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Cloning and expression of 16kDa protein of Johne's disease as diagnostic antigen.

Paratuberculosis (Johne'sdisease), chronic granulomatous enteritis of domestic ruminants (1) and wild animals (2,3) caused by acid-fast, slow growing, fastidious Mycobacterium avi Freum subspecies paratuberculosis (MAP). It is an enzootic disease on the B list of the Office des International Epizootes ('OIE' and causes huge economic losses to the dairy industry worldwide.

Detection of MAP, especially during the often-lengthy subclinical phase of the disease, remains difficult due to intermittent shedding of small numbers of bacteria and a lack of effective diagnostic reagents (4). The fecal culture test requires 12-16 weeks for cultivation and its sensitivity level is estimated to be approximately 38% (5). So there is a need to improve the sensitivity or specificity of currently used diagnostic tests. Development of sensitive serological tests for the rapid identification of infected animals at subclinical stage requires expression and characterization of early secreted proteins of MAP as important antigens for the diagnosis of paratuberculosis (6,7). Literature mining indicates the present work as the first of its kind involving epitopic region from MAP 0862 from the MAP genome strain k10 encoding immunoreactive 16kDa protein.

MATERIALS AND METHODS

Materials and reagents

All the chemicals and biological used in the present study were of molecular biology grade. Agarose, Ammonium persulphate, Acryl amide, Bovine serum albumin, Bisacryl amide, b-mercapto-Life technology USA ethanol, Bromophenol blue, Calcium Chloride, Coomassie brilliant blue 250, Magnesium Chloride, Sodium Chloride, Potassium Acetate, Tween-20, Isopropyl thiogalactosepyranoside (IPTG), X-gal, lysozyme, Sodium dodecyl sulphate, Diaminobenzidine, TEMED, Glycine, Ethidium Bromide, Proteinase K, RNase, Triton X-100, PMSF, Trypan blue, Fetal calf serum, GMEM, Goat anti-rabbit IgG HRP conjugate, Rabbi t anti-goat IgG HRP conjugate, Dialysis tube and Antibiotics (ampicillin and kanamycin) were obtained from, Sigma Chemicals, USA. Potassium chloride, Disodium hydrogen orthophosphate, Potassium dihydrogen orthophosphate, Hydrogen peroxide, Certrimide, Agar, CTAB, Glucose and Glycerol, Sodium nitrite and Orthophosphoric acid, Sodium carbonate, Sodium bicarbonate, Isopropanol, Phenol, Chloroform, Ethylene diamine tetra-acetic acid, Boric acid, Glacial ace tic acid, Isoamyl alcohol, Sodium hydroxide, Sodium acetate, Sodium dihydrogen phosphate, Magnesium sulphate were purchased from Qualigens& Merck, India respectively. QIAGEN, Germany supplied QIAEX II Gel Extraction Kit while Difco, USA supplied Luria- Bertani Media, SOB Media, and Middlebrook 7H10 agar and OADC salt. Restriction endo-nucleases and T4 DNA ligase were procured from New England Biolabs, UK and MBI Fermentas, Germany respectively. All other reagents were acquired from commercial sources.

Oligonucleotide primers

A set of specific oligonucleotide primers 862 F and 862 Rfrom (locus tag MAP 0862 region884392-884892), were synthesized by integrated DNA technologies, inc.Coralville, IA USA, basedon sequence information of MAP strain k10 complete genome GeneBankAccession No:AE016958 Linkers with restriction endonuclease sites BamH1 at 5' end and Pst1 at 3' end were included in the forward and reverse primers respectively.

MAP PPG 5 Forward 5'-GTA GGA TCC ATG CTC GGC TAC GTT CTC-3'27mer

MAP PPG 6 Reverse 5'-TAT CTG CAG TCA AAA CGT GGG GAC CG-3'26mer

Mycobacterial strain and antigen

Bacterial strain of M. a. paratuberculosis (MAP) strain 316F were obtained from Biological Products Division of IVRI, Izzatnagar and later maintained at Gene Expression lab, Division of Animal Biotechnology. IVRI, Izzatnagar. Antigen against MAP culture filtrate and clinical sera from the goat affected with paratuberculosis were available in the lab.

Plasmid and host strains

Prokaryotic expression vector pQE-30 was purchased from QIAGEN, Germany

Laboratory animals

New Zealand white rabbits were obtained from LaboratoryAnimal Resource Section, IVRI, Izzatnagar. Standard prescribed guidelines for care and use of laboratory animals were followed during the experimentation with these animals.

METHODS

Culture and growth of M. a. paratuberculosisand E. coli

MAP organisms were grown on Middlebrook 7H10 agar enriched with 0.1% glycerol v/v and 10% oleic acid dextrose catalase (OADC) with additional supplementation of Mycobactin J (2mg/l) while E. coli cells were grown in Luria Bertani (LB) medium with shaking at 180 rpm. E. coli M15 cells containing pQE-30 vector were grown in presence of kanamycin @ 50 mg/ml and ampicillin @ 50 mg/ml. Both organisms were grown at 37.c.

Isolation of genomic DNA from M. a. paratuberculosis

The genomic DNA from MAP was isolated by the method of Portillo et al. (1991) with slight modifications. The bacterial colonies scrapped from two months old Middle brook 7H10 agar slants were washed thrice with 1X TE and resuspended in 500 ml of 1X TE. Lysozyme was added to the final concentration of 5 mg/ml. After incubation at 37[degrees]C for two hrs, SDS and proteinase K were added (@1% and 250 [micro]g/ml respectively) and incubated further at 65[degrees]C for 30 min. To this, added 80 [micro]l of 5M NaCl followed by addition of 64 [micro]l of CTAB/NaCl solution and vortexed. The suspension was again incubated at 65[degrees]C for 30 min. DNA was extracted once with phenol, once with phenol: chloroform (1:1) and finally with chloroform: isoamyl alcohol (24:1). The aqueous phase containing DNA was pelleted by centrifugation and washed with 80% ethanol, dried and re-dissolved in 200 [micro]l of 1x TE. And ethanol precipitation. Contaminating RNA was removed from DNA by incubating with 100 [micro]g/ml RNase. The treatment was given for one hour at 37[degrees]C, followed by phenol: chloroform extraction and ethanol precipitation. The DNA was quantitatively estimated using the following formula, by recording the absorbance at 260nm wavelength:

Concentration of dsDNA ([micro]g/ml) = A260 X 50 X dilution factor Agarose gel electrophoresis

The DNA preparations were analyzed on 1% agarose gel and visualized under UV transillumination and documented by photography in Uvitech Gel Documentation System (Austria).

Polymerase chain reaction

The PCR was carried out in 25 [micro]l reaction volume using 1 [micro]l of genomic DNA (10 ng) as template, 2.5 pl of PCR buffer, 1 [micro]l of MgCl2 (1.5 mM), 1 [micro]l (25 pM) of each primers, 1 [micro]l of dNTP mix (200 [micro]M of each dNTP) and 1 U of Taq DNA polymerase. The volume was made up to 25 [micro]l by adding DNase free water. The thermal cycling steps were carried out in PTC-200 thermo-cycler MJ Research Inc, U SA with initial denaturation at 94[degrees]C for 5 min followed by 30 cycles with denatuaration at 94[degrees]C for 1 min, annealing of 1 min at 58[degrees]C for 441bp followed by extension at 72[degrees]C for 30 Seconds and final extension at 72[degrees]C for 1 min. Size of the amplified product was confirmed by using DNA molecular weight marker in a 1.2% agarose gel and quantified by spectrophotometric analysis. The DNA fragment of 441 bp was eluted from agarose gel using QIAEXII gel extraction kit.

Cloning of 441bpin prokaryotic (E. coli) expression vector pQE-30 Ligation

Plasmid pQE 30 was isolated from E.colicells by small scale alkaline lysis method followed by the ligation of eluted PCR product of 441 bp as follows:

a. pQE-30: 1 [micro]l (50ng)

b. 441 bp PCR product: 1 [micro]l (50ng)

c. 2X ligation master mix: 5 [micro]l

d. nuclease free water: 3 [micro]l

The 10 [micro]l ligation mixture was kept at 16[degrees]C overnight and the resulting plasmid was designated as pQE 441 which was stored at -20[degrees]C.

Transformation and Screening of recombinant pQE 441

10 [micro]l ligation mixture was diluted to 200 [micro]l TCM (1X), to which, 200 [micro]l of the competent cells were added with gentle mixing and left at 0 [degrees]C on ice for 1hr. Heat shock was given to this mixture for 2min at 45[degrees]C and then was rapidly chilled on ice for 10 min. Then 600 [micro]l of SOC medium was added and incubated at 37[degrees]C for 1hr with shaking. The transformed cells were spread on LB agar plate containing ampicillin (100 [micro]g/ml) and kanamycin (25 [micro]g/ml). Plates were incubated at 37[degrees]C overnight and later stored at 4[degrees]C.

Screening of recombinant pQE441

About twenty randomly selected colonies were picked up containing pQE 441 clones and grown on LB broth containing Ampicillin and Kanamycin and incubated at 37[degrees]C overnight in a shaker incubator at 180 rpm. Plasmid DNA was extracted & positive clones were identified by Colony PCR and restriction endonuclease analysis.

Expression and purification of the recombinant 16kDa (His 16) Protein

Fresh E. coli culture harbouringpQE (OD600 of 0.6 was induced with 1mM IPTG to express the recombinant 16protein and grown again for 4 hr at 37[degrees]C in a shaker incubator at 180 rpm. The E. coli cells were pelleted by centrifugation at 6,000 rpm for 10 min.& frozen at -20[degrees]C overnight followed by thawing at room temperature for 15min. These pellets were resuspended in 1/25th volume of lysis buffer (buffer B-8M urea, 0.1M NaH2PO4, 1M TrisCl-pH 8.0) and incubated with agitation for 1hr at RT. Removed the cell debris by centrifugation at 10,000 x g for 20-30min at RT (20-25oC) and about 600 [micro]l of the cleared lysate supernatant containing the 6X His-tagged protein was loaded onto a pre-equilibrated (with 600 [micro]l of buffer B) Ni-NTA spin column and centrifuged for 2min at 700 x g. The column Ni-resin bound 6X His-tagged proteins was washed twice with 600 [micro]l of wash buffer (buffer C 8M urea, 0.1M NaH2PO4, 1M TrisCl-pH 6.3) and eluted twice with 200 [micro]l of elution buffer (buffer E -8M urea, 0.1M Na[H.sub.2]P[O.sub.4], 1M TrisCl-pH 4.5).

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE)

SDS-PAGE was carried out on a vertical slab mini apparatus (Atto, Japan). After electrophoresis the gel was stained with Coomassie brilliant blue G250 anddistained in solution containing methanol and acetic acid. The fraction containing the purified recombinant 16kDa protein was resolved and approximate molecular weight of the protein was determined by comparison with protein molecular weight marker in Uvitec gel documentation system using Uvipro software.

Dialysis of the recombinant 16kDa

The selected fractions of each protein were pooled and extensively dialyzed at 4[degrees]C against PBS (pH 7.4) in dialysis tubing with 8 kDa molecular weight cut-off, and stored in aliquots at -20[degrees]C until used.

Hyperimmunization for raising antisera

New Zealand white 8-10 weeks old rabbits were immunized to raise antibody against the purified His-16by inoculating with 150 [micro]g of the immunogen in 0.5ml PBS (pH 7.4) along with equal volumes of incomplete Freund's adjuvant (IFA) subcutaneously. After three weeks, subsequent boosters of 100 [micro]g immunogens were given intramuscularly at weekly intervals. The rabbits were bled a week after third booster and sera were separated and stored at -20[degrees]C in aliquots.

Western blotting

Samples were electrophoresed as before and the protein bands were transferred to nitrocellulose membrane by semi dried blotting according to Bjerrum and Schafter-Nielsen (1986) utilizing transblot buffer (Tris Glycine, pH 8.2 with 20% methanol). Non specific sites were blocked with 5% skim milk powder in PBS-T (phosphate buffer saline pH 7.4 containing 0.05% Tween 20) for 2hr at 37[degrees]C. The membrane was washed three times with PBS-T and the blot was incubated for 2hr at 37[degrees]C with polyclonal hyperimmune sera raised in rabbit against His-16and His-16 proteins (1:500 in PBS-T). After washing the blot with PBST, they were further incubated for 1hr with HRP labeled goat anti-rabbit IgG (diluted to 1:15000 in PBS-T). After washing, the membrane was dipped in substrate solution (0.1% diaminobenzidine and 0.03% hydrogen peroxide in 50mM Tris, pH 7.6). After the development of sufficient colour, the reaction was stopped by washing the blot in PBS (pH 7.4).

Dot blot assay

20 [micro]l of His-16proteins were spotted on a nitrocellulose membrane as a small dot, dried at room temperature and processed to confirm the reactivity of the proteins with the hyperimmune sera raised against the recombinant 16kDa protein, MAP culture filtrate and also from goat with clinical paratuberculosis as per the procedure described for western blotting.

Sequence analysis

DNA sequencing of the plasmid pQE 441 was carried out commercially.

RESULTS

PCR amplification of 441bpM. a. paratuberculosis

PCR amplification of 441 bp was carried out using MAP 862F and R specific primers, atannealing temperature 58[degrees]C in 34 cycles amplified a product of 441 bp size band (Fig. 1, lane 1).

Screening and confirmation of recombinant pQE 441

Isolated Plamid DNA from randomly selected 15 colonies (from pQE 441) was double digested with BamHI/PstI restriction enzymes released a fragment of 441 bp(Fig. 2, lane 2).

Expression of recombination 16kDa proteins in E.coli

IPTG induced E.colicells harbouring the recombinant plasmids pQ E441 12% SDS-PAGE resolved a predominant band of 16kDa corresponding to the recombinant 16kDa (His 16) protein (Fig. 3, lane 3, 4).

Purification of the His-16 protein

The purified protein analyzed by 12% SDS-PAGE, showing the monomeric band of 16kDa (Fig. 5, lane 4) with a concentration of 15 mg/L at flask level.

Immunogenicity of the His-16 protein

The pooled polyclonal sera raised in rabbits against purified His-16protein, reacted with 4 hour IPTG induced culture of E.coli M15 harbouringpQE 441as well as recombinant 16kDa protein on western blot. (Fig. 4, Lane 4)

Dot ELISA

The His-16was also recognized by the polyclonal serum again MAP culture filtrates and also by the serum from a goat naturally infected with MAP (Fig. 5, Lane 4).

DNA sequence and deduced amino acid sequence analysis

The nucleotide and protein sequence analysis of plasmid PQE 441 containing 441 bp gene fragments of MAP 316F strain revealed about 100% homology with retrieved ORFsof the MAP k10 strain by laser gene software DNA star. The predicted primary protein composed of 166 amino acids with a mature protein of 16kDa. Analysis of the deduced amino acids sequence of MAP16kDa according to Kyte and Doolittle (1982) algorithm showed presence of three hydrophobic regions.

DISCUSSIONS

Johne's disease with a potential of causing economic losses worldwide could not be eradicated successfully due to the lack of simple and specific diagnostic tests for the early detection prior to the appearance of disease signs' such as fecal shedding of MAP in the environment (4,8). Early diagnosis is important to identify and remove potential fecal shedders of MAP to prevent the spread of John's disease, and requires the development of sensitive and specific diagnostic tests. Indeed fecal culture the current gold standard tests, which can detect both clinical and subclinical stages is time consuming, requires more than 15 weeks and also labour intensive. Poor specificity of the intradermal skin test and interferon gamma (IFN-?) release assay, agarose gel immuno diffusion (AGID), complement fixation (CF) and ELISA use a complex ill defined mixtures of proteins derived from either, whole cell or fractionated extracts of MAP (9,10,11). To date a number of antigens have also been identified but the data about the characterization of proteins secreted early post infections from MAP are still lacking. Recently Bannatineet. al (12) constructed the partial protein array and examined the antibody profiles of subclinically MAP infected animals and study has focused on the detecting antigens eliciting early antibody response. Since, proteins produced by the MAP are in very low concentration, purification becomes highly cumbersome and uneconomical. Therefore, for high level expression of proteins, E. coli has been widely used for the expression of large number of genes at level sufficient for structural and biochemical analysis and even for product development (13). Therefore, in the present work keeping in view the importance of identifying MAP specific antigens through the cloning and hetrologous expression of 441 BP coding sequences found in the MAP genome encoding 39.7kDa proteins was chosen for expression and immuno-reactivity. Based on the sequence information of two genes encoding 16kDa from MAP strain k10, complete genome Gene Bank Accession No. AE016958 (tag MAP 0862 and MAP 1087 coding for hypothetical proteins) and also the information about multiple cloning sites of the expression vector pQE-30, restriction sites BamHI and PstI were incorporated into the oligonucleotide primers to facilitate directional cloning. The resulting plasmid pQE 441 containing an open reading frame encoding successively 6X histidinepolypeptide, 16kDa. The recombinant pQE 441 clones were confirmed by release of the insert by double digestion with BamHI and PstI restriction enzymes and on induction with IPTG for 4 hours appearance of 16kDa protein band. Expression of the recombinant proteins was induced by IPTG which bind to the lac repressor protein, inactivating it leading to transcription of sequences downstream of the promoter. Using pQE-30 vector, Goswamiet al. (14) and Basagoudanavaret al. (15) have got maximum expression of the recombinant protein after 4-6 hrs post induction. Therefore, it was decided to purify the recombinant proteins 4 hours post induction from E. coli culture. The presence of 6X histidine tag of 840 Dalton at the N-terminal of the recombinant proteins facilitates single step affinity purification, which is poorly immunogenic and may not interfere with the protein immunogenicity its functional structure; hence the tag was not removed by protease cleavage. The polyclonal antisera raised in rabbit against the recombinant His-16proteins strongly reacted with the E. coli expressed recombinant 16kDa and 16 kDa proteins on immunoblot. This has not only confirmed the heterologous expression but also suggested that the recombinant proteins retained their antigenicity. Reactivity of the His-16proteins with polyclonal sera against culture filtrate of MAP on Dot ELISA revealed the secretory nature of the recombinant proteins. Further reaction of these recombinant proteins with clinical sera from par tuberculosis affected goat suggested that these proteins have retained their antigen city even after purification. The result of these studies suggested that these recombinant proteins will further be useful in the development of diagnostic reagents for subclinical detection par tuberculosis in animals. While further studies using sera from experimentally as well as naturally infected animals are needed to lay down the solid foundation towards the development of feasible ELISA based assay for subclinical detection of MAP infected animals.

ACKNOWLEDGMENTS

The authors are thankful to the Director, Indian Veterinary Research Institute for providing the facilities to carry out the work.

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(11.) Eda, S., Elliott, B., Scott, M.C., Waters, W.R., Bannantine, J.P., Whitlock, R.H. and Speer, C.A. New method of serological testing for Mycobacteriumavium subspparatuberculosis (Johne's disease) by flow cytometry. Foodborne Pathol. Dis, 2005; 2: 250-262.

(12.) Bannantine, J.P., Paustian, M.L., Waters, W.R., Stabel, J.R., Palmer, M.V, Li, L. and Kapur, V, Pro.ling bovine antibody responsesto Mycobacteriumavium subsp. paratuberculosis infection by using protein arrays. Infect. Immun., 2008; 76: 739-749.

(13.) Rosenberg, I. M., Recombinant protein techniques. In: proteinanalysis and purifications, Rosenberg, I. M. (e.d.), Boston, M.A.: Birkauser, Publications, 1996.

(14.) Goswami, P.P., Rupa, P., Parehar, N.S. and Garg, L.C., Molecularcloning of colostrumsperfringes epsilon toxin gene and its highlevel expression in E. coli. Biochem. Res. Commun, 1996; 226: 735-740.

(15.) Basagoudanavar, S.H., Goswami, P.P., Tiwari, V, Pandey, A.K. andSingh, N., Heterologous expression of a gene encoding 35kDa protein of Mycobacterium aviumparatuberculosisin Escherichiacoli. Vet. Res. Commun., 2004; 28: 209-224.

Dheeraj Pal and P.P. Goswami

Gene Expression Laboratory, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh-243122, India.

(Received: 29 April 2015; accepted: 11 July 2015)

* To whom all correspondence should be addressed. Mob.: + 919457432866; E-mail: dheerajvet0199@rediffmail.com
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Author:Pal, Dheeraj; Goswami, P.P.
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Sep 1, 2015
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