Construction of multi-epitope gene of periodic Brugia malayi and its expression in eukaryotic cells / 中华地方病学杂志

Objective:To construct a eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 of periodic Brugia malayi cysteine protease inhibitor/glyceraldehyde 3-phosphate dehydrogenase (CPI502/GAPDH720) multi-epitope gene and observe its protein expression in Hela cells. Methods:Primers were designed according to the predicted gene sequences of T cell epitopes. The target gene fragment was amplified by reverse transcription (RT)-PCR using plasmid pGEM-T-CPI621 as template. The fragment was cloned into prokaryotic expression plasmid pET-28a(+) to construct prokaryotic expression plasmid pET28a(+)-BmCPI502. The BmCPI502 gene and BmGAPDH720 gene were amplified by RT-PCR, respectively. The gene fragments of pcDNA3.1(+), BmCPI502 and BmGAPDH720 were digested by double enzyme digestion and ligated with the target gene. The eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 was constructed. The recombinant plasmid was transfected into Hela cells and verified by RT-PCR to obtain the desired target bands. The expression product was detected by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).Results:The recombinant plasmid pET28a(+)-BmCPI502 was obtained, and the 502 bp specific fragment was identified by enzyme digestion, which was in line with the expected value; the eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 of the multi-epitope gene was successfully constructed, and the fragment size was in line with the expected value. The eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 was transfected into Hela cells and the recombinant protein was stable expressed. SDS-PAGE analysis showed that the relative molecular weight ( Mr × 10 3) of the recombinant protein was about 50. Conclusions:The eukaryotic expression plasmid of pcDNA3.1(+)-BmCPI502/BmGAPDH720 of periodic Brugia malayi multi-epitope gene is successfully constructed, and the corresponding recombinant protein is obtained in eukaryotic cells. This study has laid a foundation for further study of the purification and biological activity of the recombinant protein.

Polarization of bone marrow-derived macrophages induced by recombinant Trichinella spiralis cysteine protease inhibitors in vitro / 中国血吸虫病防治杂志

Objective To investigate the regulatory role of recombinant Trichinella spiralis cysteine protease inhibitors (rTs-Cys) in induction of polarization of bone marrow-derived macrophages (BMDMs) in vitro. Methods BMDMs were captured and cultured in conditioned medium for 7 days. Then, mature BMDMs were harvested and assigned into four groups. Cells in Group A (negative control) were given 10 ng/mL IFN-γ combined with 100 ng/mL LPS, cells in Group B (positive control) were treated with IL-4 and IL-10 (at 10 ng/mL both), and cells in Group C (recombinant protein alone) were stimulated with 1 μg/mL rTs-Cys, while cells in Group D (protein co-culture) were simultaneously treated with 1 μg/mL rTs-Cys, 10 ng/mL IFN-γ and 100 ng/mL LPS. Cells and culture supernatant were collected 24 hour post-treatment, and the proportions of F4/80+, CD11b+, CD206+ and CD11c+ cells were detected by flow cytometry. The levels of interleukin IL-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-10 and transforming growth factor-β (TGF-β) in the cell culture supernatant were measured by ELISA and the CD86+ and CD206+ phenotypes were identified by immunofluorescent staining. Results Flow cytometry detected no significant difference in the proportion of F4/80+ CD11b+ CD11c+ cells among the four groups (F = 46.184, P < 0.001), and a lower proportion of F4/80+ CD11b+ CD11c+ cells was seen in groups C and D than in group A (all P values < 0.001). There was a significant difference in the proportion of F4/80+ CD11b+ CD206+ cells among the four groups (F = 11.032, P < 0.001), and a greater proportion of F4/80+ CD11b+ CD206+ cells was seen in groups C and D than in group A (all P values < 0.01). Immunofluorescent staining showed higher CD206+ expression and lower CD86+ expression in groups C and D than in Group A. There were significant differences in the IL-6 and (F = 3.950, P < 0.001) and TNF-α (F = 205.827, P < 0.001) levels in the cell culture supernatants among the four groups, and significantly lower IL-6 and TNF-α levels were measured in groups C and D than in Group A (both P < 0.05). There were significant differences in the IL-10 and (F = 8.274, P < 0.001) and TGF-β (F = 13.559, P < 0.01) levels in the cell culture supernatants among the four groups, and greater IL-10 and TGF-β levels were measured in Group C than in Group A (both P values < 0.01). In addition, the TGF-β level was significantly higher in Group D than in Group A (P < 0.05); however, there was no significant difference in the IL-10 level between groups D and A (P > 0.05). Conclusion rTs-Cys may induce the polarization of BMDMs to antiinflammatory M2 macrophages in vitro and inhibit the activation of M1 macrophages.

Expression of cystatin A in esophageal squamous cell carcinoma and its clinical significance / 国际生物医学工程杂志

Objective To investigate the expression and clinical significance of cysteine protease inhibitor A(CSTA) in esophageal squamous cell carcinoma. Methods A total of 59 patients with esophageal cancer who underwent esophagectomy or endoscopic submucosal tumor dissection were enrolled. The esophageal squamous cell carcinoma and normal esophageal tissues were collected and clinical pathological data were collected. The expression of CSTA mRNA and protein in cancer tissues and normal tissues was determined by real-time quantitative fluorescent polymerase chain reaction (RTFQ-PCR) and immunohistochemistry. The expressions of CSTA and Ki-67 mRNA and protein in cancer tissues and normal tissues were determined by RTFQ-PCR and Western Blot. Results Compared with normal, the expression of CSTA mRNA and protein in esophageal squamous cell carcinoma tissues was significantly lower, and the difference was statistically significant (all P<0.05). In squamous cell carcinoma, the CSTA-positive expression is often associated with Ki-67 expression, whereas normal esophageal tissue has CSTA expression but no Ki-67 expression. Squamous cell carcinoma with CSTA-positive expression had higher tumor pT stage and tumor grade (all P<0.05). Conclusions The expression of CSTA in cancer tissues of patients with esophageal squamous cell carcinoma is significantly lower than that in normal tissues. The CSTA-positive expression in esophageal squamous cell carcinoma is related to the pT clasification and tumor grade. The CSTA test for esophageal squamous cell carcinoma can provide a basis for clinical treatment.

Effect of cystatin from Schistosoma japonicum on DSS-induced ulcerative colitis in mice / 中国血吸虫病防治杂志

Objective To investigate the effect of cysteine protease inhibitor derived from S chistosoma japonicum(SjCys-tatin)on dextran sodium sulfate(DSS)-induced acute ulcerative colitis in mice.Methods Eighteen C57BL/6 mice were ran-domly divided into three groups:a control group treated with PBS(Group A),a DSS-induced-colitis group treated with PBS(Group B),and a DSS-induced-colitis group treated with SjCystatin(Group C).Colitis was induced in mice by giving 3％DSS orally for 7 days.During this period,the mice were daily injected with 10μg of SjCystatin or PBS only as a control intraperitone-ally.The mice were monitored daily for their clinical manifestations and given scores based on disease activity index(DAI).The severity of colonic inflammation was monitored by the macroscopic score and pathological change.The cytokine profile including TNF-α,IL-4,IL-6 and IL-10 in the supernatants of colon homogenate was detected by ELISA.Results Compared with Group A(0.50 ± 0.28),the DAI score increased significantly in Group B(9.30 ± 1.30)(F=86.86,P<0.01),with remarkable path-ological damages seen in colon tissues.and the levels of TNF-α and IL-6 were(321.33±67.01)and(403.58 ±180.51)pg/mL.The DAI score significantly reduced in Group C(6.67±1.57)as compared to Group B(F=86.86,P<0.01),with improve-ments in the macroscopic and microscopic pathology in mouse colon specimens.As compared to Group B,the levels of TNF-α [(188.14 ± 40.14)pg/mL] and IL-6 [(209.71 ± 48.47)pg/mL] significantly decreased(F=17.46 and 9.89,both P<0.01).Con-clusion SjCystatin has a significantly inhibitory effect for alleviating DSS-induced acute ulcerative colitis in C57BL/6 mice.

Evaluation of diagnostic indicators in chronic renal failure / 国际检验医学杂志

Objective To evaluate the clinical values of endogenous creatinine clearance rate(Ccr) ,serum creatinine (SCr) ,urea nitrogen(Urea) ,serum cystatin C(s‐Cys‐C) ,serum retinol binding protein(s‐RBP) ,Urine total protein (u‐Pro) ,urine albumin and creatinine ratio(u‐Alb/Cr) ,urine RBP(u‐RBP) ,urine Cys‐C(u‐Cys‐C) ,u‐NAG and et al in the diagnosis of chronic renal failure (CRF) ,find suitable and effective detection combinations to increase the diagnostic accuracy of CRF .Methods SCr ,Urea ,s‐Cys‐C , s‐RBP ,u‐Pro ,u‐Alb/Cr ,u‐RBP ,u‐Cys‐C ,u‐NAG were detected respectively in 206 hospitalized patients and Ccr values were calcu‐lated at the same time .By using Excel and SPSS19 .0 softwares ,the data were analysed .Combined detections included two and four items combined detections .Results Youden index(YI) of serum Cys‐C was 0 .59 .Area under the curve in the receiver operating characteristic(ROC) of Cys‐C was 0 .872 which was the highest of all the single detection items .Combined detection of SCr and s‐Cys‐C got the highest YI (0 .60) .Combination of four items(Urea ,SCr ,s‐RBP ,s‐Cys‐C) had the highest positive predictive value (100 .00% ) .Combination of u‐RBP and u‐Cys‐C had the highest negative predictive value(100 .00% ) .Conclusion Combined detec‐tion was more favorable for CRF diagnosis .Combination detection of SCr and s‐Cys‐C was the most valuable detection for the diag‐nosis of CRF .Among single item detections ,s‐Cys‐C detection had better sensitivity and specificity ,and diagnostic efficiency than other detection items .U‐RBP and u‐Cys‐C could be used to exclude renal impairment due to its noninvasive sampling .

THE EFFECT OF CYSTEINE PROTEASE INHIBITOR OF SNAKE VENOM(SV-CYSTATIN) ON MOUSE MELANOMA CELLS INVASION IN VITRO AND IN VIVO / 解剖学报

Objective To investigate the effect of cysteine protease inhibitor of snake venom(sv-cystatin) on invasiveness and metastasis of melanoma cells.Methods The recombinant plasmid pcDNA3.1/sv-cystatin was constructed and transferred into mouse melanoma cell line B16F1 by lipofection technology.The positive clones were screened by G418 and the sv-cystatin expression was detected by RT-PCR and Western blotting.The ability of tumor cell invasion was identified by Boyden chamber in vitro and tumor invasion animal model in vivo.The ability of tumor cell proliferation and adhension was also determined by MTT assay.Results Expression of sv-cystatin was detected in B16F1/sv-cys cells after gene-transfection.Transfection of sv-cystatin significantly decreased the invasion and migration of B16F1/sv-cys cells along with obviously inhibited the experimental pulmonary metastasis in vivo,but the proliferation and adhension capacity of B16F1/sv-cys cells had no change.Conclusion Transfection of sv-cystatin gene into mouse melanoma cell line results in the suppression of the invasion potential in vitro and in vivo.