A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats.

BACKGROUND: Current options to treat clinical relapse in inflammatory central nervous system (CNS) conditions such as cerebral ischemia-reperfusion injury are limited, and agents that are more effective are required. Disruption of the blood-brain barrier is an early feature of lesion formation that correlates with clinical exacerbation and facilitates the entry of inflammatory medium and inflammatory cells. Interleukin-1 receptor antagonist (IL-1RA) is a naturally occurring anti-inflammatory antagonist of the interleukin-1 (IL-1) family. The broad-spectrum anti-inflammatory effects of IL-1RA have been investigated against various forms of neuroinflammation. However, the effect of IL-1RA on blood-brain barrier disruption following ischemia-reperfusion has not been reported. METHODS: In this study, we investigated the effects of IL-1RA and a novel protein (IL-1RA-PEP) that was fused to IL-1RA with a cell penetrating peptide, on blood-brain barrier integrity, in male rats subjected to transient middle cerebral artery occlusion. RESULTS: After intravenous administration, IL-1RA-PEP (50 mg/kg) penetrated cerebral tissues more effectively than IL-1RA. Moreover, it preserved blood-brain barrier integrity, attenuated changes in expression and localization of tight junction proteins and matrix metalloproteinases, and enhanced angiogenesis in ischemic brain tissue. Further study suggested that the effects of IL-1RA-PEP on preserving blood-brain barrier integrity might be closely correlated with the p65/NF-κB pathway, as evidenced by the effects of the inhibitor JSH-23. CONCLUSIONS: Collectively, our results demonstrated that IL-1RA-PEP could effectively penetrate the brain of rats with middle cerebral artery occlusion and ameliorate blood-brain barrier disruption. This finding might represent its novel therapeutic potential in the treatment of the cerebral ischemia-reperfusion injury.

Transient receptor potential vanilloid 2 (TRPV2), a potential novel biomarker in childhood asthma.

BACKGROUND: The presence of transient receptor potential vanilloid 2 (TRPV2) in human peripheral blood cells may suggest a role under pathological conditions. The aim of this study was to explore the relationship between the expression profile of TRPV2 gene and childhood asthma in the north of China. The effects of allergens exposure on the expression of TRPV2 gene were also investigated. METHODS: Sixty asthmatics children confirmed by physician diagnosis and 60 healthy children as a control group were recruited. Serum total IgE and specific IgE were measured. Using quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), TRPV2 was detected in total RNA extracted from peripheral blood lymphocytes. Student's t-test and chi-square test were used to analyze the relationship between TRPV2 transcript and different parameter variables on susceptibility of childhood asthma. Multiple logistic regression was used to analyze the associations between TRPV2 gene and allergens. RESULTS: The expression level of TRPV2 gene was increased 2.6 times in asthmatic children compared with controls (p < .01). The up-regulation of TRPV2 gene and sensitization to one of three the allergens-spring pollen, dust mite, and dog and cat hair-were correlated with childhood asthma. In addition, the hypersensitivity to spring pollen, cockroach, and dust mite and up-regulation of TRPV2 gene expression may be the risk factors for the childhood asthma in Beijing. CONCLUSIONS: The increased expression of TRPV2 gene in peripheral lymphocytes is closely correlated with childhood asthma in the north of China. This study provides a potential new biomarker of childhood asthma and lays the basis for further clarification of the pathogenesis underlying asthma.

Expression, purification and characterization of recombinant human angiogenin in Pichia pastoris.

The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.

Hepatoprotective effects of IL-22 on fulminant hepatic failure induced by d-galactosamine and lipopolysaccharide in mice.

Interleukin-22 (IL-22), a member of the IL-10 cytokine family that is produced by activated Th22, Th1 and Th17 cells as well as natural killer cells, plays an important role in increase of innate immunity, protection from damage and enhancement of regeneration. Here, we examined the effects of IL-22 on acute liver failure model induced by d-galactosamine (GalN) and lipopolysaccharide (LPS). Administration of recombinant human IL-22 (rhIL-22) reduced the death rate markedly and prevented mice from severe hepatic injury, as evidenced by decreased serum alanine aminotransferase (ALT) and total bilirubin (T.Bil) activity as well as improved histological signs in liver. Furthermore, IL-22 treatment decreased the hepatic malondialdehyde (MDA) contents and increased the reduced glutathione levels. Serum tumor necrosis factor α (TNF-α) level and hepatic caspase-3 activity were significantly lower in mice administrated with IL-22. Moreover, IL-22 treatment significantly enhanced activation of STAT3 and up-regulated the expression of Bcl-xL, heme oxygenase-1 (HO-1) and redox factor-1 (Ref-1) in the liver injury induced by GalN/LPS. Collectively, these data indicate that IL-22 can provide critical protection against GalN/LPS-induced liver injury through anti-apoptotic, anti-oxidant and anti-inflammatory actions.

Interleukin-22 protects against acute alcohol-induced hepatotoxicity in mice.

The protective effects of interleukin-22 (IL-22) on acute alcohol-induced liver injury were investigated. Mice were gavaged with 7 doses of alcohol (56% wt/vol, 15.2 mL/kg of body weight for each dose) over the 24 h, and IL-22 (0.5 mg/kg BW) was given to the mice by injection into the tail vein 1 h after alcohol administration. The results indicated that acute alcohol administration caused prominent hepatic microvesicular steatosis and an elevation of serum transaminase activities, induced a significant decrease in hepatic glutathione in conjunction with enhanced lipid peroxidation, and increased hepatocyte apoptosis as well as hepatic TNF-alpha production. IL-22 treatment attenuated these adverse changes induced by acute alcohol administration. The protective effects of IL-22 on alcohol-induced hepatotoxicity were due mainly to its anti-inflammatory, anti-oxidant, and anti-apoptotic features.

Thr-114 is an important functional residue of fibroblast growth factor 10 identified by structure-based mutational analysis.

Fibroblast growth factor 10 (FGF10) plays important roles in vertebrate limb development, lung branching morphogenesis, and epidermis regeneration. The receptor (FGFR2b) binding specificity is an essential element in regulating the diverse functions of FGF10. Analyzing the FGF10:FGFR2b complex we found that Thr-114 in beta4 of FGF10 could form specific interactions with D3 of FGFR2b. To investigate the role of Thr-114 played on functions of FGF10, two mutants of FGF10 were constructed, named TA (Thr-114-->Ala) and TR (Thr-114-->Arg), respectively. The biological activity assays showed that the receptor-binding affinity, the stimulating growth effect on rat tracheal epithelium (RTE) cells, and the inducing ability in receptor phosphorylation of both mutants were decreased, which were consistent with the interaction analysis of the TA:FGFR2b and TR:FGFR2b complexes. These results suggested that Thr-114 is a crucial functional residue for FGF10, and mutating Thr-114 to Ala or Arg would lead to great decrease in receptor-binding affinity and biological activity of FGF10.

The protective role of IL-1Ra on intestinal ischemia reperfusion injury by anti-oxidative stress via Nrf2/HO-1 pathway in rat.

BACKGROUND: Intestinal ischemia reperfusion injury is a frequent clinical damage, in which the oxidative stress and inflammation play an important role. Interleukin-1 receptor antagonist (IL-1Ra) is a natural anti-inflammatory factor, however, its effect on intestinal ischemia reperfusion injury remains unclear. METHODS: The rat model of intestinal I/R was induced by occlusion (for 60 min) and reopening (for 60 min) of superior mesenteric artery. The rats were randomly divided into the following 5 groups: sham-operation(S), model (I/R),10 mg/kgIL-1Ra + I/R (C1),20 mg/kgIL-1Ra + I/R (C2), and30 mg/kgIL-1Ra + I/R (C3). RESULTS: In this study it was the first time to confirm that IL-1Ra had a significant protection against the intestinal ischemia reperfusion injury. IL-1Ra not only effectively inhibited the expression of inflammatory factors (such as IL-1ß, IL-6 and TNF-α) and the activation of neutrophil in intestinal tissues, but also decreased the death of intestinal cells and the damages of intestinal tissues. Interestingly, besides anti-inflammation effect, it was also found that IL-1Ra possessed a significant inhibitory effect on the oxidative stress caused by ischemia/reperfusion injury. Furthermore, the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1), and the phosphorylation level of Nrf2 were greatly promoted by IL-1Ra. At the same time, IL-1Ra inhibited the mitogen-activated protein kinase (MAPKs) pathway. CONCLUSION: IL-1Ra had the protective effect against intestinal ischemia reperfusion injury, its mechanism included anti-inflammation and anti-oxidative stress in which the Nrf2/HO-1 pathway played an important role. The above-mentioned results may extend the clinical application of IL-1Ra in the treatment of intestinal ischemia reperfusion injury.

[Effects of interleukin 1 receptor antagonist on allergy asthma in rat model and its mechanism].

OBJECTIVE: To study the effects of interleukin 1 receptor antagonist (IL-Ira) on allergy asthma and its mechanism. METHODS: Thirty female SD rats underwent intraperitoneal and hypodermic injection of ovalbumin (OVA) on days 1 and 14, and then underwent spraying of OVA aerosol since day 21 for 7 days so as to provoke asthma, and then the rats were randomly divided into 3 equal groups: asthma model group, low dose IL-1ra treatment group undergoing intravenous injection of IL-1ra 6 mg/kg before each provocation (low dose treatment group), and high dose IL-1ra treatment group undergoing intravenous injection of IL-1ra 30 mg/kg before each provocation (high dose treatment group). Another 10 rats were used as normal controls. Twenty-four hours after the last provocation physiological monitoring equipment was used to detect the pulmonary function. Then the rats were killed. Bronchoalveolar lavage fluid (BALF) was collected. ELISA was used to detect the serum IgE content. The ratio of inflammatory cells from the BALF was calculated. Microscopy was conducted to observe the histopathology of lung. RT-PCR was used to examine the mRNA expression of NF-kappaB and signal transducer and activator of the transcription 6 (STAT6). RESULTS: The respiratory rate, expiratory flow, percentage of eosinophils in BALF inflammatory cells, peripheral blood IgE concentration, mRNA expression of STAT6 and NF-kappaB of the asthma group were (206 +/- 11) times/min, (77 +/- 8) microl/s, 24.8% +/- 1.3%, (72.5 +/- 8.1) ng/ml, 0.294 +/- 0.048, and 0.686 +/- 0.052 respectively, all significantly higher than those of the low dose treatment group [(183 +/- 9) times/min, (64 +/- 5) microl/s, 18.5% +/- 3.1%, (63.4 +/- 4.8) ng/ml, 0.229 +/- 0.038, and 0.613 +/- 0.062 respectively, all P < 0.05] and those of the high dose treatment group [(181 +/- 11) times/min, (57 +/- 4) microl/s, 14.7% +/- 2.1%, (41.4 +/- 7.4) ng/ml, 0.194 +/- 0.076, and 0.352 +/- 0.267, all P < 0.05]. The therapeutic effect of high dose treatment group is superior to that of low dose treatment group (P < 0.05). CONCLUSION: IL-1ra is significantly effective in treatment of allergic asthma, and its potential mechanism is through regulating both STAT6 mRNA and NF-kappaB mRNA expression simultaneously.

SAK-HV Promotes RAW264.7 cells Migration Mediated by MCP-1 via JNK and NF-κB Pathways.

Macrophage migration plays an essential role in immune system and is also involved in many pathological situations. However, the regulatory mechanism of macrophage migration remains to be elucidated due to its diverse responses to various stimuli. SAK-HV, a multifunctional protein possessing thrombolytic and lipid-lowering activity, can selectively induce the macrophage proliferation. Here, we reported SAK-HV significantly triggered RAW264.7 cells migration through its functional domain of SAK-mutant by activating both c-jun N-terminal kinases (JNK) and nuclear factor-κB (NF-κB) pathways. Meanwhile, SAK-HV upregulated the expression of some effector proteins, among which only the expression of Monocyte chemoattractant protein-1 (MCP-1) was inhibited by the blockade of JNK and NF-κB pathways. Further research showed that MCP-1 promoted migration ultimately by interacting with Chemokine (C-C motif) Receptor 2 (CCR2) in an autocrine manner. In summary, SAK-HV induced RAW264.7 cells migration through its SAK-mutant domain, during which MCP-1 chemokine mediated by JNK and NF-κB pathways played a key role. These results revealed a novel effect of SAK-HV on modulating macrophage migration and also deepened the understanding of its pharmacodynamics.

A novel IL-1RA-PEP fusion protein with enhanced brain penetration ameliorates cerebral ischemia-reperfusion injury by inhibition of oxidative stress and neuroinflammation.

Neuroinflammation and oxidative stress are involved in cerebral ischemia-reperfusion, in which Interleukin 1 (IL-1), as an effective intervention target, is implicated. Interleukin-1 receptor antagonist (IL-1RA) is the natural inhibitor of IL-1, but blood-brain barrier (BBB) limits the brain penetration of intravenously administered IL-1RA, thereby restricting its therapeutic effect against neuroinflammation. In this study, we evaluated the potential effects of anti-inflammation and anti-oxidative stress of a novel protein IL-1RA-PEP, which fused IL-1RA with a cell penetrating peptide (CPP). Studies were carried out in transient middle cerebral artery occlusion (MCAO) in rats and oxygen glucose deprivation/reoxygenation (OGD/R) in primary cortical neurons. In MCAO rat model, IL-1RA-PEP (50mg/kg) injected i.v., penetrated BBB effectively, and alleviated brain infarction, cerebral edema, neurological deficit score and motor performance as well as inhibited the inflammatory cytokines expression. Furthermore, our results firstly showed that IL-1RA-PEP also regulated the oxidases expression, decreased the levels of NO, MDA and ROS. In addition, the inhibitory effects of IL-1RA-PEP on oxidative stress and inflammation were confirmed in rat cortical neurons induced by OGD/R, it reduced ROS, IL-6 and TNF-α. Further study showed that the effects of IL-1RA-PEP were closely associated with the NF-κB and p38 pathways which were proved respectively by their inhibitors JSH-23 and SB203580. Our results indicated that IL-1RA-PEP could effectively penetrate the brain of MCAO rats, alleviated the cerebral ischemia reperfusion injury by inhibiting neuroinflammation and oxidative stress, showing a great clinical potential for stroke.

[Clonging, expression and activity determination of the recombinant human soluble B lymphocyte stimulator and its two mutants].

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) ligand superfamily that functionally involved in B cell proliferation. Here, the full length cDNA of human soluble BlyS (hsBLyS) was amplified by reverse transcription-PCR from total RNA of human peripheral blood lymphocytes and sequenced. The cloned cDNA fragments was inserted into pBV220, a thermo-sensitive expression plasmid, forming recombinant plasmid pBV220/hsBLyS. The two mutants of hsBLyS: hsBY-A (Cys(146) right curved arrow Ala(146), TGC right curved arrow GCT) and hsBY-V (Cys(146) right curved arrow Val(146), TGC right curved arrow GTT), were amplified by overlap PCR from pBV220/hsBLyS and inserted into the pBV220 to form pBV220/hsBY-A and pBV220/hsBY-V expression plasmids as the method above. The three expression plasmids were transformed into E.coli DH5alpha and it was found that the recombinant proteins were highly expressed as inclusion bodies. Pure recombinant proteins were obtained by Sephacryl-200 gel filtration and renaturation. The experimental results demonstrated that the sequences of the PCR products were identical with the published hsBLyS cDNA sequence or expected mutant sequence and the purity of the recombinant proteins obtained was relatively high. The activity of the purified recombinant proteins was very significant in the B cell survival and proliferation assay, and statistical tests indicated that the mutant rhsBY-V promoted proliferation of B lymphocyte cell remarkably better than the wild type.

[Quantitative method for detection of plasma lumbrokinase content and its assessment].

This study was aimed to set up and evaluate a quantitative method for detecting lumbrokinase level in plasma. The lumbrokinase was used to immunize rabbit and BALB/c mouse for preparation of rabbit or mouse-derived polyclonal antibodies, and then the standard curves were drawn up by detecting the lumbrokinase diluted in PBS using the double antibody sandwich ELISA. This method further was analyzed for its specificity, precision and recovery rate. This established double antibody sandwich ELISA was used to assay the lumbrokinase in human plasma, and the assayed results were assessed. The results showed that a double antibody sandwich ELISA for the detection of lumbrokinase has been established. And the standard curve fitting R value > 0.99, the precision assessment showed that the measured values of coefficient of variation (CV) in 3 batches were all < 15%; recovery assessment in 3 batches showed that all the measured recovery rates were > 80%; the quantitative low limit was assessed as 5 ng/ml (precision CV < 15%, recovery rate > 85%). It is concluded that this method is consistent with the criteria stipulated by the Pharmacopeia, which provides a reliable measurement method for quantitative detection of plasma lumbrokinase in clinical trials.

[Influence of long-used staphylokinase derivative on hemoagglutinative and fibrinolytic systems].

This study was aimed to explore the influence of staphylokinase derivative (SAKD) on the hemoagglutinative and fibrinolytic systems, and to determine the safety of the staphylokinase derivative in application. The normal and model rats each 30 were divided into normal saline, SAKD and rSAK groups. The hemorrhage, bleeding time (BT), blood platelet count (BPC), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fg), D-dimer (D-D), plasminogen (PLG) and plasmin inhibitor activity (PI) were detected before and after the administration with staphylokinase derivative 0.5 mg/kg body weight, once three days for consecutive 15 days. The results indicated that one case of normal rats with SAKD and two cases of high fat diet model group had mild hemorrhage, all of which showed automatic hemostasis; and 3 cases in rSAK group had mild hemorrhage. And the platelet counting, D-D, PLG and PI in all groups did not significantly change. The rats of high fat diet group treated with SAKD showed the significant extension of APTT, PT and TT times, and the decrease of Fg time (p < 0.05). All the experimental results demonstrated that the influence of SAKD on the hemagglutination of the normal animals was lower, however, which can improve the high-hemagglutination status of the rats with high fat diet. It is concluded that the SAKD at the dosage of this study has the higher safety, which can alleviate the high hemagglutination symptoms of the rats with high fat diet.

[Construction and identification of the eukaryotic expression vector pIRES2-EGFP-IL-1ra-Fcepsilon].

The cDNA of IgE constant domain of rat was cloned from the spleen of allergy asthma rat by RT-PCR. The IL-1ra segment was obtained from intermediate vector pBV220-IL-1ra. By overlap extension PCR, the fusion gene IL-1ra-Fcepsilon was cloned, then inserted into the eukaryotic expression plasmid pIRES2-EGFP to obtain a recombinant expression plasmid pIRES2-EGFP-IL-1ra-Fcepsilon. The recombinant expression plasmid was transfected into 293T cells using lipofectamin and instillated into the rat lung through trachea. The expression of IL-1ra-Fcepsilon was identified by Western blot, RT-PCR, and this protein could inhibit the activity of IL-1 in vitro. Green fluorescent protein could be detected in the transfected 293T cells and the rat lungs at different times. The research paved the way for the gene therapy of allergy asthma.

[Soluble high-expression, purification and bioassay of IGFBP-3].

cDNA for Insulin-like growth factor binding protein 3 was cloned and constructed a prokaryotic expression vector--pET-DsBA-IGFBP3. The construct was transformed into E. coli BL21 (DE3)plysS. The induced fusion protein (D-IGFBP3) was expressed successfully in soluble form. We obtained D-IGFBP3 the purify of which is over 95% after purification by His affinity chromatography. The product was identified by Western-blot. The cell assay showed that the obtained fusion protein can inhibit the growth of MCF-7 and bind with IGF-I in vitro.

[Construction and characterization of soluble HLA-A*0201-PR1 complex].

This study was aimed to construct the soluble HLA-A*0201-PR1 complex for preparation of HLA-A*0201-PR1 tetramer. The recombinant HLA-A*0201-BSP (BirA substrate peptide) fusion protein as heavy chain and beta(2)-microglobulin (beta(2) m) as light chain were expressed highly as insoluble aggregates in Escherichia coli and then purified with gel filtration, and the final purity reached above 90%. The two subunits were refolded to form an HLA-A*0201-peptide complex by dilution method in the presence of an antigenic peptide PR1, a HLA-A2-restricted peptide from proteinase 3 (aa 169 - 177, VLQELNVTV). Refolded HLA-A*0201-PR1 complex was biotinylated using a BirA enzyme and purified by anion exchange chromatography on a Q-Sepharose (fast flow) column. The extent of reconstitution of the HLA-A*0201-PR1 complex was analyzed by HPLC gel filtration. The refolded and biotinylated products were detected by Western blot and ELISA with monoclonal antibody BB7.2 that recognized the natural conformations of HLA-A2 and streptavidin. The results showed that the refolded complex was composed of HLA-A*0201-BSP aggregate, HLA-A*0201-PR1 complex and beta(2) m, and reconstitution yields of 18% with PR1 was obtained. Refolded HLA-A*0201-PR1 complex could be confirmed by practical immunological method and biotinylated efficiently. It is concluded that the refolding and biotinylation of HLA-A*0201-PR1 complex is successfully obtained. This work provides the basis for the preparation of HLA-A*0201-PR1 tetramer.

[Cloning and expression of HLA-A*0201-BSP].

High-yield production of HLA-A*0201 heavy chain is a prerequisite to the preparation of HLA-A2 tetramer. The present study was aimed to construct the expression vector of recombinant HLA-A*0201-BSP fusion gene for preparing HLA-A2 tetramers. The extracellular region HLA*0201 was cloned by RT-PCR from HLA-A2(+) donor, and a 15-amino acid biotin-protein ligase (BirA) substrate peptide (BSP) for BirA-dependent biotinylation was added to the COOH-terminus of HLA-A*0201 heavy chain. Then the fusion gene was cloned into pBV220 vector at EcoRI and Bam HI sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pBV220-HLA-A*0201-BSP was transformed to the competent cells of E.coli DH5alpha. The results showed that the HLA-A*0201-BSP fusion protein was successfully expressed in the form of inclusion body and amounted to over 28% of total cell proteins via induction at 42 degrees C. After washed with triton X-100 and urea, the inclusion body was dissolved with 8 mol/L urea and then purified with Sepharcyl S-300 HR, and the final purity reached above 90%. It is concluded that the HLA-A*0201-BSP fusion gene was cloned successfully and expressed efficiently in E.coli DH5alpha. This work establishes a convenient approach for purification of large quantity of recombinant HLA-A*0201-BSP. This provides the basis for the preparation of HLA-A2 tetramers.

[Cloning of Human beta2-microglobulin gene and efficient expression in Escherichia coli].

Human beta(2)-microglobulin (beta(2)m) is the light chain of major histocompatibility complex (MHC) class I molecule. High-yield production of this protein is a prerequisite to the preparation of MHC class I tetramer. The present study was aimed to obtain recombinant human beta(2)m expressed in Escherichia coli (E. coli) for preparing MHC class I tetramers. For cloning of human beta(2)m gene, a pair of specific primers was designed based on the published sequence of this gene. A 300 bp specific DNA fragment corresponding to the encoding region of beta(2)m lack of the signal peptide sequence was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was inserted into the IPTG-inducible expression plasmid pET-17b by Nde I and Bam H I sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pET-beta(2)m was transformed to the competent cells of E. coli BL21 (DE3). The results showed that beta(2)m was expressed in the form of inclusion body and amounted to over 32% of total cell proteins after IPTG induction. After washing with triton X-100 and urea, the inclusion body was dissolved with 4 mol/L urea and then purified with Sephacryl S-200 HR, and the final purity reached above 95%. The denatured protein was renatured by dilution method. Western blot assay indicated that the monoclonal antibody against human native beta(2)m could react specifically with the recombinant protein. In conclusion, the human beta(2)m gene was cloned successfully and expressed efficiently in E. coli BL21 (DE3). This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta(2)m. This provides the basis for the preparation of MHC tetramers.