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This study was aimed to investigate the SLC25A38 expression in pediatric patients with acute lymphoblastic leukemia (ALL) and its clinical significance. A total of 23 newly diagnosed ALL pedictric patients were enrolled in test group, 10 pediatric patients with non-hematologic malignancies were selected as control group. The expression in protein and mRNA levels of SLC25A38 were detected by Western blot and real-time PCR respectively. The results showed that the SLC25A38 protein was positive in 8 of 23 pediatric ALL patients (34.78%), while no positive case was found in 10 controls. The relative expression level of SLC25A38 mRNA was 0.4673 ± 0.05344 in SLC25A38-protein positive group of ALL patients, while that was 1.296 ± 0.2517 in SLC25A38-protein negative group of ALL patients. The expression level of SLC25A38 mRNA in SLC25A38-protein positive group was significantly lower than that in negative group (P = 0.001) . No statistically significant difference was found in comparison of SLC25A38-protein negative group of ALL patients with the control group (P = 0.1097). The analysis of clinical data showed that there were significantly differences in sex, immunophenotype, initial peripheral white blood cell count and LDH between the SLC25A38-protein positive and SLC25A38-protein negative groups (P < 0.05). It is concluded that as a novel protein, SLC25A38 highly expressed in pediatric ALL patients, indicating that SLC25A38 may serve as a molecular marker and potential therapeutic target for acute lymphoblastic leukemia in children.

Aim: To perform a comprehensive investigation into the potential correlation between circulating myeloid-derived suppressor cells (MDSCs) and Th17 cells in esophageal cancer (ECA)., Methods: A total of 31 patients newly diagnosed with ECA and 26 healthy subjects were included in the current study. The frequencies of MDSCs and Th17 cells in peripheral blood were determined by flow cytometry. The mRNA expression of cytokines, arginase 1 (Arg1) and inducible NO synthase (iNOS) in peripheral blood mononuclear cells (PBMCs) and plasma Arg1 were assessed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively., Results: There was an increased prevalence of MDSCs in the peripheral blood from ECA patients (15.21% ± 2.25%) when compared with healthy control (HC) (1.10% ± 0.12%, P < 0.0001). The plasma levels of Arg1 in ECA patients were significantly higher than those in HC (28.28 ± 4.10 ng/mL vs 9.57 ± 1.51 ng/mL, P = 0.0003). iNOS mRNA levels in the peripheral blood of ECA patients also showed a threefold increase compared with HC (P = 0.0162). The frequencies of Th17 cells (CD4⁺IL-17A⁺) were significantly elevated in ECA patients versus HC (3.50% ± 0.33% vs 1.82% ± 0.19%, P = 0.0001). Increased mRNA expression of IL-17 and ROR-γt was also observed in ECA patients compared with HC (P = 0.0041 and P = 0.0004, respectively), while the mRNA expression of IL-6 and tumor necrosis factor-α (TNF-α) showed significant decreases (P = 0.0049 and P < 0.0001, respectively). No obvious correlations were found between the frequencies of MDSCs and Th17 cells in the peripheral blood from ECA patients(r = -0.1725, P = 0.3534). Arg1 mRNA levels were positively correlated with levels of IL-6 (r = 0.6404, P = 0.0031) and TNF-α (r = 0.7646, P = 0.0001). Similarly, iNOS mRNA levels were also positively correlated with levels of IL-6 (r = 0.6782, P = 0.0007) and TNF-α (r = 0.7633, P < 0.0001)., Conclusion: This study reveals the relationship between circulating MDSCs and Th17 cells, which may lead to new immunotherapy approaches for ECA based on the associated metabolites and cytokines.

Aim: To investigate the effect of IL-17 on the expression of collagen I/III in cardiac fibroblasts and analyze its molecular mechanism., Methods: Cardiac fibroblasts were isolated from 7-14-day-old BALB/c mice and cultured in DMEM with 10% fetal bovine serum (FBS). The cells were collected after IL-17 treatment for 0, 24, 48, 72 h. IL-17 receptors on cardiac fibroblasts were detected by PCR; the collagen I/III expression was analyzed by immunofluorescence; the PKCβ, Erk1/2, NF-κB phosphorylation were investigated by Western blotting., Results: IL-17RA/C was expressed on cardiac fibroblasts; after 24 h of IL-17 stimulation, the collagen I/III expression obviously increased; Western blotting showed that PKCβ, Erk1/2 and NF-κB were phosphorylated on 30, 45, 45 min, respectively., Conclusion: IL-17 could induce the expression of collagen I/III in cardiac fibroblasts, which might be related with PKCβ-ERK1/2-NF-κB phosphorylation.

Aim: To investigate whether cardiac fibroblasts (CFs) treated by LPS can actively secrete high-mobility group box protein 1 (HMGB1) and to analyze the correlation between HMGB1 releasing and the accumulation of collagen type I , III ., Methods: CFs were isolated from the heart of 7-14-day-old BALB/c mice and cultured in DMEM with 10% fetal bovine serum (FBS). We collected the CFs and cell supernatants after treated by LPS for 0, 6, 12, 24, 36, 48 h, respectively. The mRNA and protein expression levels of HMGB1, collagen 1a1 (col1a1) and collagen 3a1 (col3a1) in CFs after LPS stimulation were detected by RT-PCR and Western blotting, respectively. The intracellular localization of HMGB1 in treated CFs was investigated by immunofluorescence., Results: After 0-6 h of LPS stimulation, the mRNA levels of HMGB1, col1a1, col3a1 had no significant changes; but increased obviously at 12, 24, 36, 48 h. HMGB1 was found in the cell supernatant by Western blotting after 24 h LPS stimulation, and its expression decreased following the first rise in CFs. Meanwhile, immunofluorescence showed HMGB1 translocation from nucleus to cytoplasm. The levels of col1a1 and col3a1 were up-regulated in CFs after stimulation., Conclusion: LPS can induce HMGB1 translocation from nucleus to cytoplasm and across cellular membrane to the outside of CFs at a time-dependent manner. Col1a1 and Col3a1, which are closely associated with myocardial fibrosis, were obviously up-regulated by LPS stimulation, which indicates that actively released HMGB1 might contribute to myocardial fibrosis following the endotoxin induced-sepsis.

Aim: To construct eukaryotic co-expression vector of Porphyromonas gingivalis outer membrane protein ragB and mouse glucocorticoid-induced tumor necrosis factor receptor ligand (mGITRL) and to analyze its immunogenicity in vivo., Methods: The ragB gene was obtained from pMD18-T-ragB, and then cloned into the eukaryotic expression vector pIRES and pIRES-mGITRL, respectively. The eukaryotic expression vectors: pIRES-ragB and pIRES-ragB-mGITRL were identified by double enzyme digestion and DNA sequencing, then transfected into COS7 cells by Lipofectamine(TM);2000. The expressions of ragB or mGITRL in COS7 cells were detected by Western blotting. The mice were immunized with the recombinant pIRES-ragB-mGITRL plasmid. The serum antibody level was determined by ELISA., Results: pIRES-ragB and pIRES-ragB-mGITRL plasmids were successfully constructed. Western blotting showed that the targeted gene was over-expressed in COS7 cells and skeletal muscle cells, respectively. The high titers of antibodies against RagB were detected in mouse serum., Conclusion: The construction of pIRES-ragB-mGITRL co-expression vector provides the experimental basis for Porphyromonas gingivalis vaccine research, prevention and treatment of periodontitis.

Aim: To explore the infiltration pathogenesis of CD4(+);T cells following the spinal nerve ligation., Methods: Healthy adult male SD rats were randomly divided into the spinal nerve ligation group (Tx), sham operation group (S), control group (C). the 50& mechanical paw withdrawal threshold ( 50&MWT ) was determined by up-down method; CD4(+);T cells infiltration was assessed by FACS; the mRNA levels of CCL2, CCL5 and CXCL10 were quantitated by RT-qPCR; serum cytokines were tested by ELISA kits., Results: After 3 days since operation, 50&MWT of Tx group was significantly reduced (P<0.01) comparing with S group, C group; on day 14, 50&MWT was up to the minimum value; whereas S group and C group were no difference (P>0.05). After 7 days since operation, CD4(+);T cells infiltration into lumbar segments of the spinal cord in the Tx group increased significantly (P<0.01), and the CCL2, CCL5mRNA expression increased (P<0.05); on day 14, the CD4(+);T cells infiltration in Tx group was higher than S group, C group; but there was no statistical significance. On day 7 and 14 days, serum levels of cytokines were no difference in the three groups., Conclusion: Following spinal nerve ligation, high expression of chemokine promoted peripheral CD4(+);T cells to infiltrate into spinal cord; and the infiltrated CD4(+);T cells maintained the neuropathic pain.

Objective: To observe the expression of Toll-like receptor 8 (TLR8) in human cervical cancer cell-line HeLa cells, and the effects of TLR8 agonist CL075 on the survival and proliferation of HeLa cells., Methods: PCR and RT-PCR were used to detect the expression of TLR8 in 13 cancer cell lines, and the expression of COX-2, Bcl-2, VEGF mRNA in the HeLa cells stimulated by TLR8 agonist CL075 were also measured by RT-PCR. Immunofluorescence technique was used to determine the exact location of TLR8 in the cells. The percentage of viable cells was determined by trypan blue exclusion after the HeLa cells were stimulated with TLR8 agonist CL075 (0.1 µg/ml, 0.5 µg/ml, 1.0 µg/ml, 2.5 µg/ml), and cell cycle and apoptosis were analyzed by flow cytometry, and the proliferation was measured by MTT., Results: Compared with the other cancer cell lines, the expression of TLR8 in HeLa cells was the highest (703.7 ± 20.6). After stimulation by CL075, the cells had a remarkable increase of the percentage of cells in G(2)/M + S phases. In the control group, the percentage of cells in G(2)/M +S phases was (39.02 ± 2.33)%, whereas after stimulated with 1.0 µg/ml CL075, the percentage of cells in G(2)/M + S phases reached the highest ratio (57.67 ± 1.73)%, and the percentage of cells in G(2)/M + S phases had a less decrease after 2.5 µg/ml CL075 stimulation and the percentage was (56.14 ± 3.73)%. After the CL075 treatment, there was no significant changes of apoptosis compared with that of the control cells (P > 0.05), but after DDP treatment the apoptosis had a significant change (P < 0.01). After stimulation by 1.0 µg/ml CL075 for 24 h, no significant difference (P > 0.05) was found by MTT test, but a significant difference was found at 48 h and 72 h (P < 0.01). An increased expression of COX-2, Bcl-2 and VEGF mRNA was observed in HeLa cells after stimulation by TLR8 agonist CL075 for 24 h and 48 h (P < 0.05)., Conclusions: Expression of TLR8 is significantly increased in HeLa cells. The proportion of cells at different phases has a significant change after CL075 stimulation, which may up-regulate the proliferation of HeLa cells. These data suggested that TLR8 agonist may influence the tumor development and TLR8 may become a potential target in the treatment for cervical cancer.

Toll-like receptors (TLRs) are found on the membranes of pattern recognition receptors and not only play important roles in activating immune responses but are also involved in the pathogenesis of inflammatory disease, injury and cancer. Furthermore, TLRs are also able to recognize endogenous alarmins released by damaged tissue and necrosis and/or apoptotic cells and are present in numerous autoimmune diseases. Therefore, the release of endogenous TLR ligands plays an important role in initiating and driving inflammatory diseases. Increasing data suggest a role for TLR signaling in rheumatoid arthritis, which is an autoimmune disease. Although their involvement is not comprehensively understood, the TLRs signaling transducers may provide potential therapeutic targets.

Aim: To explore the effect of lincomycin (lin) on the immune function of dendritic cell (DCs) line DC2.4., Methods: Three experimental groups, namely, DC2.4 cells group, DC2.4 Cells+LPS group and DC2.4 cells +LPS +lin group were established (LPS and lin were 500 ng/mL). The morphological changes in each group were observed under inverted microscope. The MHC class II , CD86 and CD80 on the DC2.4 cells were detected by flow cytometer. The effects of lipopolysaccharide (LPS) and LPS +lin on the DC2.4 cells immuno-stimulatory capacity were evaluated by allogeneic mixed leukocytes reaction (MLR) between DC2.4 cells and T cells. ELISA was adopted to quantitate the level of IFN-γin the supernatant of cultured DC2.4 cells and T cells in each group., Results: DC2.4 cells showed typical morphology of immature DCs. When stimulated with LPS or LPS combined with lin, DC2.4 cells exhibited typical maturate DCs modality. LPS of 500 ng/mL could significantly up-regulate the expression of MHC class II , CD86 and CD80 on DC2.4 cells and also augment stimulatory action of DC2.4 cells on T cells proliferation and secretion of IFN-γ. However, compared with LPS alone, the treatment of lin (500 ng/mL) combined with LPS down-regulated the immmuno-regulation function of DC2.4 cells., Conclusion: Lin can partly inhibit the immuno-regulation function of maturate DC2.4 cells.

Aim: To explore the effects of oral type II collagen (CII) on the morphology, cytokine expressions of Peyer's patches(PP)and the levels of serum specific IgG, IgA, IgM., Methods: CII was orally administrated to Kunming mice in continuous 10 days at different dosage. The CII or adjuvant immunization was given at 11 d and 21 d. The blood and Peyer's patches were collected at 11 d, 21 d and 31 d. The PP hyperplasy was observed by light microscope after HE staining. The fluorescent real time RT-PCR was used to detect the mRNA expressions of IL-17, TNF-α, IFN-γ and TGF-β1 in PP lymph node. The serum specific IgG, IgA, IgM contents were detected by ELISA., Results: After oral administration of CII for 10 d, the PP lymph node hyperplasia was active and the cap-shape structure could be seen clearly in high dose group, the serum IgA could be detected, the gene expressions of IL-17, TNF-α and IFN-γ were inhibited. After the CII initial immunity, the IgA, IgM, IL-17 levels were descended and TGF-β1 level was increased in the experiment groups as compared with control group(P < 0.05 or P < 0.01). After the CII booster, IgA was notably increased in high dose group(P < 0.05), in experiment groups IgM was still suppressed (P < 0.05 or P < 0.01) and TGF-β1 levels were higher than control group(P < 0.05). In adjuvant immunization groups the cytokine expressions were similar to CII immunization groups, the differences of serum specific IgG, IgA, IgM could not be observed as compared with control group., Conclusion: The oral administration of CII can increase the serum specific IgA and suppress the gene expressions of IL-17, TNF-α, IFN-γ in the Peyer's patches. It can still have inhibitory action on the serum specific IgA, IgM and IL-17 gene expressions after CII immunization. The results indicate that the changes of the serum specific antibodies and cytokine gene expressions play an important role on treating rheumatoid arthritis by oral CII to induce immune tolerance.

Aim: To express the mouse glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) protein with Bac-to-Bac baculovirus expression system., Methods: GITRL gene was obtained by double digestion using EcoR I and Sal I and cloned into the baculovirus transfer vector pFastBacHTA. Then the pFastBacHTA was transformed into competent 10BacTM E.coli cells. The transposition occurred between pFastBacHTA and bacmid and a recombinant bacmid was obtained. The positive clones were picked out and the recombinant bacmid was isolated, and then complete transfected into Tn cells for producing complete recombinant baculovirus. The baculoviral stock was amplified and the GITRL protein was expressed., Results: The presence of GITRL gene containing the recombinant bacmid was verified by PCR and gene sequencing. The cytopathic effect (CPE) displayed in the transfected Tn cells assumed that the transfection was successful. Western blot analysis showed that the molecular weight of mGITRL protein was about 20 KD., Conclusion: The GITRL protein is expressed successfully with Bac-to-Bac baculovirus expression system, which may lay the foundation for further study on biological activity and function of GITRL protein.

Aim: To transfect Hlx into mouse dendritic cell line DC2.4 and observe the effect of hlx on function of dendritic cells., Methods: The eukaryotic expression vector PIRES2-EGFP/Hlx was transfected into DC2.4 by liposomes. The transfection efficiency was identified through FACS. RT-PCR and Real-time PCR were used to test the transcription level of Hlx in DC2.4. Forty-eight hours after transfection, DC2.4 cells were studied for cytokine production, cell phenotype, phagocytosis, unilateral mixed lymphocyte reaction., Results: The pIRES2-EGFP/Hlx vector was transfected into DC2.4 with the transfection efficiency of up to 60%. Highly expressed Hlx in DC2.4 increased the expression of maturation makers including CD80 and CD86, and major histocompatibility complex-II. Functional assay showed that over-expression of Hlx in DC2.4 increased the interleukin-12 transcription and decreased DC endocytosis. The Hlx modified DC2.4 highly expressed IL-10 and TGF-β at the same time. Furthermore, it was shown that in a unilateral mixed lymphocyte reaction model, Hlx modified DC2.4 inhibited proliferation of lymphocytes., Conclusion: Transient over-expression of Hlx in DC2.4 promotes DC2.4 maturation and up-regulates IL-12, IL-10 and TGF-β expression. However, the Hlx modified DC2.4 cells functionally appear as regulatory dendritic cells.

Aim: To express human HMGB1 B box protein and obtain monoclonal antibodies (mAbs) against HMGB1 B box for further study of the function of human HMGB1 protein., Methods: pET28-HMGB1 B box plasmid transfected the DH5α, then expressed. And the extracted protein was purified by protein purification system. BALB/c mice were immunized with recombinant human HMGB1 B box protein. Hybridoma cell lines secreting mAb against human HMGB1 B box protein were screened by ELISA and subcloning approach. The characteristics of these mAbs were identified by ELISA and Western blot., Results: Two hybridoma cell lines (1D2F4E3 and 2D4E3A2) stable secreting specific mAbs were successfully obtained.Western blot exhitited the two mAbs binded specifically to human HMGB1 B box protein. The immunoglobulin (Ig) class of two mAbs belonged to IgG, their titers were 1×10(6);, and the A(450); of mAb1D2F4E3, 2D4E3A2 were 0.324±0.093, 0.296±0.085, respectively., Conclusion: Two of high specificity mAbs against human HMGB1 B box protein have been successfully prepared, which laid the foundation for further study of biological function of human HMGB1 protein.

Aim: To construct recombinant adenovirus vector pAdEasy-GFP-GITRL and detect the viral titer., Methods: GITRL gene was obtained by double digestion using Bgl II and Sal I, and cloned into the baculovirus transfer vector(pAdtrack-CMV), then the recombinant adenovirus vector (pAdtrack-CMV-GITRL) was digested by restrictive endoenzyme Pme I. The linear recombinant adenorirus vector and pAdEasy-1 were cotransfected into HEK293 cells by co-precipitate of calcium phosphate. Recombinant adenovirus was packaged and purified in HEK293A cells., Results: Recombinant adenovirus vector pAdEasy-GFP-GITRL was constructed successfully and high titer of recombinant adenovirus was obtained (2.0 x 10⁹ pfu/mL). Western blotting analysis also revealed the expression of GITRL by recombinant adenovirus vector., Conclusion: The construction of recombinant adenovirus vector pAdEasy-GFP-GITRL and recombinant adenovirus will facilitate the potential GITRL gene therapy.

Objective: To detect the expression levels of transcription factors and associated cytokines of Th17 and Treg cells in peripheral blood mononuclear cells (PBMC) of patients with gastric cancer, and explore the possible pathological mechanism of these cells involved in the development of gastric cancer., Methods: The mRNA levels of RORgammat, FoxP3 in PBMC were determined by quantitative real-time PCR (QRT-PCR) from 57 patients with gastric cancer, 31 patients with benign gastric illness and 40 healthy people. The concentration of IL-17, IL-23, TGF-beta, IL-10 in plasma were detected by enzyme linked immunosorbent assay (ELISA)., Results: Compared with healthy volunteers, patients with gastric cancer showed higher levels of RORgammat and FoxP3 in PBMC (P < 0.05). The ratio of FoxP3/RORgammat in gastric cancer group was higher than that in the volunteer group and benign gastric illness group (P < 0.05). The ratio of FoxP3/RORgammat was higher in advanced disease than early disease (P < 0.05). The expressions of IL-17, IL-23, TGF-beta and IL-10 were higher in patients with gastric cancer than that in healthy volunteers (P < 0.05). In addition, The expression of TGF-beta and IL-10 were significantly increased in the advanced disease group than that in the early group (P < 0.05), but IL-17 and IL-23 was not significantly changed between the two groups (P > 0.05)., Conclusion: There are higher levels of Th17 and Treg cells in gastric cancer patients, and it also shows a persistent predominant tendency of Treg cells and a reduced tendency of Th17 cells in advanced disease. Detecting the expression of Th17/Treg transcription factor and related cytokines would contribute to the diagnosis and prediction of the disease development and prognosis.

Aim: To explore the expression of Toll-like receptor 8(TLR8)in rheumatoid arthritis induced by chicken II collogen in mice and analyze the relation of TLR8 to IL-10, IL-17A and IL-1beta., Methods: Twelve DBA/1J mice were randomly divided into model group(T) and negative group(NC). The paw swelling was seen at the day of 27 post the first immunization with chicken II collogen. The serum TNF-alpha was detected by ELISA and inflammatory cell infiltration was examined by HE. Real-time PCR was used to analysis the expression of IL-10, IL-17A, IL-1beta and TLR8 in spleen mononuclear cells., Results: Severe inflammation was detected in model group mice by histological analysis, which was not seen in negative group. The serum TNF-alpha in model group was enhanced compared with that in negative group(P<0.01). The expression levels of IL-10, IL-17A, TLR8 and IL-1beta in spleen mononuclear cells from model group were increased compared with those from negative group(P<0.01, P<0.05). Statistical analysis showed that there was negative correlation between the expression of TLR8 and IL-10(P<0.01), and there was no correlation between the expression of TLR8 and IL-17A or IL-1beta(P>0.05)., Conclusion: The expression of TLR8 was increased in rheumatoid arthritis-mice induced by chicken II collogen, and there was negative correlation between the expression of TLR8 and the expression of IL-10.

Aim: To construct the MyD88-Pseudomonas aeruginosa epitope vaccine and study its expression in eukaryotic cells., Methods: To design and synthesize an epigene containing three B cell epitopes of OprF and one foreign "promiscuous" T cell epitope by overlapping extension PCR. tPA signal encoding sequence was amplified by PCR and then it was inserted into the 5' terminus of the epigene to construct tPA-OprF. tPA-OprF and MyD88 were cloned into the expression vector pIRES and the recombinant plasmid pIRES-tPAOprF-MyD88 was constructed. The recombinant plasmid was transfected into COS-7 cells by electroporation. The expression protein of tPA-OprF and MyD88 was detected by Western blot., Results: The recombinant plasmid pIRES-tPA-OprF-MyD88 was successfully constructed. Western blot analysis indicated the tPA-OprF fusion protein was expressed in supertanant of COS-7 cells and MyD88 protein in COS-7 cells., Conclusion: The recombinant plasmid pIRES-tPA-OprF-MyD88 has been successfully constructed and tPA-OprF and MyD88 protein can be highly expressed in transfected cells. It may be used as a potential candidate of preventive vaccine of pseudomonas aeruginosa.

Aim: Runx3, a type of Runt family member, plays an important role in immune regulation. In this study, we cloned and analyzed the cDNA encoding human Runx3 from T lymphocyte, expressed Runx3 protein in E.coli system, and studied the relation between Runx3 and some immune disorders or tumors., Methods: The CD8(+) T were isolated from human peripheral blood with MACS, Runx3 cDNA was amplified by RT-PCR and cloned into pMD19-T vector, and recombinant was transformed into competent cells DH5alpha and recombinant sequencing were performed. The identical was subcloned into pQE30 vector and expressed in E.coli M15. The fusion protein was identified by Western blot., Results: The 1,248 bp fragment amplified by RT-PCR was the same as the anticipated one in size and encodes 415 amino acids. Runx3 protein was gained., Conclusion: Human Runx3 gene was cloned and expressed in E.coli system successfully, which brought a foundation for further research on its biological function.

Aim: To express and purify the extracellular region of human glucocorticoid-induced tumor necrosis factor (hGITR(aa27-165)), and to prepare and identify the polyclonal antibody (pAb) against this fusion protein., Methods: The 429 bp DNA sequence of hGITR(aa27-165) was obtained from pGEM T-hGITR by PCR and then it was inserted into pQE30 plasmid to construct the prokaryotic expression plasmid pQE30-hGITR(aa27-165). pQE30-hGITR(aa27-165) was transformed into E.coli. The target fusion protein was expressed with the induction of Isopropyl beta-D-1-thiogalacto- pyranoside (IPTG) and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hGITR(aa27-165) was obtained from the rabbits immunized with hGITR(aa27-165). The titer of pAb was detected by ELISA and the specificity of pAb was identified by Western blot., Results: The prokaryotic expression plasmid pQE30-GITR(aa27-165) was constructed successfully. The culture condition in which the target fusion protein was highly expressed was found out: the optimal concentration of IPTG was 0.5 mmol/L, the culture temperature was 30 degree centigrade and the culture time was 6 h. The GITR(aa27-165) fusion protein was effectively expressed in E.coli as inclusion body. Soluble protein was obtained through denaturation and refolding procedure. The fusion protein was purified by Profinity IMAC Ni-Charged Resin affinity column with above 90% purity. The anti-GITR(aa27-165) pAb was prepared by immunizing the rabbits with the purified target fusion protein. ELISA showed the titer of pAb was 1:1.6 x 10(5). Western blot analysis confirmed anti-GITR(aa27-165) pAb was of good specificity., Conclusion: The fusion protein hGITR(aa27-165) with high purity has been obtained and the anti-hGITR(aa27-165) pAb with high titer and good specificity has been prepared. Our study may be conductive to further research into the molecular mechanism of action between human GITR and GITRL.

Aim: To construct the recombination adeno-associated virus (rAAV) carrying murine forkhead box P3 (FoxP3) gene, and then detect the expression in NIH3T3 cells., Methods: The recombination adeno-associated virus (rAAV) vector containing internal ribosome entry site which could coordinate the expression of FoxP3 gene and enhance green fluorescent protein gene was constructed. HEK293 cells were transfected by the transfection cocktail containing the constructed rAAV vector, trans plasmid and helper plasmid by the calcium phosphate method. The infectious rAAV carrying FoxP3 gene (rAAV/FoxP3) suspensions were harvested and purified by heparin affinity chromatography. The purity and titre of rAAV were detected by SDS-PAGE electrophoresis and real-time PCR, respectively. NIH3T3 cells were transfected by rAAV, the transfection efficiency was analyzed by flow cytometry and FoxP3 mRNA expression was detected by real-time PCR., Results: The titre of the infectious rAAV was 5x 10(12) vg/mL by real-time PCR analysis. After NIH3T3 cells were transfected, the transfection efficiency of the infectious rAAV/FoxP3 was up to 92.88% and high level of FoxP3 mRNA was detected., Conclusion: rAAV carrying FoxP3 gene was successfully constructed and expressed in NIH3T3 cells, which will be of benefit to further researches on the function of FoxP3.