the classical interleukin ( il)-1 family cytokines , il-1 and il-1 , as well as il-18 , are related by mechanism of origin , receptor structure , and signal transduction pathways utilized . il-33 ( also named il-1f11 ) , which belongs to il-1 family , is most closely related in structure to il-18 and il-1 . il-33 is also described as a nuclear factor expressed in high endothelial venules ( nf - hev ) . the human il-33 gene is located on chromosome 9p24.1 , while its mouse counterpart can be found on the syntenic chromosome 19qc1 region . the il-33 cdna sequences encode 270 and 266 amino acid polypeptides for human and mouse , respectively , corresponding to full - length proteins with calculated masses of 30 and 29.9  kda . the proteins do not contain a signal sequence for secretion , similar to the il-1 , il-1 , and il-18 genes [ 4 , 5 ] . the precursor protein ( pre - il-33 ) was cleaved by caspase-1 into a mature 18-kda protein ( il-33 ) in in vitro experiments using a recombinant protein . il-33 signals via the il-1 receptor - related protein st2 [ 2 , 6 ] and il-1 receptor accessory protein   activate nf-b and mitogen - activated protein kinases to induce t helper type 2 ( th2)-associated cytokines [ 2 , 8 , 9 ] . administration of il-33 to mice induces production of th2 cytokines and severe pathological changes in mucosal organs . moreover , the ability of il-33 to induce th2 responses has functional relevance in the context of intestinal helminth infection . il-33 can promote survival , adhesion , and cytokine production in human mast cells , but the mechanism behind its involvement in inflammatory conditions remains unclear . it is also a key mediator of immune inflammatory hypernociception normally associated with a th1 type of response , revealing a hitherto unrecognized function of il-33 in a key immune pharmacological pathway that may be amenable to therapeutic intervention .   in this study , the cloning and expression of mouse il-33 gene in escherichia coli ( e. coli ) , purification of recombinant protein , and generation of polyclonal antibody against il-33 are described . the prepared antibody can be useful for the study of expression and distribution of il-33 in various tissues at protein level and for the elucidation of its biofunctions and regulation mechanism in inflammatory diseases . e. coli strain bl21 ( de3 ) and ni - nta his - bind resins were obtained from novagen ( madison , wi ) . hotstar taq dna polymerase and qiaquick gel extraction kit were purchased from qiagen ( valencia , ca ) . primescript first strand cdna synthesis kit , restriction endonucleases xhoi , ndei , t4 dna ligase , isopropyl--d - thiogalactoside ( iptg ) , dna marker d2000 , and protein marker were from takara ( dalian , china ) . lysozyme , complete freund 's adjuvant ( cfa ) , and incomplete freund 's adjuvant ( ifa ) were obtained from sigma ( st . louis , mo ) . total cellular rna was extracted from spinal cord of balb / c mouse according to trizol reagent instructions . the coding region of mouse il-33 mature protein was amplified by reverse transcription polymerase chain reaction ( rt - pcr ) . the first strand of cdna was synthesized using the primescript first strand cdna synthesis kit , and the pcr reactions were carried out with a ptc-200 peltier thermal cycler ( mj research , waltham , ma ) . primer 1 ( 5-ggaattccatatg acattgagcatccaaggaac-3 ) and primer 2 ( 5-ccgctcgaggattttcgagagcttaaaca-3 ) were synthesized by invitrogen ( shanghai , china ) . primer 1 was synthesized with an ndei site ( shown in bold ) and an additional atg for initiation of translation in italicized style . the amplification profile included one initial hot - start denaturation step at 94c for 15  minutes , followed by 30 cycles of the following conditions : 94c for 30 seconds , 57c for 30 seconds , and 72c for 1  minute , and a final extension at 72c for 10  minutes . plasmid pet-44 ( novagen , darmstadt , germany ) was digested with ndei and xhoi restriction enzymes . the large fragment was cut and purified from agarose gel using the qiaquick gel extraction kit . a recombinant plasmid was constructed by inserting the amplified fragments ( also digested with ndei and xhoi ) into the ndei and xhoi sites of pet-44 and transformed into e. coli strain bl21 ( de3 ) . the transformants ( pet-44-mil-33 ) were confirmed by colony pcr , restriction enzyme digestion , and dna sequencing . expression in bacteria results in the producing of recombinant il-33 protein containing a hexahistidine tag in the c - terminus . a single colony of successfully transformed e. coli was picked up and cultured overnight at 37c in luria bertani ( lb ) medium , supplemented with 100  g / ml ampicillin . the culture mixture was then inoculated to fresh lb medium ( 1  :  100 dilution ) containing ampicillin and grown at 37c under continuous shaking , until the absorbance at 600  nm reached 0.60.8 . to optimize the expression conditions , il-33 expression was induced by adding 1  mm iptg to the transformed e. coli and the bacteria were incubated at 25c for a period of 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , and 9  hours , respectively . the degree of expression was evaluated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) . to compare the protein dissolubility , the bacteria with pet-44-mil-33 after 7  hours of induction at 25c , the cells were harvested by centrifugation at 8,000   g for 20  minutes at 4c . the supernatant was discarded and the pellet was resuspended in lysis buffer consisting of 300  mm nacl , 50  mm nah2po4 , 10  mm imidazole , ph 8.0 , and lysed by using lysozyme , and incubated for 30  minutes at room temperature ( rt ) with gentle shaking to assure thorough cell lysis . the cell lysate was further disrupted by sonication on ice with six 10-second pulses at high intensity with a 10  seconds cooling period between each burst . the suspension was centrifuged at 12,000   g for 30  minutes to remove insoluble debris . the supernatant was added to an ni - nta spin column pre - equilibrated with the lysis buffer , and then they were gently shaked on ice for 60  minutes . the column was then washed with native wash buffer ( 300  mm nacl , 50  mm nah2po4 , 20  mm imidazole , ph 8.0 ) . finally , the bound protein was eluted with native elution buffer ( 300  mm nacl , 50  mm nah2po4 , 250  mm imidazole , ph 8.0 ) and analyzed with 12% sds - page . fractions were desalted and concentrated by an ultrafiltration device ( millipore corporation , bedford , ma ) . the new zealand white rabbits were given intradermal injections of il-33 protein ( 500  g / rabbit ) mixed with cfa in 1 : 1 ratio . after 2 week , the rabbits were boosted subsequently 4 times with protein ( 200  g / rabbit ) mixed with ifa in 1 : 1 ratio at 2-weeks interval . before every immunization , blood samples were taken from the marginal vein of the rabbit ear , centrifuged , and the sera were obtained to determine the antibody titer by enzyme - linked immunosorbent assay ( elisa ) . one week after the last injection , antisera of the rabbits were collected and purified by saturated ammonium sulfate ( sas ) and then protein a sepharose affinity chromatography ( amersham biosciences , piscataway , nj ) following manufacturer 's instructions . the titer of antiserum was determined by an indirect elisa . for each well of the 96-well elisa plate , 200  ng of il-33 protein was diluted in 100  l of sodium bicarbonate solution and incubated overnight at 4c . after three washes with phosphate - buffered saline-(pbs- ) tween buffer ( 0.05% tween 20 in pbs ) , the wells were blocked using 100  l of 3% bovine serum albumin for 1 hour at 37c and then incubated with 100  l polyclonal antibodies against il-33 with different dilution ( from 1  :  100 to 1  :  64,000 ) for 1.5 hours . the wells were incubated with 50  l / well horseradish peroxidase-(hrp- ) conjugated goat antirabbit immunoglobulin ( igg , 1  :  250 dilution ) for 1 hour at 37c after thoroughly washed . after final washes , 100  l / well tetramethyl benzidine ( tmb ) solution was added for color development . after 10  minutes , the reaction was stopped by 100  l / well 2  m  h2so4 and the absorbance was measured at 450  nm using a plate reader ( molecular devices ) . ratio of antiserum versus control serum absorbency greater than 2.1 was recognized as positive . in order to confirm that the polyclonal antibodies are able to recognize il-33 protein from tissues , we also examined the il-33 protein expression of plasmid pcdna3.1-mil-33 after delivery through hydrodynamics - based gene injection in balb / c mice [ 14 , 15 ] . the plasmid pcdna3.1-mil-33 was constructed as forenamed , but whole mouse il-33 cdna was amplified by pcr and then inserted into mammalian expression plasmid pcdna3.1(+ ) ( invitrogen ) . briefly , slides were dried in an oven ( 60c ) before removing paraffin in several changes of xylene . slides were hydrated through a series of graded alcohols to water , followed by incubation with 0.5% hydrogen peroxide for 5  minutes . sections were incubated with 5% bovine serum albumin ( bsa ) in pbs for 10  minutes at rt to block nonspecific binding . after rinsing with pbs , slides were incubated overnight at 4c with rabbit anti - il-33 polyclonal antibody ( 1  :  1,000 dilution in pbs containing 5% bsa ) . after rinsing with pbst ( pbs containing 0.01% triton x-100 ) , tissue sections were incubated for 40  minutes at rt with hrp - conjugated goat antirabbit igg ( 1  :  100 ) and rinsed with pbst . subsequently , slides were stained with diaminobenzidine ( dab ) and counterstained with hematoxylin , dehydrated , and mounted . in order to express recombinant il-33 in e. coli , the mature protein coding region of il-33 was generated by pcr and was cloned between ndei and xhoi sites of pet-44 vector containing the coding sequence for a hexahistidine - coding sequence in the c - terminal part . amplification of the il-33 gene by pcr produced a single amplified 480  bp dna fragment , coding for a mature il-33 protein of 160 amino acids . the final pet-44-mil-33 construct is able to express the recombinant il-33 protein in fusion with a hexahistidine tag and allow for metal affinity purification . after induction with iptg , e. coli bl21 ( de3 ) transformed with pet-44-mil-33 produced a protein of approximately 18  kda as shown in figure 1 . the size of the protein matched well with its theoretical molecular weight . to determine the optimal induction period , the bacteria were incubated with iptg at 25c for 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , and 9  hours , respectively . the results showed that while the incubation period was below 7  hours , the yield of the   ~18  kda protein was increased as the induction time prolonged ; but when the incubation time exceeded 7  hours , the yield of the product was not significantly raised ( figure 1 ) . mmol / l , and the bacteria were cultivated at 25c for 7 hours . induced bl21 ( de3 ) cells at 25c can express the il-33 protein ( figure 2 , lane 5 ) . in contrast , noninduced bl21 ( de3 ) cells did not express this fusion protein ( figure 2 , lane 2 ) . the soluble il-33 was expressed at high levels at 25c ( figure 2 , lane 3 ) , but at low levels at 37c ( data not shown ) . this was most likely due to growth at 37c , which causes some protein to accumulate as inclusion bodies , whereas incubation at 25c decreases the rate of protein synthesis and leads to soluble protein . the target il-33 protein appeared as a single band on sds - page ( figure 2 , lane 1 ) , which is in agreement with the molecular weight reported . polyhistidine tags form high - affinity complexes with immobilized divalent metal ions ( such as ni or co ) , even in the presence of high concentrations of chaotropic agents ( e.g. , urea or guanidine hydrochloride ) , thereby allowing isolation of tagged protein from a crude cellular extract . his - tagged il-33 exists in the bacterial cells in two forms : either soluble protein or inclusion bodies . the soluble protein was incubated with the ni - nta agarose slurry to allow binding of his - tagged il-33 . n - terminal sequencing of the purified il-33 revealed that the first 20 residues were identical to the mouse il-33 sequence deduced from the dna ( tlsiqgtslltqspaslsty ) . after immunizing rabbits with recombinant il-33 according to standard protocol , anti - il-33 serum was purified by sas precipitation and protein a affinity chromatography . the igg fractions showed two bands , a heavy chain ( 50 kda ) and a light chain ( 25  kda ) on sds - page gel after passing through the protein a - sepharose column ( figure 3 ) . the antibody at different dilutions ( 100- to 64,000-fold ) was reacted with an equal amount of the il-33 protein ( 0.2  g ) . the antibody titer is defined as the highest dilution of serum at which the a450 ratio ( a450 of postimmunization sera / a450 of preimmunization sera ) is greater than 2.1 . the antibody titer was found to be approximately 1  :  32,000 ( figure 4 ) . il-33 is a  dual - function  cytokine that may function as both an intracellular heterochromatin - associated nuclear factor and a potent proinflammatory cytokine . after immunohistochemistry was performed using the anti - il-33 polyclonal antibody in mouse lung and liver tissues , there were brown staining in nuclei or cytoplasm ( figures 5(b ) , 5(d ) , 5(f ) , 5(h ) ) . furthermore , there was a significant increase of il-33 expression in hydrodynamics - injected mice with il-33 cdna plasmid compared with normal mice ( figure 5 ) . bronchial epithelial cells in lung and liver cells revealed the strongest staining with anti - il-33 antibody in hydrodynamics injected mice ( figures 5(h ) , 5(f ) ) . no staining was detected in tissues where no antibody or control igg was used in the primary layer ( igg control , figures 5(a ) , 5(c ) , 5(e ) , 5(g ) ) . our results demonstrated that the endogenous il-33 protein in tissues could be specifically recognized by our prepared anti - il-33 polyclonal antibodies . we have developed a procedure for the efficient expression and purification of il-33 using pet-44 vector and ni - nta affinity chromatography . furthermore , we report the production of specific anti - il-33 antibody and the localization of endogenous il-33 . it has been observed that il-33 mrna is broadly expressed in many tissues but is more restricted at the level of cell type , which includes pulmonary smooth muscle cells , epithelial cells , dermal fibroblasts , keratinocytes , dendritic cells , and activated macrophages . il-33 is also expressed in endothelial cells from chronically inflamed rheumatoid arthritis synovium and crohn 's disease intestine [ 3 , 18 ] and atherosclerotic tissues . however , very little information is available on the expression of il-33 protein in liver and lung tissues . to our knowledge this is the first report so far of production of polyclonal antibody against full - length mouse il-33 and il-33 protein expression in mouse liver and lung determined with anti - il-33 polyclonal antibodies . as il-33 induces gene expression of th2-associated cytokines and pathological changes in the lung , blockade of il-33 may be a new therapeutic strategy for bronchial asthma . we are investigating the effect of exogenously administered anti - il-33 antibody in a murine model of allergen - induced asthma   and collagen - induced arthritis . this research may contribute to elucidation of the biological mechanisms of il-33 in human inflammatory diseases in the future .
interleukin ( il)-33 is the latest member of il-1 cytokine family . in this study , the cloning , expression , purification , and polyclonal antibody preparation of mouse il-33 were described . 
 the coding region of il-33 mature protein was cloned into the prokaryotic expression vector pet-44 . the recombinant protein , il-33 containing a hexahistidine tag in the c - terminal , 
 was expressed in escherichia coli . 
 the expressed soluble protein was purified by immobilized metal - ion affinity chromatography using ni2 + -nitrilotriacetic acid agarose . 
 the rabbits were immunized with the purified recombinant protein . 
 the obtained antiserum was precipitated by saturated ammonium sulfate and then purified by protein a affinity chromatography . 
 the sensitivity and specificity of the antibodies were evaluated by enzyme - linked immunosorbent assay and immunohistochemistry . 
 the high titer ( 1 : 32000 ) polyclonal antibodies with high specificity were obtained by immunizing rabbits with the purified recombinant protein . 
 significant expression of il-33 was seen in mouse liver and lung tissues determined with the anti - il-33 . 
 the production of the polyclonal antibody against il-33 provides a good tool for studying the biofunctions of il-33 .