosis kit.
3. application according to claim 2 is characterized in that, described Rv3119 recombinant protein prepares by the following method:
(1) Much's bacillus H
37The preparation of Rv genomic DNA;
(2) pcr amplification of Rv3119 gene;
(3) the Rv3119 gene expression plasmid makes up and identifies;
(4) expression plasmid that step (3) is made up is transformed into host cell;
(5) induction expression protein Rv3119;
(6) separation and purification abduction delivering product obtains the Rv3119 recombinant protein.
4. application according to claim 3 is characterized in that, pcr amplification in the step (2), and primer is: Rv3119-F:5 '-CATGCCATGGCCAATGTGGTAG-3 '; Rv3119-R:5 '-CTTCTCGAGTGGTCTATCGCCGAC-3 '; Restriction enzyme site is respectively Nco I, Xho I.
5.Rv3119 the application of recombinant protein in preparation Diagnosis of Tuberculosis reagent.
6. one kind is detected kit lungy, it is characterized in that, described kit contains recombinant protein Rv3119.
7. one kind is detected kit lungy, it is characterized in that, described kit contains the specific antibody of recombinant protein Rv3119.
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| CN108165562A (en) * | 2017-12-01 | 2018-06-15 | 北京蛋白质组研究中心 | Mycobacterium tuberculosis H37Rv encoding gene and its application |
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Application publication date: 20130424 |