CN104805063A

CN104805063A - Mycobacterium tuberculosis latent infection related proteins, preparation and applications thereof

Info

Publication number: CN104805063A
Application number: CN201410033726.5A
Authority: CN
Inventors: 吴雪琼; 白雪娟; 梁艳; 阳幼荣; 张俊仙
Original assignee: 309th Hospital of PLA
Current assignee: 309th Hospital of PLA
Priority date: 2014-01-24
Filing date: 2014-01-24
Publication date: 2015-07-29

Abstract

The present invention relates to Mycobacterium tuberculosis latent infection related proteins, preparation and applications thereof, wherein the three Mycobacterium tuberculosis latent infection related proteins are LTBIPr1, LTBIPr2 and LTBIPr3, and the nucleotide sequences of the protein epitope are represented by the sequences 1-3 in the sequence table. According to the present invention, the genetic engineering technology is adopted to clone, express and purify Mycobacterium tuberculosis to obtain the three protein antigens related with Mycobacterium tuberculosis latent infection; the protein antigen is adopted as the tuberculosis specific cell stimulating agent to stimulate the peripheral blood mononuclear T-lymphocyte of a subject so as to make the T-lymphocyte secrete specific IFN-gamma, and then the spot number of the T-lymphocyte secreting the IFN-gamma is detected through the ELISpot technology, wherein the whole process takes two days; and according to the generated blue-purple spot number, the result is read with the naked eye or instrument, such that the Mycobacterium tuberculosis latent infection related proteins can be used for detection of whether a subject is infected with tubercle bacillus, and active tuberculosis assisted diagnosis and differential diagnosis.

Description

Mycobacterium tuberculosis latent infection associated protein and Synthesis and applications thereof

Technical field

The present invention relates to mycobacterium tuberculosis latent infection associated protein and Synthesis and applications thereof, be specifically related to three kinds of mycobacterium tuberculosis latent infection associated protein LTBIPr1, LTBIPr2 and LTBIPr3 prepared by using gene engineering technique and preparation method thereof, belong to Medical Immunology diagnostic techniques field.

Background technology

Tuberculosis is one of Infectious Diseases of harm humans health in worldwide.The immigrant of HIV/AIDS Epidemic, tuberculosis infected students and the part population reason such as to be poorly off makes European and American developed countries' incidence of tuberculosis such as the U.S. be rise trend, especially tubercule bacillus resistance problems, and human immunodeficiency virus (HIV) concurrent infection tuberculotherapy is made the matter worse especially.There is tuberculosis patient about 2,000 ten thousand in the current whole world, annual newly-increased tuberculosis patient 800 ~ 1,000 ten thousand, annual death toll about 2,000,000.

The tuberculosis epidemic situation of China and tubercle bacillus affection situation are all quite serious, and be one of 22 tuberculosis high burden countries in the whole world, tuberculosis patient numerical digit occupies the second in the world, is only second to India.2000 the 4th time national tuberculosis epidemiological random sampling survey PRELIMINARY RESULTS shows, China's erythema nodosum is 44.5%, higher than the infection rate in the world 1/3, means that China about has 5.5 hundred million people to infect tubercule bacillus.2010 the 5th time national tuberculosis epidemiological random sampling survey PRELIMINARY RESULTS shows, 15 years old and the phthisical morbidity of above crowd activity's property are 4,59/,100,000, and the pulmonary tuberculosis morbidity rate being coated with sun is 66/,100,000.Tuberculosis death toll occupies first in transmissible disease.Tuberculosis is also AIDS the infected's main causes of death, and just have 1 example to die from merging tuberculosis according in every 3 the dead AIDS patients of WHO in 1996 statistics, 45-85%HIV died is caused by diagnosis of tuberculosis is incured loss through delay.In the face of the situation of this sternness, prevention and corntrol lungy has caused the great attention of domestic and international government and scholar.

Not yet fall ill after tuberculosis latent infection (Latent tuberculosis infection, LTBI) refers to host infection mycobacterium tuberculosis, positive and without the clinical manifestation of active tuberculosis and Imageology for feature with tuberculin test.In latent infection person, untreated about has 5%-10% to develop into active tuberculosis, if simultaneously with HIV, this probability is then up to 10%, and tuberculosis latent infection person becomes the important sources of tubercular.There is investigation display: the active tuberculosis that 85%-90% newly diagnoses is developed by LTBI.More severe problem, a part of high risk population is there is in tuberculosis latent infection crowd, in its body, mycobacterium tuberculosis is in vegetative state, but not yet there is clinical manifestation, this kind of crowd is the High risk group of progress for active tuberculosis, if give supervision and Primary preventive intervention treatment to this kind of crowd, the sickness rate of tuberculosis will be reduced.Early screening, Diagnosis and Treat tuberculosis latent infection are one of most effective means of tuberculosis prevention and treatment.But the diagnosis at present for latent infection still lacks unified standard and method, therefore, the breakthrough of latent infection early diagnosis technology becomes the significant challenge faced in current Control.

The method of diagnosis latent infection mainly comprises tuberculin skin test (tuberculin skin test, TST) and IFN-γ release test (interferon gamma release assays, IGRA) both at home and abroad at present.TST and IGRA evaluates tuberculosis infection situation by the main tuberculosis immunity of detection body and cellullar immunologic response.Briefly introduce with regard to these two kinds of test methods below.

Domestic much more existing using TST as main detection means, generally by purified protein derivative (PPD) (PPD) strong positive or transfer the positive to from feminine gender in a short time and be judged as tubercule bacillus latent infection person without clinical tuberculosis evidence person.PPD its act on people know from experience excite sensitization body produce cell immune response, activated T lymphocytes, monocyte and scavenger cell, discharge a large amount of cytokine, and make these cell proliferations, gathering, coating antigen forms tubercle, delayed metamorphosis that Here it is (DTH) is reacted, and its response intensity and cellular immunization are parallel relation.PPD tuerculoderma has become a kind of tubercle bacillus affection diagnostic method the most frequently used and the easiest clinically at present, react stronger, represent that tuberculosis infection possibility is larger, China uses scleroma >=5mm for the positive always, >=20mm or have bubble, necrosis, poradenolymphitis etc. for strong positive, less than 3 years old children >=15mm is the standard of strong positive.TST feature is simple to operate, cheap, but PPD is the antigen mixture slightly carried from mycobacterium tuberculosis, comprise 200 multiple protein, it is much wherein the common antigen composition of non-tuberculous mycobacteria and bacill calmette-guerin, therefore determine TST detection specificity poor, inoculate in crowd and non-tuberculous mycobacteria infection population at BCG and easily produce false positive results.TST can only according to the strong and weak auxiliary diagnosis of the reaction of skin, and its sensitivity only has 70-80%.When TST exists check fee in addition, need experimenter to pay a return visit (72h), skin test operation and result to explain to there is the shortcomings such as subjective dependency.

In recent years, IGRA is used for m tuberculosis infection detection as the novel detection means of one.IGRA adopts enzyme linked immunosorbent assay (ELISA) or ELISpot test (ELISPOT) method quantitatively to detect person under inspection's whole blood or peripheral blood mononuclear cell to detect release reaction, for the diagnosis of tubercule bacillus latent infection to the IFN-γ of Specific Antigen of Mycobacterium Tuberculosis (ESAT6, CFP10 and TB7.7).In October, 2007, application ESAT-6, CFP-10 and TB7.7 QuantiFERON-TB Gold In-Tube(QFT-GIT as specific antigens and through constantly improveing) technology, become the detection tuberculosis infection method that FDA approval uses.In July, 2008, researched and developed by England Oxford immunological technique company, utilize ESAT6 polypeptide and CFP10 polypeptide to stimulate peripheral blood T lymphocyte respectively, then detect the T SPOT-TB test box of the periphery blood T lymphocyte number of secretion of gamma-IFN by FDA official approval.Antigen ESAT6, CFP10 and TB7.7 of applying in these two kinds of technology are selected from M. tuberculosis genes group difference section, its all expression deletion in BCG and most of environment mycobacterium, the cross reaction with these bacterial classifications can be avoided, compensate for the deficiency of TST, improve the specificity that m tuberculosis infection detects.But IGRA can not differentiate active tuberculosis and tuberculosis infection of hiding.

As previously mentioned, the method for diagnosis latent infection comprises TST and IGRA detection method both at home and abroad at present.TST is cheap, domestic showing mainly with it is main detection means, but TST is subject to the impact of bacille Calmette-Guerin vaccine and subject immune's state, easily occurs false positive and false negative result, especially Chinese neonates BCG (Bacille Calmette-Guerin) vaccination is classified as planned immunity for children project, and TST diagnoses latent infection efficiency very low.IGRA is difficult to distinguish active tuberculosis and tuberculosis infection of hiding.

Summary of the invention

The object of the invention is the deficiency for overcoming prior art, three kinds of novel mycobacterium tuberculosis latent infection associated protein LTBIPr1, LTBIPr2 and LTBIPr3 and preparation method thereof are provided, as tuberculosis cellular immunization stimulant, for detecting in peripheral blood the T lymphocyte secreting tuberculosis specificity IFN-γ, sensitivity and the specificity of the detection of tuberculosis latent infection can be improved, auxiliary diagnosis and differential diagnosis lungy.

A kind of mycobacterium tuberculosis latent infection associated protein LTBIPr1, is made up of the critical epitopes of mycobacterium tuberculosis protein antigen; The nucleotide sequence (DNA sequence dna) of LTBIPr1 Protein Epitopes is as shown in sequence in sequence table 1.

Another kind of mycobacterium tuberculosis latent infection associated protein LTBIPr2, is made up of the critical epitopes of mycobacterium tuberculosis protein antigen; The nucleotide sequence of LTBIPr2 Protein Epitopes is as shown in sequence in sequence table 2.

The third mycobacterium tuberculosis latent infection associated protein LTBIPr3, is made up of the critical epitopes of mycobacterium tuberculosis protein antigen; The nucleotide sequence of LTBIPr3 Protein Epitopes is as shown in sequence in sequence table 3.

LTBIPr1 coding phosphofructokinase (phosphofructokinase pfkB) is the antigen in latent period that mycobacterium tuberculosis anoxic is relevant.The research of contriver shows the effector T cell number discharging IFN-γ after this proteantigen stimulates latent infection crowd PBMC and is significantly higher than active tuberculosis crowd.Application LTBIPr1DNA vaccine immunity BALB/c mouse, can induce stronger humoral immunization and Th1 type cellullar immunologic response in mouse.

LTBIPr2 may encode PhiRv2 phage protein intergrase (PhiRv2prophage integrase), is positioned at BCG and lacks district RD11, is the antigen in latent period that nutritive deficiency is relevant.The research of contriver shows the effector T cell number producing IFN-γ after this proteantigen stimulates tuberculosis latent infection person peripheral blood PBMC and is significantly higher than active tuberculosis patient.

LTBIPr3 encodes a kind of imaginary albumen, is the antigen in latent period that mycobacterium tuberculosis anoxic is relevant.The research of contriver show this proteantigen stimulate latent infection crowd peripheral blood PBMC after the effector T cell number of secretion of gamma-IFN be significantly higher than pulmonary tuberculosis crowd.

The epitope of above-mentioned LTBIPr1, LTBIPr2 and LTBIPr3 tri-kinds of mycobacterium tuberculosis latent infection associated protein is cloned respectively, expresses, purifying, tuberculosis latent infection diagnosis and latent infection vaccine research in there is very large potential value.

Three kinds of mycobacterium tuberculosis latent infection associated protein LTBIPr1, LTBIPr2 and LTBIPr3 that the present invention proposes, be select the epitope of its key, cloned, express respectively by genetic engineering technique, purifying.

The present invention proposes preparation and the construction process of above-mentioned three kinds of recombinant proteins LTBIPr1, LTBIPr2 and LTBIPr3.

A construction process of mycobacterium tuberculosis latent infection associated protein LTBIPr1, comprises the steps:

(1) clone of protein gene: cloned by genetic engineering technique

Add Nhe I restriction enzyme site at LTBIPr1 upstream primer, add EcoR I restriction enzyme site at LTBIPr1 downstream primer, by pcr amplification product with after Nhe I and EcoR I double digestion, insert in the pET30aSETB plasmid vector after with Nhe I and EcoR I double digestion; Be transformed in bacillus coli DH 5 alpha recipient bacterium; Through plasmid extraction, through the two-way sequence verification of T7 and T7t, obtain nucleotide sequence and the on all four recombinant plasmid LTBIPr1/pGEM-T of design, its nucleotide sequence is as shown in sequence in sequence table 4;

Recombinant plasmid LTBIPr1/pGEM-T is transformed in e. coli bl21 (DE3) recipient bacterium; Through plasmid extraction, through T7 and/or T7t sequence verification, obtain nucleotide sequence and the on all four recombinant expression plasmid LTBIPr1/pET30aSETB of design, its nucleotide sequence, as shown in sequence in sequence table 5, shows successfully to construct the recombinant expression plasmid with mycobacterium tuberculosis latent infection associated protein LTBIPr1 epitope;

(2) qualification of recombinant protein: LTBIPr1 bacillus coli gene engineering strain (LTBIPr1/pET30aSETB) carried out induce, express, purifying, SDS-PAGE electrophoresis, qualification, ELISPOT and elisa assay, obtain the recombinant protein of the purifying conformed to the size of actual design, and there is good immunogenicity and antigenicity.The LTBIPr1 upstream primer (5 ' end is containing restriction enzyme Nhe I) adopted in the above-mentioned methods is:

5’－CTAGGCTAGCCACCATGGCGGAGCCAGCGGCGTG－3'

LTBIPr1 downstream primer (5 ' end is containing restriction enzyme EcoR I) is:

5’－CCGGAATTC TCATGGCGAGGCTTCCGGG－3'

A construction process of mycobacterium tuberculosis latent infection associated protein LTBIPr2, comprises the steps:

(1) clone of protein gene: cloned by genetic engineering technique

Add Nhe I restriction enzyme site at LTBIPr2 upstream primer, add EcoR I restriction enzyme site at LTBIPr2 downstream primer, by pcr amplification product with after Nhe I and EcoR I double digestion, insert in the pET30aSETB plasmid vector after with Nhe I and EcoR I double digestion; Be transformed in bacillus coli DH 5 alpha recipient bacterium; Through plasmid extraction, through the two-way sequence verification of T7 and T7t, obtain nucleotide sequence and the on all four recombinant plasmid LTBIPr2/pGEM-T of design, its nucleotide sequence is as shown in sequence in sequence table 6;

Recombinant plasmid LTBIPr2/pGEM-T is transformed in e. coli bl21 (DE3) recipient bacterium; Through plasmid extraction, through T7 and/or T7t sequence verification, obtain nucleotide sequence and the on all four recombinant expression plasmid LTBIPr2/pET30aSETB of design, its nucleotide sequence, as shown in sequence in sequence table 7, shows successfully to construct the recombinant expression plasmid with mycobacterium tuberculosis latent infection associated protein LTBIPr2 epitope;

(2) qualification of recombinant protein: LTBIPr2 bacillus coli gene engineering strain (LTBIPr2/pET30aSETB) carried out induce, express, purifying, SDS-PAGE electrophoresis, qualification, ELISPOT and elisa assay, obtain the recombinant protein of the purifying conformed to the size of actual design, and there is good immunogenicity and antigenicity.

The LTBIPr2 upstream primer (5 ' end is containing restriction enzyme Nhe I) adopted in the above-mentioned methods is:

5’－CTAGGCTAGCCACCATGGCGCAAACCGGCAAGC－3'

LTBIPr2 downstream primer (5 ' end is containing restriction enzyme EcoR I) is:

5’－CCGGAATTCTCACATCTCCTGGTTCTCG－3'

A construction process of mycobacterium tuberculosis latent infection associated protein LTBIPr3, comprises the steps:

(1) clone of protein gene: cloned by genetic engineering technique

Add Nhe I restriction enzyme site at LTBIPr3 upstream primer, add EcoR I restriction enzyme site at LTBIPr3 downstream primer, by pcr amplification product with after Nhe I and EcoR I double digestion, insert in the pET30aSETB plasmid vector after with Nhe I and EcoR I double digestion; Be transformed in e. coli bl21 (DE3) recipient bacterium; Through plasmid extraction, through T7 and/or T7t sequence verification, obtain nucleotide sequence and the on all four recombinant expression plasmid LTBIPr3/pET30aSETB of design, its nucleotide sequence, as shown in sequence in sequence table 8, shows successfully to construct the recombinant expression plasmid with mycobacterium tuberculosis latent infection associated protein LTBIPr3 epitope;

(2) qualification of recombinant protein: LTBIPr3 bacillus coli gene engineering strain (LTBIPr3/pET30aSETB) carried out induce, express, purifying, SDS-PAGE electrophoresis, qualification, ELISPOT and elisa assay, obtain the recombinant protein of the purifying conformed to the size of actual design, and there is good immunogenicity and antigenicity.

The LTBIPr3 upstream primer (5 ' end is containing restriction enzyme Nhe I) adopted in the above-mentioned methods is:

5’－CTAGGCTAGCCACCATGGCCACGCAACGACCGAG－3'

LTBIPr3 downstream primer (5 ' end is containing restriction enzyme EcoR I) is:

5’－CCGGAATTCTTAGACCGCAACGGCAATC－3'

Mycobacterium tuberculosis latent infection associated protein LTBIPr1 of the present invention, LTBIPr2 and LTBIPr3 can be applicable to preparation ELISpot tuberculosis infection diagnostic reagent kit respectively.Result of study shows, compare with IGRA detection kit with current commercial PPD tuerculoderma, mycobacterium tuberculosis latent infection associated protein LTBIPr1 prepared by the present invention, LTBIPr2 and LTBIPr3, in the diagnosis of tuberculosis latent infection, all there is higher sensitivity and specificity, and with other antigen, there is complementary advantage, can be used for hiding tuberculosis infection diagnosis, differentiate that active tuberculosis and latent tuberculosis infect, auxiliary diagnosis and differential diagnosis lungy.

The present invention is different with IGRA detection kit two kinds of detection methods from above-mentioned PPD tuerculoderma, adopt mycobacterium tuberculosis under anoxic and hungry environment, latent infection associated protein LTBIPr1, LTBIPr2 and LTBIPr3 that specific up-regulation is expressed stimulate person under inspection's periphery blood T lymphocyte as specific antigens, it is made to secrete specificity IFN-γ, then by ELISpot technology for detection, whole process needs 2 day time, person under inspection can be understood whether infect tubercule bacillus, whether be in the latent infection phase, and auxiliary diagnosis and differential diagnosis lungy.

Feature of the present invention and technique effect:

Recombinant protein LTBIPr1, LTBIPr2 and LTBIPr3 that the present invention is correlated with by genetic engineering technique clone, expression, purifying three kinds of mycobacterium tuberculosis latent infections, as tuberculosis cellular immunization stimulant, for detecting in peripheral blood the T lymphocyte secreting tuberculosis specificity IFN-γ, these recombinant proteins can be used for detecting tuberculosis latent infection, and there is no such detection reagent at present.

The present inventor studies proves that the sensitivity of recombinant protein LTBIPr1, LTBIPr2 and LTBIPr3 Detection of antigen tuberculosis latent infection that application three kinds of mycobacterium tuberculosis latent infections are correlated with is respectively 81.1%, 54.05%, 61.5%, and specificity is respectively 82.9%, 81.40% and 82.4%.

Mycobacterium tuberculosis restructuring LTBIPr1, LTBIPr2 and LTBIPr3 proteantigen of the present invention can be mass-produced, and advantage of lower cost.

Three kinds of recombinant proteins of the present invention can be used as one of associating antigen of novel tuberculosis infection detection, for detecting the T lymphocyte secreting tuberculosis specificity IFN-γ in different tuberculosis infection state person peripheral blood, can be used for the cellular immunology diagnosis of tuberculosis infection.

Accompanying drawing explanation

Fig. 1 is 1% agarose electrophoresis figure of the LTBIPr1DNA fragment of pcr amplification.

Fig. 2 is SDS-PAGE result before and after LTBIPr1/pET30aSETB colibacillus engineering IPTG induces.

Fig. 3 is the expression-form of LTBIPr1/pET30aSETB colibacillus engineering and the SDS-PAGE result of purifying.

Fig. 4 is 1% agarose electrophoresis figure of the LTBIPr2DNA fragment of pcr amplification.

Fig. 5 is SDS-PAGE result before and after LTBIPr2/pET30aSETB colibacillus engineering IPTG induces.

Fig. 6 is the expression-form of LTBIPr2/pET30aSETB colibacillus engineering and the SDS-PAGE result of purifying.

Fig. 7 is 1% agarose electrophoresis figure of the LTBIPr3DNA fragment of pcr amplification.

Fig. 8 is SDS-PAGE result before and after LTBIPr3/pET30aSETB colibacillus engineering IPTG induces.

Fig. 9 is the expression-form of LTBIPr3/pET30aSETB colibacillus engineering and the SDS-PAGE result of purifying.

Embodiment

Embodiment 1

One, engineering bacteria, abduction delivering purification Identification recombinant protein LTBIPr1 is built by genetic engineering technique

1, according to the pair of primers of the sequence 1 Design and synthesis amplification LTBIPr1 epitope in sequence table

Upstream primer (5 ' end is containing restriction enzyme Nhe I)

5’－CTAGGCTAGCCACCATGGCGGAGCCAGCGGCGTG－3'

Downstream primer (5 ' end is containing restriction enzyme EcoR I)

5’－CCGGAATTCTCATGGCGAGGCTTCCGGG－3'

Amplified fragments: 1035bp

2, pcr amplification LTBIPr1 gene

With above-mentioned upstream and downstream primer, under the effect of Taq archaeal dna polymerase, with Mycobacterium tuberculosis H37Rv DNA for template, amplification LTBIPr1 gene.After 94 DEG C of 10min warm starts, add archaeal dna polymerase and carry out PCR, response procedures: 94 DEG C of 1min, about 65 DEG C 1min, 72 DEG C of 1min, circulate 30 times; Last 72 DEG C extend 7min.In the amplification of DNA fragments of 1% agarose gel electrophoresis qualification 1035bp.Fig. 1 is the agarose electrophoresis figure of the LTBIPr1DNA fragment of pcr amplification, and in figure, the DNA band of left side swimming lane display is exactly the position of PCR primer on 1% agarose electrophoresis glue of the 1035bp of LTBIPr1 gene amplification; Right lanes is Beijing CoWin Bioscience Co., Ltd. molecular weight standard DM5000DNA Marker, and band is respectively 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp.

3, goal gene fragment is reclaimed:

After agarose gel electrophoresis terminates, under UVA Radiation, on glue, cut the agar block that will reclaim DNA with clean knife blade, put into aseptic centrifuge tube.The specification sheets reclaimed in test kit with reference to agarose DNA reclaims goal gene fragment, quantitatively, be stored in-20 DEG C for subsequent use.

4. goal gene is connected with pGEM-T

With reference to Promega company pGEM-T vector System-I description of product, PCR primer after purifying be connected with cloning vector pGEM-T, 10 μ l reaction systems are as follows:

Mixing is placed on 4 DEG C of refrigerator reaction 12h.75 DEG C of deactivation 10min, directly transform after ice bath.

5. connect the conversion of product:

The connection product 5 μ l of goal gene fragment and pGEM-T is added in the centrifuge tube containing 100 μ l bacillus coli DH 5 alpha competent cells, ice bath 0.5h; Put into 42 DEG C of water bath heat-shocked 90s, take out ice bath 2min rapidly; Add LB nutrient solution 400 μ l, put 37 DEG C of constant-temperature tables and cultivate 1h; Add X-Gal60 μ l, IPTG4 μ l, mixing, the LB that taking-up 200-400 μ l coats containing penbritin (60ug/ml) is dull and stereotyped.Be inverted dull and stereotyped, put 37 DEG C of constant incubators and cultivate 14h.

6. the extraction of plasmid:

Screen according to blue hickie, random picking white colony 6, be inoculated in 5ml respectively and contain in the LB substratum of 60 μ g/ml penbritins, put 37 DEG C of shaking culture and spend the night; According to " molecular cloning " alkaline lysis method, extract plasmid in a small amount, quantitatively, put-20 DEG C and store for future use.

7. identify recombinant plasmid:

(1) pcr amplification qualification

The thallus DNA selecting 6 white colonies is template, and above, downstream primer carries out pcr amplification qualification; 1% agarose gel electrophoresis, if DNA molecular amount standard is reference, positive recombinant plasmid called after LTBIPr1/pGEM-T.

(2) sequencing

Select a clone through above-mentioned qualification and send order-checking.The sequencing result of LTBIPr1/pGEM-T is consistent with the genome sequence of design, and its nucleotide sequence is as follows, and in sequence table as shown in sequence 4, wherein, GCTAGC is Nhe I restriction enzyme site, and GAATTC is EcoR I restriction enzyme site.

GCAGTAGGTGCCTATAGAATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTCTA ATGACGGAGCCAGCGGCGTGGGACGAAGGCAAGCCGCGAATCATCACTTTGACCATGAACCCCGCCTTGGACATCACGACGAGCGTCGACGTGGTGCGCCCGACCGAGAAAATGCGTTGTGGCGCACCTCGCTACGATCCCGGCGGCGGCGGTATCAATGTCGCCCGCATTGTGCATGTCCTCGGCGGTTGCTCGACAGCACTGTTCCCGGCCGGCGGGTCGACCGGGAGCCTGCTGATGGCGCTGCTCGGTGATGCGGGAGTGCCATTTCGCGTCATTCCGATCGCGGCCTCGACGCGGGAGAGCTTCACGGTCAACGAGTCCAGGACCGCCAAGCAGTATCGTTTCGTGCTTCCGGGGCCGTCGCTGACCGTCGCGGAGCAGGAGCAATGCCTCGACGAACTGCGCGGTGCGGCGGCTTCGGCCGCCTTTGTGGTGGCCAGTGGCAGCCTGCCGCCAGGTGTGGCTGCCGACTACTATCAGCGGGTTGCCGACATCTGCCGCCGATCGAGCACTCCGCTGATCCTGGATACATCTGGTGGCGGGTTGCAGCACATTTCGTCCGGGGTGTTTCTTCTCAAGGCGAGCGTGCGGGAACTGCGCGAGTGCGTCGGATCCGAACTGCTGACCGAGCCCGAACAACTGGCCGCCGCACACGAACTCATTGACCGTGGGCGCGCCGAGGTCGTGGTGGTCTCGCTTGGATCTCAGGGCGCGCTATTGGCCACACGACATGCGAGCCATCGATTTTCGTCGATTCCGATGACCGCGGTTAGCGGTGTCGGCGCCGGCGACGCGATGGTGGCCGCGATTACCGTGGGCCTCAGCCGTGGCTGGTCGCTCATCAAGTCCGTTCGCTTGGGAAACGCGGCAGGTGCAGCCATGCTGCTGACGCCAGGCACCGCGGCCTGCAATCGCGACGATGTGGAGAGGTTCTTCGAGCTGGCGGCCGAACCCACCGAAGTCGGGCAGGATCAATACGTTTGGCACCCGATCGTTAACCCGGAAGCCTCGCCATGA CGGAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGCCCAATCGCCCCCTAAG

8. by restriction enzyme Nhe I and EcoR I double digestion LTBIPr1/pGEM-T plasmid DNA, electrophoresis in 1% sepharose, cut the LTBIPr1 gene fragment of 1035bp and the pET30aSetB plasmid DNA fragment of 5.344kb, reclaim kits with agarose gel electrophoresis.Quantitatively, LTBIPr1 and pET30aSetB plasmid DNA fragment, by the mixed in molar ratio of 2 ︰ 1, under the catalysis of T4DNA ligase enzyme, is spent the night in 16 DEG C of connections, get next day and connect product 5 μ l transformation of E. coli BL21(DE3) competent cell, put 37 DEG C of thermostat containers and hatch 14h.Transferred species is in 5ml containing in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins respectively to select 6 clones, and put 37 DEG C of constant temperature oscillator overnight incubation, according to " molecular cloning " alkaline lysis method, a small amount of extracts plasmid, quantitatively, puts-20 DEG C and stores for future use.

9. the qualification of recombinant expression plasmid

(1) pcr amplification qualification

The thallus DNA selecting 6 white colonies is template, and above, downstream primer carries out pcr amplification qualification; 1% agarose gel electrophoresis, if DNA molecular amount standard is reference, positive recombinant plasmid called after LTBIPr1/pET30aSETB.

(2) sequencing

Select a clone through above-mentioned qualification and send order-checking.The sequencing result of LTBIPr1/pET30aSETB is consistent with the genome sequence of design, and its nucleotide sequence is as follows, and in sequence table as shown in sequence 5, wherein, GCTAGC is Nhe I restriction enzyme site, and GAATTC is EcoR I restriction enzyme site.

TAGCTCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTC TCATGGCGAGGCTTCCGGGTTAACGATCGGGTGCCAAACGTATTGATCCTGCCCGACTTCGGTGGGTTCGGCCGCCAGCTCGAAGAACCTCTCCACATCGTCGCGATTGCAGGCCGCGGTGCCTGGCGTCAGCAGCATGGCTGCACCTGCCGCGTTTCCCAAGCGAACGGACTTGATGAGCGACCAGCCACGGCTGAGGCCCACGGTAATCGCGGCCACCATCGCGTCGCCGGCGCCGACACCGCTAACCGCGGTCATCGGAATCGACGAAAATCGATGGCTCGCATGTCGTGTGGCCAATAGCGCGCCCTGAGATCCAAGCGAGACCACCACGACCTCGGCGCGCCCACGGTCAATGAGTTCGTGTGCGGCGGCCAGTTGTTCGGGCTCGGTCAGCAGTTCGGATCCGACGCACTCGCGCAGTTCCCGCACGCTCGCCTTGAGAAGAAACACCCCGGACGAAATGTGCTGCAACCCGCCACCAGATGTATCCAGGATCAGCGGAGTGCTCGATCGGCGGCAGATGTCGGCAACCCGCTGATAGTAGTCGGCAGCCACACCTGGCGGCAGGCTGCCACTGGCCACCACAAAGGCGGCCGAAGCCGCCGCACCGCGCAGTTCGTCGAGGCATTGCTCCTGCTCCGCGACGGTCAGCGACGGCCCCGGAAGCACGAAACGATACTGCTTGGCGGTCCTGGACTCGTTGACCGTGAAGCTCTCCCGCGTCGAGGCCGCGATCGGAATGACGCGAAATGGCACTCCCGCATCACCGAGCAGCGCCATCAGCAGGCTCCCGGTCGACCCGCCGGCCGGGAACAGTGCTGTCGAGCAACCGCCGAGGACATGCACAATGCGGGCGACATTGATACCGCCGCCGCCGGGATCGTAGCGAGGTGCGCCACAACGCATTTTCTCGGTCGGGCGCACCACGTCGACGCTCGTCGTGATGTCCAAGGCGGGGTTCATGGTCAAAGTGATGATTCGCGGCTTGCCTTCGTCCCACGCCGCTGGCTCCGTCA T CATACCATGATGATGATGATGATGAGAACCCCGCATATGTATATCTCCTTCTTAAAGTAAACAAAATTATTCTAGAGGGAAGACC

Two, the abduction delivering of LTBIPr1 engineering bacteria and qualification

LTBIPr1 colibacillus engineering (LTBIPr1/pET30aSETB) is inoculated in 5ml containing in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins, put 37 DEG C of constant temperature oscillator overnight incubation, then contain in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins to 10ml by 1% transferred species, put 37 DEG C of constant temperature oscillators and be cultured to OD ₆₀₀when being 0.8, add IPTG, induction 3-4hr.Distilled water is added the precipitation sample of 1ml bacterium liquid, piping and druming mixing, again 1 × loading buffer 50 μ l is added the precipitation sample from 1ml bacterium liquid, piping and druming mixing, 100 DEG C of boiling water bath 5min, the centrifugal 10min of 12000g, get supernatant 20 μ l and carry out SDS-PAGE, deposition condition is: spacer gel constant voltage 80v, separation gel constant voltage 120v, treat bottom tetrabromophenol sulfonphthalein electrophoresis to gel, stop electrophoresis.With coomassie brilliant blue R250 staining fluid dyeing 6h; Destainer decolouring is clear to band, and observe whether there is target protein band of expression, the results are shown in Figure 2, the recombinant protein molecular weight of expression is about 36.85kDa, induces 4-6 hour expression amount maximum.Fig. 2 is SDS-PAGE result before and after pET30aSETB-LTBIPr1 colibacillus engineering IPTG induces, wherein:

M: protein molecular weight standard (Beijing Tian Gen biochemical technology company limited pre-dyed albumen Marker);

1: genetic engineering bacterium IPTG latter 1 hour of induction;

2: genetic engineering bacterium IPTG latter 2 hours of induction;

3: genetic engineering bacterium IPTG latter 3 hours of induction;

4: genetic engineering bacterium IPTG latter 4 hours of induction;

5: genetic engineering bacterium IPTG latter 5 hours of induction;

6: genetic engineering bacterium IPTG latter 6 hours of induction;

Before 7: genetic engineering bacterium IPTG induction.

Three, the purifying of LTBIPr1 recombinant protein

After bacterium ultrasonication will be induced, test kit specification sheets purifying LTBIPr1 albumen pressed by the Ni Sepharose6Fast Flow resin adopting GE Healthcare Life Sciences company to produce, the 36.85kDa albumen of the visible purifying of SDS-PAGE electrophoresis is a band, recombinant protein is expressed with inclusion bodies, the results are shown in Figure 3.Fig. 3 is the expression-form of LTBIPr1/pET30aSETB colibacillus engineering and the SDS-PAGE result of purifying, wherein:

M: protein molecular weight standard (Beijing Tian Gen biochemical technology company limited pre-dyed albumen);

Before 1: genetic engineering bacterium IPTG induction;

After 2: genetic engineering bacterium IPTG induction;

3: the isolated supernatant of high speed centrifugation (soluble fractions) sample after the ultrasonication of genetic engineering bacterium expression thalline;

4: the isolated precipitation of high speed centrifugation (insolubility part) sample after the ultrasonication of genetic engineering bacterium expression thalline;

5: protein sample after purifying.

Result shows that the recombinant protein of purifying obtained conforms to the size of actual design, and has good immunogenicity and antigenicity.

Embodiment 2

One, by the engineering bacteria of genetic engineering technique construction expression LTBIPr2 albumen:

1, according to the pair of primers of the sequence 1 Design and synthesis amplification LTBIPr2 epitope in sequence table

Upstream primer (5 ' end is containing restriction enzyme Nhe I)

5’－CTAGGCTAGCCACCATGGCGCAAACCGGCAAGC－3'

Downstream primer (5 ' end is containing restriction enzyme EcoR I)

5’－CCGGAATTCTCACATCTCCTGGTTCTCG－3'

Amplified fragments: 1143bp

2, pcr amplification LTBIPr2 gene

With above-mentioned upstream and downstream primer, under the effect of Taq archaeal dna polymerase, with Mycobacterium tuberculosis H37Rv DNA for template, amplification LTBIPr2 gene.After 94 DEG C of 10min warm starts, add archaeal dna polymerase and carry out PCR, response procedures: 94 DEG C of 1min, about 60 DEG C 1min, 72 DEG C of 1min, circulate 30 times; Last 72 DEG C extend 7min.In the amplification of DNA fragments of 1% agarose gel electrophoresis qualification 1143bp.Fig. 4 is the agarose electrophoresis figure of the LTBIPr2DNA fragment of pcr amplification, and in figure, the DNA band of left side swimming lane display is exactly the position of PCR primer on 1% agarose electrophoresis glue of LTBIPr2 gene amplification; Right lanes is Beijing CoWin Bioscience Co., Ltd. molecular weight standard DM5000DNA Marker, and band is respectively 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp.

3, goal gene fragment is reclaimed:

4. goal gene is connected with pGEM-T

5. connect the conversion of product:

6. the extraction of plasmid:

7. identify recombinant plasmid:

(1) pcr amplification qualification

The thallus DNA selecting 6 white colonies is template, and above, downstream primer carries out pcr amplification qualification; 1% agarose gel electrophoresis, if DNA molecular amount standard is reference, positive recombinant plasmid called after LTBIPr2/pGEM-T.

(2) sequencing

Select a clone through above-mentioned qualification and send order-checking.The sequencing result of LTBIPr2/pGEM-T is consistent with the genome sequence of design, and its nucleotide sequence is as follows, and in sequence table as shown in sequence 6, wherein, GCTAGC is Nhe I restriction enzyme site, and GAATTC is EcoR I restriction enzyme site.

TAGAATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTCTA GTGACGCAAACCGGCAAGCGTCAGAGACGCAAATTCGGTCGCATCCGACAGTTCAACTCCGGCCGCTGGCAAGCCAGCTACACCGGCCCCGACGGCCGCGTGTACATCGCCCCCAAAACCTTCAACGCCAAGATCGACGCCGAAGCATGGCTCACCGACCGCCGCCGCGAAATCGACCGACAACTATGGTCCCCGGCATCGGGTCAGGAAGACCGCCCCGGAGCCCCATTCGGTGAGTACGCCGAAGGATGGCTGAAGCAGCGTGGAATCAAGGACCGCACCCGCGCCCACTATCGCAAACTGCTGGACAACCACATCCTGGCCACCTTCGCTGACACCGACCTACGCGACATCACCCCGGCCGCCGTGCGCCGCTGGTACGCCACCACCGCCGTGGGCACACCGACCATGCGGGCACACTCCTACAGCTTGCTGCGCGCAATCATGCAGACCGCCTTGGCCGACGACCTGATCGACTCCAACCCCTGCCGCATCTCAGGCGCGTCCACCGCCCGCCGCGTCCACAAGATCAGGCCCGCCACCCTCGACGAGCTGGAAACCATCACCAAAGCCATGCCCGACCCCTACCAGGCGTTCGTGCTGATGGCGGCATGGCTGGCCATGCGCTACGGCGAGCTGACCGAAT TACGCCGCAAAGACATCGACCTGCACGGCGAGGTTGCGCGGGTGCGGCGGGCTGTCGTTCGGGTGGGCGAAGGCTTCAAGGTGACGACACCGAAAAGCGATGCGGGAGTGCGCGACATAAGTATCCCGCCACATCTGATACCCGCCATCGAAGACCACCTTCACAAACACGTCAACCCCGGCCGGGAGTCCCTGCTGTTCCCATCGGTCAACGACCCCAACCGTCACCTAGCACCCTCGGCGCTGTACCGCATGTTCTACAAGGCCCGAAAAGCCGCCGGCCGACCAGACTTACGGGTGCACGACCTTCGACACTCCGGCGCCGTGTTGGCTGCATCCACCGGCGCCACACTGGCCGAACTGATGCAGCGGCTAGGACACAGCACAGCCGGCGCCGCACTCCGCTACCAGCACGCCGCCAAGGGCCGGGACCGCGAAATCGCCGCACTGTTAAGCAAACTGGCCGAGAACCAGGAGATGTGA CGGAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCG

8. by restriction enzyme Nhe I and EcoR I double digestion LTBIPr2/pGEM-T plasmid DNA, electrophoresis in 1% sepharose, cut the LTBIPr2 gene fragment of 1143bp and the pET30aSetB plasmid DNA fragment of 5.344kb, reclaim kits with agarose gel electrophoresis.Quantitatively, LTBIPr2 and pET30aSetB plasmid DNA fragment, by the mixed in molar ratio of 2 ︰ 1, under the catalysis of T4DNA ligase enzyme, is spent the night in 16 DEG C of connections, get next day and connect product 5 μ l transformation of E. coli BL21(DE3) competent cell, put 37 DEG C of thermostat containers and hatch 14h.Transferred species is in 5ml containing in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins respectively to select 6 clones, and put 37 DEG C of constant temperature oscillator overnight incubation, according to " molecular cloning " alkaline lysis method, a small amount of extracts plasmid, quantitatively, puts-20 DEG C and stores for future use.

9. the qualification of recombinant expression plasmid

(1) pcr amplification qualification

The thallus DNA selecting 6 white colonies is template, and above, downstream primer carries out pcr amplification qualification; 1% agarose gel electrophoresis, if DNA molecular amount standard is reference, positive recombinant plasmid called after LTBIPr2/pET30aSETB.

(2) sequencing

Select a clone through above-mentioned qualification and send order-checking.The sequencing result of LTBIPr2/pET30aSETB is consistent with the genome sequence of design, and its nucleotide sequence is as follows, and in sequence table as shown in sequence 7, wherein, GCTAGC is Nhe I restriction enzyme site, and GAATTC is EcoR I restriction enzyme site.

CCGCGTCCCGGGCTCGGGATGCGTCATTCCCTCTGAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATG GTGACGCAAACCGGCAAGCGTCAGAGACGCAAATTCGGTCGCATCCGACAGTTCAACTCCGGCCGCTGGCAAGCCAGCTACACCGGCCCCGACGGCCGCGTGTACATCGCCCCCAAAACCTTCAACGCCAAGATCGACGCCGAAGCATGGCT CACCGACCGCCGCCGCGAAATCGACCGACAACTATGGTCCCCGGCATCGGGTCAGGAAGACCGCCCCGGAGCCCCATTCGGTGAGTACGCCGAAGGATGGCTGAAGCAGCGTGGAATCAAGGACCGCACCCGCGCCCACTATCGCAAACTGCTGGACAACCACATCCTGGCCACCTTCGCTGACACCGACCTACGCGACATCACCCCGGCCGCCGTGCGCCGCTGGTACGCCACCACCGCCGTGGGCACACCGACCATGCGGGCACACTCCTACAGCTTGCTGCGCGCAATCATGCAGACCGCCTTGGCCGACGACCTGATCGACTCCAACCCCTGCCGCATCTCAGGCGCGTCCACCGCCCGCCGCGTCCACAAGATCAGGCCCGCCACCCTCGACGAGCTGGAAACCATCACCAAAGCCATGCCCGACCCCTACCAGGCGTTCGTGCTGATGGCGGCATGGCTGGCCATGCGCTACGGCGAGCTGACCGAATTACGCCGCAAAGACATCGACCTGCACGGCGAGGTTGCGCGGGTGCGGCGGGCTGTCGTTCGGGTGGGCGAAGGCTTCAAGGTGACGACACCGAAAAGCGATGCGGGAGTGCGCGACATAAGTATCCCGCCACATCTGATACCCGCCATCGAAGACCACCTTCACAAACACGTCAACCCCGGCCGGGAGTCCCTGCTGTTCCCATCGGTCAACGACCCCAACCGTCACCTAGCACCCTCGGCGCTGTACCGCATGTTCTACAAGGCCCGAAAAGCCGCCGGCCGACCAGACTTACGGGTGCACGACCTTCGACACTCCGGCGCCGTGTTGGCTGCATCCACCGGCGCCACACTGGCCGAACTGATGCAGCGGCTAGGACACAGCACAGCCGGCGCCGCACTCCGCTACCAGCACGCCGCCAAGGGCCGGGACCGCGAAATCGCCGCACTGTTAAGCAAACTGGCCGAGAACCAGGAGATGTGA GAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTATGAC

Two, the abduction delivering of LTBIPr2 engineering bacteria and qualification

LTBIPr2 colibacillus engineering (LTBIPr2/pET30aSETB) is inoculated in 5ml containing in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins, put 37 DEG C of constant temperature oscillator overnight incubation, then contain in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins to 10ml by 1% transferred species, put 37 DEG C of constant temperature oscillators and be cultured to OD ₆₀₀when being 0.8, add IPTG, induction 3-4hr.Distilled water is added the precipitation sample of 1ml bacterium liquid, piping and druming mixing, again 1 × loading buffer 50 μ l is added the precipitation sample from 1ml bacterium liquid, piping and druming mixing, 100 DEG C of boiling water bath 5min, the centrifugal 10min of 12000g, get supernatant 20 μ l and carry out SDS-PAGE, deposition condition is: spacer gel constant voltage 80v, separation gel constant voltage 120v, treat bottom tetrabromophenol sulfonphthalein electrophoresis to gel, stop electrophoresis.With coomassie brilliant blue R250 staining fluid dyeing 6h; Destainer decolouring is clear to band, and observe whether there is target protein band of expression, the results are shown in Figure 5, the recombinant protein molecular weight of expression is about 40.76kDa, induces 3-6 hour expression amount maximum.Fig. 5 is SDS-PAGE result before and after pET30aSETB-LTBIPr2 colibacillus engineering IPTG induces, wherein:

Before 1: genetic engineering bacterium IPTG induction;

2: genetic engineering bacterium IPTG latter 1 hour of induction;

3: genetic engineering bacterium IPTG latter 2 hours of induction;

4: genetic engineering bacterium IPTG latter 3 hours of induction;

5: genetic engineering bacterium IPTG latter 4 hours of induction;

6: genetic engineering bacterium IPTG latter 5 hours of induction;

7: genetic engineering bacterium IPTG latter 6 hours of induction.

Three, the purifying of LTBIPr2 recombinant protein

After bacterium ultrasonication will be induced, test kit specification sheets purifying LTBIPr2 albumen pressed by the Ni Sepharose6Fast Flow resin adopting GE Healthcare Life Sciences company to produce, the 40.76kDa albumen of the visible purifying of SDS-PAGE electrophoresis is a band, recombinant protein is expressed with inclusion bodies, the results are shown in Figure 6.Fig. 6 is the expression-form of pET30aSETB-LTBIPr2 colibacillus engineering and the SDS-PAGE result of purifying, wherein:

Before 1: genetic engineering bacterium IPTG induction;

After 2: genetic engineering bacterium IPTG induction;

5: protein sample after purifying.

Embodiment 3

One, by the engineering bacteria of genetic engineering technique construction expression LTBIPr3 albumen

1, according to the pair of primers of the sequence 1 Design and synthesis amplification LTBIPr3 epitope in sequence table

Upstream primer (5 ' end is containing restriction enzyme Nhe I)

5’－CTAGGCTAGCCACCATGGCCACGCAACGACCGAG－3'

Downstream primer (5 ' end is containing restriction enzyme EcoR I)

5’－CCGGAATTCTTAGACCGCAACGGCAATC－3'

Amplified fragments: 378bp

2, pcr amplification LTBIPr3 gene

With above-mentioned upstream and downstream primer, under the effect of Taq archaeal dna polymerase, with Mycobacterium tuberculosis H37Rv DNA for template, amplification LTBIPr3 gene.After 94 DEG C of 10min warm starts, add archaeal dna polymerase and carry out PCR, response procedures: 94 DEG C of 1min, about 60 DEG C 1min, 72 DEG C of 1min, circulate 30 times; Last 72 DEG C extend 7min.In the amplification of DNA fragments of 1% agarose gel electrophoresis qualification 378bp.Fig. 7 is the agarose electrophoresis figure of the LTBIPr3DNA fragment of pcr amplification, and in figure, the DNA band of left side swimming lane display is exactly the position of PCR primer on 1% agarose electrophoresis glue of the 378bp of LTBIPr3 gene amplification; Right lanes is Beijing CoWin Bioscience Co., Ltd. molecular weight standard DM5000DNA Marker, and band is respectively 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp.

3, goal gene fragment is reclaimed:

4. reclaim product with restriction enzyme Nhe I and EcoR I double digestion LTBIPr3DNA, electrophoresis in 1% sepharose, cut the LTBIPr3 gene fragment of 378bp and the pET30aSetB plasmid DNA fragment of 5.344kb, reclaim kits with agarose gel electrophoresis.Quantitatively, LTBIPr3 and pET30aSetB plasmid DNA fragment, by the mixed in molar ratio of 2 ︰ 1, under the catalysis of T4DNA ligase enzyme, is spent the night in 16 DEG C of connections, get next day and connect product 5 μ l transformation of E. coli BL21(DE3) competent cell, put 37 DEG C of thermostat containers and hatch 14h.Transferred species is in 5ml containing in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins respectively to select 6 clones, and put 37 DEG C of constant temperature oscillator overnight incubation, according to " molecular cloning " alkaline lysis method, a small amount of extracts plasmid, quantitatively, puts-20 DEG C and stores for future use.

5. the qualification of recombinant expression plasmid

(1) pcr amplification qualification

The thallus DNA selecting 6 white colonies is template, and above, downstream primer carries out pcr amplification qualification; 1% agarose gel electrophoresis, if DNA molecular amount standard is reference, positive recombinant plasmid called after LTBIPr3/pET30aSETB.

(2) sequencing

Select a clone through above-mentioned qualification and send order-checking.The sequencing result of LTBIPr3/pET30aSETB is consistent with the genome sequence of design, and its nucleotide sequence is as follows, and in sequence table as shown in sequence 8, wherein, GCTAGC is Nhe I restriction enzyme site, and GAATTC is EcoR I restriction enzyme site.

GGTGAAACGGTCATTCCCTCTAGAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCGGGGTTCTCATCATCATCATCATCATGGTATG ATGTCCACGCAACGACCGAGGCACTCCGGTATTCGGGCTGTTGGCCCCTACGCATGGGCCGGCCGATGTGGTCGGATAGGCAGGTGGGGGGTGCACCAGGAGGCGATGATGAATCTAGCGATATGGCACCCGCGCAAGGTGCAATCCGCCACCATCTATCAGGTGACCGATCGCTCGCACGACGGGCGCACAGCACGGGTGCCTGGTGACGAGATCAC TAGCACCGTGTCCGGTTGGTTGTCGGAGTTGGGCACCCAAAGCCCGTTGGCCGATGAGCTTGCGCGTGCGGTGCGGATCGGCGACTGGCCCGCTGCGTACGCAATCGGTGAGCACCTGTCCGTTGAGATTGCCGTTGCGGTCTAA GAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGCCGATTTCGGCCTATTGGTTAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTACA

Two, the abduction delivering of LTBIPr3 engineering bacteria and qualification

LTBIPr3 colibacillus engineering (LTBIPr3/pET30aSETB) is inoculated in 5ml containing in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins, put 37 DEG C of constant temperature oscillator overnight incubation, then contain in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins to 10ml by 1% transferred species, put 37 DEG C of constant temperature oscillators and be cultured to OD ₆₀₀when being 0.8, add IPTG, induction 3-4hr.Distilled water is added the precipitation sample of 1ml bacterium liquid, piping and druming mixing, again 1 × loading buffer 50 μ l is added the precipitation sample from 1ml bacterium liquid, piping and druming mixing, 100 DEG C of boiling water bath 5min, the centrifugal 10min of 12000g, get supernatant 20 μ l and carry out SDS-PAGE, deposition condition is: spacer gel constant voltage 80v, separation gel constant voltage 120v, treat bottom tetrabromophenol sulfonphthalein electrophoresis to gel, stop electrophoresis.With coomassie brilliant blue R250 staining fluid dyeing 6h; Destainer decolouring is clear to band, and observe whether there is target protein band of expression, the results are shown in Figure 8, the recombinant protein molecular weight of expression is about 13.04kDa, induces 3-5 hour expression amount maximum.Fig. 8 is SDS-PAGE result before and after pET30aSETB-LTBIPr3 colibacillus engineering IPTG induces, wherein:

Before 1: genetic engineering bacterium IPTG induction;

2: genetic engineering bacterium IPTG latter 1 hour of induction;

3: genetic engineering bacterium IPTG latter 2 hours of induction;

4: genetic engineering bacterium IPTG latter 3 hours of induction;

5: genetic engineering bacterium IPTG latter 4 hours of induction;

6: genetic engineering bacterium IPTG latter 5 hours of induction;

7: genetic engineering bacterium IPTG latter 6 hours of induction.

Three, the purifying of LTBIPr3 recombinant protein

After bacterium ultrasonication will be induced, test kit specification sheets purifying LTBIPr3 albumen pressed by the Ni Sepharose6Fast Flow resin adopting GE Healthcare Life Sciences company to produce, the 13.04kDa albumen of the visible purifying of SDS-PAGE electrophoresis is a band, recombinant protein is expressed with inclusion bodies, the results are shown in Figure 9.Fig. 9 is the expression-form of LTBIPr3/pET30aSETB colibacillus engineering and the SDS-PAGE of purifying, wherein:

Before 1: genetic engineering bacterium IPTG induction;

After 2: genetic engineering bacterium IPTG induction;

5: protein sample after purifying.

Describe application of the present invention in detail below in conjunction with specific embodiment, the antigen bulk in each embodiment all obtains by above-mentioned preparation method, but should not form the restriction to the scope of the present invention.

Embodiment 4

By constructed by embodiment of the present invention 1-3, LTBIPr1, LTBIPr2 and LTBIPr3 recombinant protein of purifying is applied to cellular immunology lungy diagnosis.Stimulator antigen using it as Enzyme linked immunospot (ELISPOT), be applied to the inspection of clinical samples, obtain good diagnosis effect, with current commercial IFN-γ release test detection kit combined utilization, active tuberculosis and tuberculosis latent infection can be differentiated, considerably improve the specificity of diagnosis of tuberculosis.ELISPOT trace routine is as follows:

1. experimental specimen: the peripheral blood sample selecting 206 routine anticoagulant heparins altogether, in this research, all experimenter's HIV antibody are feminine gender.

(1) tuberculosis group: active tuberculosis patient 94 example made a definite diagnosis through iconography, laboratory examination and antituberculosis therapy clinically, the mean age is 45.96 years old (12 years old-89 years old), the male sex 53 people, women 41 people.Comprise pulmonary tuberculosis 73 example, outer tuberculosis 21 example of lung, has tuberculous pleuritis, tuberculous pericarditis, tuberculous meningitis, urinary tuberculosis etc.43 examples that antigen LTBIPr1 stimulates are applied, 41 examples that LTBIPr2 stimulates, 34 examples that LTBIPr3 stimulates in 94 routine cases of tuberculosis.

(2) normal healthy controls group: comprise 112 examples through iconography, laboratory examination and the normal healthy controls group made a definite diagnosis clinically and to enlist the new recruit of health check-ups, the mean age be 19 years old (17-21 year).What application LTBIPr1 and LTBIPr2 stimulated is respectively 112 examples, 62 examples that LTBIPr3 stimulates.Through CFP10-ESAT6ELISPOT tests positive (SFC >=13) this kind of crowd as tuberculosis latent infection group; CFP10-ESAT6ELISPOT detects and is negative (<13 of SFC) this kind of crowd as the normal group without tuberculosis latent infection.

2. laboratory apparatus and reagent

Instrument: spot scanner s5Versa Analyzer (Cellular Technology Ltd., Ohio, USA) and analysis software analyzed with (CTL Analyzer LLC).

Tuberculosis latent infection associated protein LTBIPr1, LTBIPr2 and LTBIPr3 prepared by reagent: embodiment 1-3, the Recombinant CFP 10-ESAT6 fusion proteins that this laboratory is provided for oneself, human peripheral lymphocyte parting liquid (Tianjin Hao ocean biological products limited liability company), MABTECH company commercial IFN-γ ELISPOT detection kit, 96 orifice plates (Millipore), washings (PBST, PBS-0.05% tween, pH7.4), complete cell culture fluid gibco(life technologies).

3. experimental technique

(1) bag quilt:

1) on 96 pore membrane plates, every hole adds 100 μ l IFN-γ mono-clonal capture antibodies, 4 DEG C of refrigerator overnight;

2) discard coating buffer, PBS washes plate 2 times, 1min/ time, pats dry;

3) every hole adds the PBS that 200 μ l contain 2%BSA and closes, and hatches 1h for 37 DEG C;

4) discard confining liquid, with RPMI-1640 rinse once, stand-by.

(2) counting lymphocyte is separated:

1) collection of specimens: asepsis injector extracts personnel to be tested's peripheral blood 4-5ml, adds containing heparin sodium anticoagulant blood-collecting pipe, turns upside down 5-6 time, makes anti-freezing liquid and blood mixing; Place room temperature and be less than 6 hours, do not place and give freezing or refrigeration.

2) peripheral blood mononuclear cell is separated and counting:

A. get the 3ml anticoagulation cirumferential blood of mixing, slowly join in the sterile centrifugation tube containing 3ml lymphocytes separating solution, form sharp interface, the at room temperature centrifugal 25min of 2500rpm.

B. lymphocyte cloud and mist layer is drawn to being totally placed with in the centrifuge tube of 1640 with aseptic dropper.

C. the RPMI-1640 8ml being preheated to 37 DEG C is added, after blowing and beating suspendible gently with dropper, in the centrifugal 10min of room temperature 2000rpm;

D. abandoning supernatant, add be preheated to 37 DEG C RPMI-1640 to 6ml, resuspended sedimentation cell, in the centrifugal 8min of room temperature 1500rpm;

E. supernatant discarded washing lotion, adds the serum free medium that 0.25ml is preheated to 37 DEG C, blows and beats suspendible cell gently with dropper.

F. get 10 μ l cell suspensions and add blood counting chamber, count under the microscope, count every ml suspension cell quantity, with serum free medium diluting cells suspension to 2.5 × 10 being preheated to 37 DEG C ⁶cell/L.

(3) immunodotting detects:

Following reagent is added in 96 hole filter membrane plates of above-mentioned bag quilt:

A.50 μ l serum free medium is to each negative control hole;

B.50 μ l phytohaemagglutinin is to each Positive control wells;

C.50 μ l recombinant proteins of Mycobacterium tuberculosis is to each detect aperture, and albumen working concentration is 60 μ g/ml;

1) every hole adds 100 μ l and contains 2.5 × 10 ⁵the suspension of individual PBMC.

2) 96 hole filter membrane plates are put into moistening incubator 37 DEG C, 5%CO2 fixed temperature and humidity incubator cultivates 18-20 hour.

3) taken out by 96 hole filter membrane plates, abandon culture supernatant, the frozen water lysing cell adding 200 μ l precoolings (places 5min for-20 DEG C in 10 minutes, then 5min is placed for 4 DEG C), with the PBS of 200 μ l1 × PBST(containing 0.05% tween 20) wash plate 7 times, 1min/ time, pat dry after washing plate for the last time.

4) add IFN-γ and detect antibody 100 μ l/ hole (diluting with PBS1:1000 is 1 μ g/ml), hatch 1 hour for 37 DEG C, abandon how anti-liquid, with the PBS of 200 μ l1 × PBST(containing 0.05% tween 20) wash plate 5 times, 1min/ time, pat dry after washing plate for the last time.

5) the streptavidin 100 μ l(PBS1:1000 adding alkali phosphatase enzyme mark dilutes) 37 DEG C hatch 30 minutes, discard liquid, with the PBS of 200 μ l1 × PBST(containing 0.05% tween 20) wash plate 5 times, 1min/ time, pat dry after washing plate for the last time.

6) every hole adds 100 μ l BCIP/NBT substrate solutions, and under being positioned over room temperature, black out colour developing grows to suitable size to spot in 7-15min minute.

7) there is bluish voilet spot at the bottom of visible plate, by distillation washing plate 3 stopped reactions, plate is put into 37 DEG C of baking ovens and dry.

8) CTL company enzyme connection spot analysis instrument is used to carry out the painted spot of analysis of accounts.

4. result

Experimental result is in table 1, through LTBIPr1, after LTBIPr2 and LTBIPr3 albumen stimulates, the SFCs that tuberculosis patient peripheral blood is separated secretion of gamma-IFN in mononuclearcell is respectively 7.41 ± 19.00, 2.40 ± 4.46, 0.68 ± 1.53, the SFCs of normal group secretion of gamma-IFN is respectively 35.99 ± 57.40, 3.6 ± 11.74, 3.03 ± 7.99, compared with tuberculosis patient and normal group these two groups, the IFN-γ horizontal cell number that latent infection component is secreted is more, SFCs is respectively 109.92 ± 107.87, 33.49 ± 62.25, 31.19 ± 56.05(P<0.01).Result shows: LTBIPr1, LTBIPr2 and LTBIPr3 tri-restructuring latent infection albumen is higher to the crowd recognition degree being in latent state in m tuberculosis infection person, compared with existing latent infection diagnostic techniques, mycobacterium tuberculosis specific proteins constructed by the present invention, has obvious advantage in the sensitivity diagnosed tuberculosis latent infection and specificity.

The present invention adopts genetic engineering technique to obtain three kinds of proteantigen LTBIPr1, LTBIPr2 and the LTBIPr3s relevant to mycobacterium tuberculosis latent infection from mycobacterium tuberculosis clone, expression, purifying, as the special cell stimulatory agents of tuberculosis for stimulating person under inspection's peripheral blood single core T lymphocyte, it is made to secrete specificity IFN-γ, then by the T lymphocyte spot number of ELISpot technology for detection secretion of gamma-IFN, whole process needs 2 day time.Utilize the present invention, according to the bluish voilet spot number produced with the naked eye or use instrument sentence read result, can be used for detection person under inspection and whether infect tubercule bacillus, special operations diagnosis and differential diagnosis lungy.Therefore, the present invention is applied to preparation ELISpot tuberculosis infection diagnostic reagent kit, and in the diagnosis of latent tuberculosis infection, the auxiliary diagnosis aspect of active tuberculosis is with a wide range of applications.

。

Claims

1. a mycobacterium tuberculosis latent infection associated protein, is characterized in that: described mycobacterium tuberculosis latent infection associated protein is that the nucleotide sequence of LTBIPr1, LTBIPr1 Protein Epitopes is as shown in sequence in sequence table 1.

2. a mycobacterium tuberculosis latent infection associated protein, is characterized in that: described mycobacterium tuberculosis latent infection associated protein is that the nucleotide sequence of LTBIPr2, LTBIPr2 Protein Epitopes is as shown in sequence in sequence table 2.

3. a mycobacterium tuberculosis latent infection associated protein, is characterized in that: described mycobacterium tuberculosis latent infection associated protein is that the nucleotide sequence of LTBIPr3, LTBIPr3 Protein Epitopes is as shown in sequence in sequence table 3.

4. a construction process for mycobacterium tuberculosis latent infection associated protein, comprises the steps:

(1) Nhe I restriction enzyme site is added at LTBIPr1 upstream primer, EcoRI restriction enzyme site is added at LTBIPr1 downstream primer, by pcr amplification product with after Nhe I and EcoR I double digestion, insert in the pET30aSETB plasmid vector after with Nhe I and EcoR I double digestion; Be transformed in bacillus coli DH 5 alpha recipient bacterium; Through plasmid extraction, through the two-way sequence verification of T7 and T7t, obtain nucleotide sequence and the on all four recombinant plasmid LTBIPr1/pGEM-T of design;

Recombinant plasmid LTBIPr1/pGEM-T is transformed in e. coli bl21 (DE3) recipient bacterium; Through plasmid extraction, through T7 and/or T7t sequence verification, obtain nucleotide sequence and the on all four recombinant expression plasmid LTBIPr1/pET30aSETB of design;

(2) LTBIPr1 bacillus coli gene engineering strain carried out induce, express, purifying, SDS-PAGE electrophoresis, qualification, ELISPOT and elisa assay, obtain the recombinant protein of the purifying conformed to the size designed.

5. the construction process of mycobacterium tuberculosis latent infection associated protein according to claim 4, is characterized in that: described LTBIPr1 upstream primer is:

5’－CTAGGCTAGCCACCATGGCGGAGCCAGCGGCGTG－3'

Described LTBIPr1 downstream primer is:

5’－CCGGAATTC TCATGGCGAGGCTTCCGGG－3'。

6. a construction process for mycobacterium tuberculosis latent infection associated protein, comprises the steps:

(1) Nhe I restriction enzyme site is added at LTBIPr2 upstream primer, EcoRI restriction enzyme site is added at LTBIPr2 downstream primer, by pcr amplification product with after Nhe I and EcoR I double digestion, insert in the pET30aSETB plasmid vector after with Nhe I and EcoR I double digestion; Be transformed in bacillus coli DH 5 alpha recipient bacterium; Through plasmid extraction, through the two-way sequence verification of T7 and T7t, obtain nucleotide sequence and the on all four recombinant plasmid LTBIPr2/pGEM-T of design;

Recombinant plasmid LTBIPr2/pGEM-T is transformed in e. coli bl21 (DE3) recipient bacterium; Through plasmid extraction, through T7 and/or T7t sequence verification, obtain nucleotide sequence and the on all four recombinant expression plasmid LTBIPr2/pET30aSETB of design;

(2) LTBIPr2 bacillus coli gene engineering strain carried out induce, express, purifying, SDS-PAGE electrophoresis, qualification, ELISPOT and elisa assay, obtain the recombinant protein of the purifying conformed to the size designed.

7. the construction process of mycobacterium tuberculosis latent infection associated protein according to claim 6, is characterized in that: described LTBIPr2 upstream primer is:

5’－CTAGGCTAGCCACCATGGCGCAAACCGGCAAGC－3'

Described LTBIPr2 downstream primer is:

5’－CCGGAATTCTCACATCTCCTGGTTCTCG－3'。

8. a construction process for mycobacterium tuberculosis latent infection associated protein, comprises the steps:

(1) Nhe I restriction enzyme site is added at LTBIPr3 upstream primer, EcoRI restriction enzyme site is added at LTBIPr3 downstream primer, by pcr amplification product with after Nhe I and EcoR I double digestion, insert in the pET30aSETB plasmid vector after with Nhe I and EcoR I double digestion; Be transformed in e. coli bl21 (DE3) recipient bacterium; Through plasmid extraction, through T7 and/or T7t sequence verification, obtain nucleotide sequence and the on all four recombinant expression plasmid LTBIPr3/pET30aSETB of design;

(2) LTBIPr3 bacillus coli gene engineering strain carried out induce, express, purifying, SDS-PAGE electrophoresis, qualification, ELISPOT and elisa assay, obtain the recombinant protein of the purifying conformed to the size designed.

9. the construction process of mycobacterium tuberculosis latent infection associated protein according to claim 8, is characterized in that: described LTBIPr3 upstream primer is:

5’－CTAGGCTAGCCACCATGGCCACGCAACGACCGAG－3'

Described LTBIPr3 downstream primer is:

5’－CCGGAATTCTTAGACCGCAACGGCAATC－3'。

10. the application of the mycobacterium tuberculosis latent infection associated protein according to any one of claim 1-3 in preparation ELISpot tuberculosis infection diagnostic reagent kit.