Patent Application
20070224608
The present invention relates to a recombinant 12 kDa protein useful for the detection of respiratory allergies. The invention particularly relates to detection of the respiratory allergies caused by fungal spores and grass pollen using the said protein.
The term “allergy” coined by Von Pirquet (1906) is defined as altered immunologic reactivity to foreign particles. The foreign agents causing altered immunologic reactivity are called allergens, which includes a broad spectrum of substances e.g. proteins, glycoprotein, lipoproteins etc derived from diverse sources such as pollens, fungal spores, insects, dust mites, animal danders, foods, etc. Pollen grains and fungal spores are the main constituents of the aerospora. They are significant cause of allergic diseases afflicting more than 25% of the atopic subjects. These foreign substances can trigger the release of mediators from immune system leading to inflammatory and other allergic reactions.
Allergy is detected clinically by skin testing and ELISA. The fungal extracts generally used for skin testing are complex mixture of proteins, carbohydrates, pigments, toxins, etc. They contain both relevant and non-relevant components that might sensitize the patient and eventually evoke anaphylaxis. Another factor that adds complexities to diagnosis of fungal allergy, is cross-reactivity among allergens from different sources. Cross reactivity is due to the presence of similar protein components and/or epitopes shared by different fungal species. Studies on cross reactivity have shown antigenic/allergenic relationship among species of fungi such as Curvularia, Cladosporium, Fusarium, Pencillium and Aspergillus [1]. Curvularia lunata has been shown to be an important allergy causing fungi also responsible for life threatening Allergic bronchopulmonary aspergillosis (ABPA) like symptoms in many patients [2].
Due to complexities involved in standardizing the large number of extracts, use of recombinant allergens have now begun for diagnostic purposes, e.g. use of rAsp f 1, rAsp f 3 and rAsp f 6 has made it possible to diagnose differentially between ABPA and A. fumigatus sensitized asthmatics, (Hemmann et al). For three reasons, diagnosis with recombinant allergen is advantageous over heterogeneous crude allergen extract [3,4]. First, it provides pure, standardized and consistent allergen preparations. Secondly, skin tests with these preparations are free of false positive or false negative i.e. non-specific reactions. Thirdly, it allows substantial evidence of patient's specific reactivity against a particular allergen and thus helps in better understanding of the disease causing components. Isolation and purification of recombinant cross-reactive allergens would lead to a better and easier way of diagnosis. Since, using cross-reactive allergens would reduce the number of extracts used for skin testing [5]. The recombinant form of these cross-reactive allergens would improve sensitivity of such diagnosis [6]. An important criterion for such applications is that there should be equivalent immuno-biochemical properties of the recombinant allergen and its native counterpart. Many recent reports have suggested the same e.g. Grendelmeier P S et al have reported [7] that Art v 1 restores its properties after making it recombinant. Similarly, a recent report [8] from Jeong KY group have shown the similar properties of recombinant and native Bla g 7, an allergen from German cockroach.
The main object of the invention is thus to provide a recombinant 12 K-Da protein useful for the detection of respiratory allergies.
Another object of the present invention is to provide a method for detection of respiratory allergies using the said recombinant 12 kDa protein.
Still another object of the invention is to provide novel primers for sequencing and expression of the disclosed 12 kDa protein by recombinant methods.
Yet another object of the invention is the expression and purification of recombinant 12 kDa protein.
The invention discloses the detection of respiratory allergies using a recombinant 12-kDa protein. The present invention is based on the fact that there is a need of a single cross-reactive protein capable of replacing large number of extracts used for detection of raised IgE levels in allergy by ELISA, immunoblotting and the likes. It is further based on the realization that such a cross-reactive protein will reduce the number of pricks, a patient gets during allergy skin testing, thus providing a single representative of large number of allergen extracts used. It is further realized that production of such a protein by recombinant methods can lead to its availability in pure form and bulk amounts required for routine diagnosis. In extension to the fact mentioned above, the resemblance of such a recombinant protein to its native form is an additional benefit forming the basis of its use clinically.
Accordingly, the present invention provides a recombinant 12 kDa fungal protein useful for detection of respiratory allergies, the said protein exhibiting the following characteristics:
The disclosed recombinant 12-kDa protein is highly cross-reactive in grasses and fungi as tested by ELISA inhibition. EC50 required for 50% loss of IgE binding activity is in the range of 1-1.5 ng.
In an embodiment of invention, the cDNA library of fungus was constructed in commercially available λZAP vector and the like.
In still another embodiment, the fungus for cDNA library was selected from Curvularia lunata [MTCC 2030], Alternaria alternate [MTCC 1362], Epicoccum nigrum [MTCC 2129] and Fusarium solani [MTCC 1756].
In yet another embodiment of the invention, the screening of cDNA library for locating the protein of interest was carried out with pooled sera of patients allergic to Curvularia lunata and the like.
In still another embodiment of the invention, the mRNA sequence SEQ ID 1 (NCBI ACCESSION NO. AY034827) and its coding sequence (CDS) SEQ ID 2 (NCBI ACCESSION NO. AY034827) were obtained using known primers.
In still another embodiment of the invention, the protein sequence obtained by translating the coding sequence SEQ ID 3 (NCBI ACCESSION NO. AAK67492) was computationally compared with known sequences available in databank using ClustalW and BLAST and the like.
In yet another embodiment of the invention, novel primers of SEQ ID NOS. 4 and 5 were designed for sub-cloning the SEQ ID NO. 2.
In still another embodiment of the invention, the protein of SEQ ID 3, was expressed in E.coli prokaryotic expression vector and the like.
In still another embodiment of the invention, the purification of the recombinant protein was carried out using two steps comprising metal affinity chromatography and Gel exclusion chromatography and the like.
In yet another embodiment of the invention, the said protein resolved as 12 kDa protein on SDS-PAGE, was recognized by commercial and raised antibodies.
In still another embodiment of the invention, the allergenic properties of the recombinant protein were assessed by ELISA, immunoblot, ELISA inhibition and the like.
In still another embodiment of the invention, the native form of the disclosed allergen was purified using two-step method comprising cation exchange chromatography using CM cellulose and the like and gel exclusion chromatography using Sephadex G50 and the like.
In still another embodiment of the invention, the disclosed recombinant allergen was compared to its native counterpart by physiochemical viz. CD and absorption spectra and like and immunological methods viz. immunoblot, ELISA, ELISA inhibition and the like.
In still another embodiment of the invention, the cross-reactivity of the disclosed recombinant allergen was compared with fungi viz. A. alternata, E. purpurascens, F. solani, C. albicans and the like by ELISA, Immunoblot, ELISA inhibition and the like.
In still another embodiment of the invention, the cross-reactivity of the disclosed recombinant allergen was checked with grass pollen viz. Lolium perenne, Poa pretense, Phleum pretense, Imperata cylindrica Pennisetum sp., Rye grass, Zea Mays and Cenchrus and the like by immunoblot, ELISA and ELISA inhibition using pooled and individual allergic sera as well as commercial and raised antibodies against disclosed protein.
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To establish the presence of r-12 kDa protein in different grasses, ELISA and immunoblot of various Indian and Western grasses was done. Grass extracts used were Lolium perenne, Poa pretense, Phleum pretense, Imperata cylindrica, Pennisetum sp., Rye grass, Zea Mays and Cenchrus. Protein extracts were coated on microtiter plate and ELISA was done as earlier, using antibodies raised in mouse. Anti-mouse IgG peroxidase labeled as secondary antibody. These extracts were separated on SDS-PAGE and transferred on nitrocellulose membrane and immunoblot performed as earlier using antibody raised in mouse against r 12 kDa protein. It showed the presence of r-12 kDa protein in grass extracts viz. Lolium perenne (Lol p), Poa pretense (Poa p), Phleum pretense (Phl p), Imperata cylindrica (Imp c), Pennisetum sp., Zea Mays and Cenchrus. Further, allergenicity of recombinant 12 kDa protein is demonstrated in
‘+’ = severity of the skin test reaction
The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention.
One hundred mg of 4 day old CL spore mycelium mass was crushed under liquid nitrogen to obtain a fine paste. Added 1 ml of TRI zol reagent and crushed again. The paste was allowed to thaw at RT and 0.2 ml of chloroform was added to it. After gentle shaking, it was incubated for 3 m at RT and centrifuged at 12000 rpm for 15 m at 4° C. The upper aqueous layer was separated and 0.5 ml isopropanol was added and kept at −20° C. for overnight. It was centrifuged at 12000 rpm at 4° C. The pellet washed with 75% ethanol followed by centrifugation at 7500 rpm at 4° C. The pellet obtained was air dried and dissolved in 0.5% SDS. The quality of total RNA was checked on formaldehyde gel.
From purified total RNA, double oligo (dT) selection was performed to obtain poly (A) mRNA for cDNA library construction. The concentration of total RNA was adjusted to 0.55 μg/μl with DEPC treated DW and the volume was made upto 640 μl. The oligo dT washed with 1.5 ml washing buffer 1 (supplied with the kit). The salt concentration of the RNA sample was adjusted to 0.5 M by adding 64 μl of 5 M NaCl and was allowed to hybridize at RT for 10 m.
The unbound RNA was expelled and the column washed with 1.5 ml of washing buffer 1 followed by washing with buffer 2 (supplied with the kit). The poly(A) mRNA was eluted with 0.5 ml preheated (65° C.) DEPC treated DW. To the eluted 500 μl mRNA, 2 μl of 50 μg/ml glycogen, 50 μl of 7.5 M ammonium acetate and 1000 μl of chilled ethanol were added. Precipitation of RNA was carried out at −20° C. for overnight. The sample was centrifuged at 3000 rpm for 30 m at 4° C. The pellet obtained washed with 75% ethanol and centrifuged at 3000 rpm for 10 m at 4° C. The pellet was dissolved in 15 μl of DEPC treated DW.
The cDNA library was synthesized using Stratagene ZAP-cDNA Gigapack III Gold cloning kit. It uses a hybrid oligo dT linker primer that contains a Xho I restriction site. Messenger RNA is primed in the first strand synthesis with the linker primer. All the reagents used were provided by commercial cDNA synthesis kit. The various steps involved in the construction of the library are described below:
First strand cDNA: Messenger RNA was used as template to synthesize first strand cDNA. The reaction mixture contained 5 μg mRNA, 5 μl of 10× first strand buffer, 3 μl of 10 mM first strand methyl nucleotide mixture, 2 μl of linker primer (1.4 μg/μl) and 1 μl of RNase block (Ribonuclease inhibitor 400 U/μl) in 50 μl volume. The reaction mixture was incubated for 10 m at RT and 1.5 μl of reverse transcriptase (Moloney murine Leukemia virus reverse transcriptase, 50 U/μl) was added. The reaction was carried out at 37° C. for 1 h.
Second strand synthesis: To the first strand mix, 20 μl of 10× second strand buffer, 6 μl of second strand dNTP mixture (10 mM), 114 μl autoclaved DW, 2 μl of RNase (1.5 U/μl) and 11 μl of DNA polymerase I (9.0 U/μl) were added in a total volume of 200 μl. The reaction was carried out at 16° C. for 2.5 h and was kept on ice.
To the second strand mix, 23 μl of dNTP mix (2.5 mM) and 2 μl of cloned pfu DNA polymerase (2.5 U/μl) were added. The reaction was carried out at 72° C. for 30 m. After the completion of the incubation, 200 μl of pre saturated phenol pH-8.0: chloroform: isopropanol was added. It was mixed at RT and the upper aqueous layer was transferred into a fresh tube. To the tube, equal volume of chloroform was added and mixed. The sample was then centrifuged and the upper aqueous layer was transferred to fresh microcentrifuge tube. The DNA was precipitated by adding 20 μl of 3 M sodium acetate and 400 μl of 100% ethanol at −20° C. for overnight.
The DNA pellet obtained washed with 70% ethanol and dried. To this, 1 μl of 10× ligase buffer, 1 μl of 10 mM rATP and 1 μl of T4 DNA ligase (40 U/μl) were added. The reaction was carried out at 8° C. for overnight.
After inactivating the ligase at 70° C. for 30 m, 1 μl of 10× ligase buffer, 2 μl of 10 mM rATP, 6 μl of autoclaved DW and 1 μl of T4 polynucleotide kinase (10 U/μl) were added. The reaction was carried out at 37° C. for 30 m.
The kinase was inactivated at 70° C. for 30 m and 28 μl of Xho I buffer supplement and 3 μl of Xho I (40 U/μl) were added. The tube was incubated at 37° C. for 1.5 h. After the completion of the reaction, 5 μl of 10×STE buffer and 12 μl of 100% ethanol were added. The DNA was precipitated at −20° C. for overnight.
The DNA pellet washed, dried and resuspended in 14 μl of 1×STE buffer followed by addition of 3.5 μl of the column loading dye was added. The drip column was packed using Sepharose CL-2B gel filtration medium and washed twice with STE buffer. After this, the cDNA sample was gently loaded without disturbing the resin. The column washed with STE buffer and cDNA sample eluates were collected. From each fraction, 5 μl of sample was aliquoted and electrophoresed on an alkaline agarose gel.
The reaction mixture contained 100 ng of cDNA, 0.5 μl of 10× ligase buffer, 0.5 μl of 10 mM rATP (pH 7.5), 1 μl of UNI-ZAP XR vector (predigested, 1 μg/μl) and 0.5 μl of T4 DNA ligase (4 U/μl). The autoclaved DW was added in a total volume of 5 μl. The reaction was carried out at 12° C. for overnight.
To the packaging extract, 2 μl of ligated DNA was added. After mixing it gently, the reaction mixture was incubated at 22° C. for 2 h. After the completion of incubation, 500 μl of SM buffer and 20 μl of chloroform were added. The contents were gently mixed and centrifuged at 7000 rpm at RT for 2 m. The supernatant was titrated for a suitable library dilution to be used for immunochemical screening.
Single colony of XL-1 Blue MRF′ cells was inoculated in LB containing 10 mM MgSO4 (described in Appendix A) and 0.2% (w/v) maltose. Cells were grown at 37° C. at 220 rpm for overnight. The cells were pelleted at 4° C. at 4000 rpm for 10 m. Different library dilutions (1:10 and 1:100 v/v) were made in SM buffer. From each dilution 1 μl was taken and incubated with 200 μl of XL-1 Blue cells diluted in 10 mM MgSO4 to 0.5 O.D600. The mix was incubated at 37° C. for 15 m in a tube. After incubation, 2-3 ml of NZY top agar (melted and cooled to approx. 48° C., Appendix A) mixed with 5 μl of 0.5 M IPTG and X-gal were plated onto the NZY agar plates. The plates were then incubated at 37° C. for 6-8 h.
XL1 Blue cells were prepared as described earlier. Primary cDNA library (250 μl containing 5×104 phage particles) was incubated with 600 μl XL-1 blue cells (O.D600 0.5) at 37° C. for 15 m. After incubation, mixture of 6.5 ml NZY top agar and infected material was plated onto 150 mm NZY agar plates. The plates were incubated at 37° C. for 6-8 h. The plates were then overlaid with 10 ml SM buffer and stirred gently at 4° C. for overnight. The suspension was pooled in a sterile polypropylene tube. The plates were rinsed with an additional 2 ml of SM buffer and pooled. Chloroform 5% v/v was added, mixed well and incubated for 15 m at RT. The sample was centrifuged at 1000 rpm for 10 m at 4° C. and supernatant was transferred in a fresh tube. The sample was again centrifuged and supernatant was transferred in a fresh polypropylene tube. To this, chloroform was added to a final concentration of 0.3% v/v and stored at 4° C. The titer of the amplified library was checked as described earlier.
The cDNA library of C. lunata in UNI-ZAP lambda vector was screened with pre-absorbed pooled CL sensitive patient's sera. The cDNA library was plated after appropriate dilution in SM buffer for obtaining 200-300 plaques per 90 mm NZY agar plates. The cDNA library (1 μl of 1:105 diluted in SM) was mixed with the host E. coli XL1-Blue MRF′ cells diluted in 10 mM MgSO4 to OD600=0.5 in a sterile polystyrene falcon tube. The cells were incubated at 37° C. for 15 m to allow the phage to attach to the cells. To this, 3 ml of NZY top agar (melted and cooled to 48° C.) was added and plated immediately onto NZY agar plates. The inverted plates were incubated at 42° C. (4-6 h) until the plaques just begin to form. Soaked the numbered nitrocellulose filter with 10 mM IPTG. The dried nitrocellulose filters were placed on the agar surface in contact with the plaques, taking care to avoid air bubbles under the filter. Using a syringe needle, pierced the filter and agar at asymmetric positions to facilitate paper alignment following staining. The layered plates were incubated at 37° C. for 4 h to induce expression. The filters were removed, washed twice with TBS (2 m each) and incubated in blocking buffer for 1 h at RT. After washing twice with TBS at RT (5 m each), it was incubated with serum 1:10 v/v at 37° C. for overnight. The filters were then washed and incubated in conjugate solution 1:1000 v/v in TBS at 37° C. for 3 h. The filters were washed with TBST thrice (10 m each) and color was developed. The reaction was stopped by rinsing the membranes with distilled water twice.
The plaque showing IgE binding was cored out from the agar plate and transferred to a sterile microcentrifuge tube containing 500 μl of SM buffer and 20 μl of chloroform. Vortexed the microcentrifuge tube to release the phage particles into the SM buffer followed by incubation at 4° C. overnight (phage stock). Separate cultures of XL1 and SOLR in LB broth supplemented with 0.2% (w/v) maltose and 10 mM MgSO4 were obtained as described earlier. On the following day, XL1 Blue and SOLR cells were spun down at 6000 rpm for 5 m at 4° C. and resuspended in 10 mM MgSO4 at an OD600 of 1.0. The following components were mixed in a 15 ml sterile polypropylene tube-200 μl of XL1-Blue MRF′ cells at an OD600 of 1.0; 5 μl of phage stock and 1 μl of the ExAssist helper phage (>1×106 pfu/μl). Incubated the tube at 37° C. for 15 m, added 3 ml of LB broth and incubated for 3 h at 37° C. with shaking at 220 rpm. Heated the falcon at 65° C. for 20 m and centrifuged at 10,000 rpm at 4° C. for 10 m. Decanted the phage supernatant into sterile microcentrifuge tube. To plate excised phagemids, 200 μl of SOLR cells were mixed with 2 μl of phage supernatant followed by incubation at 37° C. for 15 m. Plated 200 μl of the cell mixture on LB-ampicillin agar plates (50 μg/ml) and incubated overnight at 37° C.
In general, a 50 μl of PCR reaction mixture contained,
Amplification Conditions:
Initial denaturation, 94° C./5 m and added the enzyme
Denaturation, 94° C./1 m
Annealing, 55° C./2 m
Extension, 72° C./2 m
Final extension, 72° C./7′ for 25 cycles.
The size of the amplified insert was determined by agarose gel electrophoresis.
Automated DNA Sequencing was performed using fluorescent dye-terminator chemistry with thermal cycle sequencing. The sequencing reaction was set up as described below: Setting up of the sequencing reaction:
Sequencing of DNA samples was performed on ABI-377, DNA Sequencer
The cDNA insert subcloned into pBluescript SK (+/−) phagemid commercial kit was expressed under lacZ promoter. The phagemid was inoculated into 250 ml LB broth with 100 μg/ml of ampicillin and incubated at 37° C. with shaking (200 rpm) until the absorbance (OD600) reached 0.2. Added IPTG at a final concentration of 1.0 mmol/L and the cultures were further grown for 5 hours at 37° C. with shaking (200 rpm). The cells were spun down at 6000 rpm for 15 min at 4° C. and suspended in 3 ml of 50 mM Tris-HCl, pH 7.5. The cells were sonicated and centrifuged at 6000 rpm for 45 min at 4° C. The supernatant was separated and was analyzed on 10% SDS-PAGE gel under reducing and denaturing conditions. After transferring the proteins onto NCM, the IgE/IgG binding activity of the fusion protein was evaluated. The patient's serum 1:10 v/v and anti CL rabbit serum 1:2000 v/v were used.
Expression of Recombinant Form of 12 kDa Protein in E.coli:
The standard PCR is typically done in 50-100 μl reaction volume and in addition to sample DNA may also contain 50 mMKCl, 10 mMTris Cl, (pH8.4), 1.5 mMMgCl2, 250 nmoles, primers, 200 μmdNTPmix, 2.5 units of Taq DNA Polymerase.
The reaction mix used generally contained:
For complete digestion of DNA the 50 μl reaction mix contained DNA appropriately diluted and 5 μl of assay buffer. The volume was made up by good quality autoclaved water. Finally the enzyme was added. Incubation was done at 37° C., for 3 hrs.
After first enzyme treatment, heat inactivation was done to stop its non-specific activity. The reaction mix was heated at 65° C. for 15 min. Precipitation was done by adding 0.6 volumes of ammonium acetate and 2.5 volumes of 100% ethanol. Incubate at −20° C., overnight, centrifuged and washed the pellet with 75% ethanol. The pellet was air-dried and then reaction was put up with second enzyme in similar way.
The most important thing considered during cloning is the reannealing of the cut ends, which leads to plasmid recircularization. This was prevented by phosphatase treatment that removes 5′phosphate group to suppress self-ligation. Ligase catalyzes formation of phosphodiester bonds between two nucleotides one with 3′ hydroxyl and other with 5′phosphate. This way only the foreign DNA insert can be ligated with the vector and self-ligation is minimized. The ligation mix (11 μl) contained:
5 μl of 2.2× reaction buffer and various ratios of vector and insert.0.5 μl of T4 DNA ligase was finally added. The reaction mix was incubated at 16° C., overnight.
Inoculated single colony of E.coli strain to be made competent, e.g. DH5α cells into a 5 ml LB tube and grown overnight at 37° C. The next day secondary culture was done (diluted 1 ml in 100 ml of culture) Grown at 37° C. 250 rpm, 2 hrs approximately for the cells to reach OD of 0.3. OD more than 0.4 leads to decrease in competence i.e. decreases the efficiency of transformation. The culture was aliquoted into prechilled polypropylene sterile tubes and left on ice. The cells need to be kept on ice subsequently. Cells were pelleted down at 3000 rpm, 4° C. for 7 min. (higher speed affects the viability of cells). The cell pellet was suspended gently in 5-6 ml of ice-cold CaCl2 solution (see in reagents). Cells were pelleted down at 2500 rpm, 4° C., and 5 min.
Cells were resuspended in ice cold CaCl2 and incubated on ice for 30-40 min. Cells were pelleted down at same speed. Cell pellet was resuspended in ice cold CaCl2. This re-suspension is final and needs to be done very well. The suspension should be kept on ice for about 1 hr. Finally the cells are aliquoted as 100 μl and stored at −70° C.
Any aliquot taken out should not be refrozen. Competence of cells is assessed by transformation with a known plasmid vector and seeing the number of colonies that appear.
No. Of transformant colonies per aliquot (μl)×105==No. of transformants per pg of DNA used for transformation.
For 100 μl of competent cells 10-20 ng of DNA usually suffices (in the volume of 10-20 μl). Competent cells with DNA were swirled gently and kept on ice for 15-20 min. This mix was then incubated at 42° C. for 2 min and immediately put on ice and kept for 5 min. This treatment is called “heat shock treatment” which actually causes DNA to enter inside the cells. The cells were revived with 300 μl of LB media and kept at 37° C., 260 rpm, 1-2 hrs. The cells were plated on LB amp plates and plates kept for overnight incubation at 37° C. Remaining part of transformation mixture can be stored at 4° C.
To confirm the expressed clone, plasmid isolation was carried out by alkaline lysis method. Inoculated single bacterial colony in 5 ml LB medium overnight containing appropriate antibiotic, e.g. here ampicillin (50 μg/ml). The cells were pelleted down at 600 rpm, 15 min 4° C. The cells were thoroughly mixed with 150 μl of TEG buffer by vortexing. Then cells were kept on ice for five-min. Added 300 μl of alkaline SDS was added. The solution becomes clear and slimy. Added ice-cold 200 μl potassium acetate and incubated on ice for half an hour. This step precipitates all genomic DNA and cell debris. Then centrifuged at 12000 rpm for 30 min, 4° C. Then the clear supernatant was taken out and 0.6 volumes of isopropanol was added and kept on ice for 10-15 min. Then centrifuged for 25 min at 12000 rpm, 4° C. The glassy pellet is difficult to see, thus care needs to be taken while rejecting the supernatant. The pellet was given a wash with 70% ethanol and then with 100% ethanol. The pellet was air dried and dissolved in water and analyzed on 1% agarose gel. The plasmids isolated were then checked with PCR and restriction digestion to confirm the insert of desired size.
The positive clone encoding 12 kDa protein was transformed into BL21 E.coli cells. The single clone was inoculated in 5 ml LB broth containing 100 μg/ml of ampicillin and incubated overnight at 37° C. with shaking (200 rpm). This culture was sub-cultured into 250 ml LB broth with 100 μg/ml of ampicillin and incubated at 37° C. with shaking (200 rpm) until the absorbance (OD600) reached 0.2. Added IPTG at a final concentration of 1.0 mmol/L and the cultures were further grown for 5 hours at 37° C. with shaking (200 rpm). The cells were spun down at 6000 rpm for 15 min at 4° C. and suspended in 3 ml of 50 mM Tris-HCl, pH 7.5. The cells were sonicated and centrifuged at 6000 rpm for 45 min at 4° C. The sonicated lysate was loaded onto equilibrated Ni-NTA slurry and incubated for an hour for binding in equilibration buffer containing 10 mMTrsi.Cl, 100 mMsodium phosphate buffer and 500 mM NaCl pH 8.5. The non-specific bound proteins were washed off using wash buffer containing 10 mMTrsi.CI, 100 mMsodium phosphate buffer and 500 mM NaCl pH 6.2. The bound protein was eluted using wash buffer containing gradient of imidazole and finally eluted at 200 mM imidazole. The protein content was estimated by known method [9] and separated 12% SDS-PAGE gel under reducing and denaturing conditions.
SDSPAGE was performed by known methods [10]
The Samples were Prepared as Follows:
30 μl eluted sample+1× sample dye (loading buffer)
Boil the samples for 10 min at 100° C. in the dry bath.
Load the samples on to the gel along with the molecular weight marker. Run the electrophoresis in 1 liter electrode buffer 1× containing 14.4 gms Glycine, 3.03 gms Tris Cl., and 1% SDS at 120V, 80 mA. The gel is stained with CBB or silver stain to visualize the protein of very little yield.
The absorption spectrum of 12 kDa protein was done to find the characteristic absorption maxima peaks of heme containing proteins and shows the peaks at 410 nm, 510 nm and 550 nm. 1 mg/ml protein was taken in a clean quartz cuvette and absorption scan carried out in the range of 210 nm-700 nm on Shimadzu UV 2100 S. the absorption maxima was recorded and the plot was scaled appropriately to fit all the peaks.
CD spectra were carried out with 1 mg/ml of each recombinant and native protein in 20 mM phosphate buffer in the far-UV range. Thermal scans in the range of 10-100° C. were also carried out to find the melting temperature (Tm) of recombinant 12 kDa protein.
The protein is transferred after SDSPAGE onto nitrocellulose membrane by electrotransfer by known methods [11]. Briefly, when the run is over, the gel is transferred on to the nitrocellulose membrane sheet, in the cassette, such that the gel is on negative side and the transfer takes place from negative to positive side. The electro transfer is carried out for about 3 hrs in the transfer buffer containing 6.9 gms Glycine, 6.6 gms Tris and 250 ml methanol the volume was made upto 1 liter with distilled water) at 80 mA. After transfer is over Ponceau staining of NCP is done to see if the transfer is appropriate. Destaining of the NCP is done using PBST solution and wash with PBS. Keep the blot (NCP) in 3% BSA solution/defatted milk (made in water or PBS) used as blocking solution at 4° C., overnight or at 37° C. at 40 rpm for 1 hour. The excess blocking reagent is washed off with PBST. The blot is then incubated with primary antibody (peroxidase conjugated monoclonal antibody could be used too). The incubation could be at 4° C. for overnight or at 37° C. for 3 hours. The excess antibody is washed off with PBST. The secondary antibody is added which has to be peroxidase conjugated and kept for 1-2 hrs at 37° C. The blot is washed by PBST and kept for developing by the addition of substrate, i.e. 15 mg diaminobenzene and 15 μlH2O2 are added freshly to acetate buffer (0.34 g in 50 ml water+39 μl acetic acid) The blot shows brown bands upon developing if the antigen looked for is present. The allergen can also be checked similarly if the primary antibody used is serum of patient allergic to that source.
Each well of the microtitre plate was coated with 1 μg of protein in 100 μl of coating buffer pH 9.6. The plate was incubated overnight at 4° C. After washing with 0.1 M PBS containing 0.1% Tween 20 (PBST), the free sites were blocked with 200 μl 3% bovine serum albumin or non fat dry milk for 1 h at RT. The plates were washed again and incubated with either 100 μl serum at 4° C. overnight. IgE binding was determined by allergic patients sera (1:10 v/v) and IgG binding by polyclonal mice sera/commercial antibodies (1:2000 v/v). The plate was washed again and incubated for 4 h at RT with 100 l antihuman-IgE peroxidase (1:1000 v/v) or anti mice IgG peroxidase (1:2000 v/v). The plate washed and color was developed using substrate containing 8 mg o-phenylene diaminebenzidine and 18 μl H2O2 in citrate buffer at 37° C. The reaction was stopped with 50 μl 5 N H2SO4 after 40 m and read at 490 nm in ELISA reader (Dynatech).
Table 1 shows the specific IgE values against recombinant 12 kDa protein in different fungal positive sera. This data demonstrates the presence of detectable specific antibodies against recombinant 12 kDa protein in different fungal sensitized patient's sera.
Table 2 shows the specific IgE antibodies against the recombinant 12 kDa protein in different grass positive sera. These patients were negative to different fungi but positive to recombinant 12 kDa protein. This shows that this 12 kDa protein contributes significantly to the grass pollen allergy also. This protein can thus be useful for detection of grass and fungal allergies without using large number of grass or fungal extracts.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1537/DEL/2005 | Oct 2005 | IN | national |
