Objective To construct human secreted apoptosis-related protein 1 (SARP1) gene yeast two-hybrid bait vector so as to study the biological functions of the SARP1 gene in the scar tissue. Methods The target gene from SARP1 gene full-length DNA segment was amplified by PCR, the upstream and downstream primers of the SARP1 gene with restriction enzymes Nde I and Sal I were designed. pGBKT7-SARP1 recombination plasmid was constructed by ligating the vector and the PCR production and identified by PCR and sequencing. Further more, pGBKT7-SARP1 was transformed into competent AH109 which contained kanamycin for selecting positive clones and screened the positive clony on the plate of SD/-Trp. The toxicity and transcriptional activation were tested by the phenotype assay. Results SARP1 was amplified and cloned into pGBKT7 successfully, SARP1 gene sequence in recombinant plasmid pGBKT7-SARP1 was verified by gel electrophoresis and DNA sequencing analysis. The sequence of inserted SARP1 gene was the same as the corresponding sequence found in GenBank database. The recombinant pGBKT7-SARP1 plasmids and empty pGBKT7 vector could form white colonies on SD/-Trp plates and none could survive on SD/-Leu plates. Conclusion The recombinant pGBKT7-SARP1 gene yeast two-hybrid bait vector is successfully constructed.
目的 观察秦绵祛风胶囊对鹌鹑高尿酸血症的影响。 方法 通过喂饲用酵母配制的造模饲料造成鹌鹑高尿酸血症模型，设秦绵祛风胶囊高、中、低3个剂量组并以苯溴马隆为阳性对照，在造模的同时连续灌胃药35 d，检测血中黄嘌呤氧化酶、尿酸、血尿素氮和三酰甘油及粪便中尿酸含量。 结果 模型动物血清中黄嘌呤氧化酶、尿酸和三酰甘油水平及粪便中尿酸含量较正常对照组明显升高。秦绵祛风胶囊各剂量组均可显著降低鹌鹑血清中的尿酸和三酰甘油水平，同时升高粪便中尿酸含量，对血清中黄嘌呤氧化酶活性影响不大。 结论 秦绵祛风胶囊具有降脂降尿酸的功能，其机制可能是通过提高动物排泄尿酸的能力，从而降低血中尿酸的含量。
目的 探讨假丝酵母菌引起泌尿道医院感染的特点，提出相应的护理干预对策，为医院泌尿道感染的预防控制提供依据。 方法 2011年6月1日－2013年3月31日，在住院患者中开展泌尿道假丝酵母菌的目标监测，分析感染的现状及相关的危险因素，并于2012年12月1日起，采取针对性的护理干预措施，并评估干预效果。 结果 共发现假丝酵母菌引起的泌尿道感染56例，占总泌尿道感染的40.29%。通过采取护理干预措施，使假丝酵母菌引起的泌尿道感染从3.17例/月降为0.50例/月（P＜0.05）。 结论 采取相应有效的护理干预措施，对减少假丝酵母菌泌尿道医院感染，保障医疗安全，具有重要的意义。
Objective To construct yeast eukaryotic expression vector carrying human endostatin (ES) cDNA. Methods The functional fragment of endostatin gene in human hepatic tissue was amplified by using RT-PCR technology, and cloned into yeast pPIC9 expression vector. The positive clone was sequenced by using automatized sequencer. Results The endostatin cDNA was successfully cloned. The positive ES clone gene in pPIC9 expression vector was sieved, and its coding sequence was identified to be as same as the previously reported sequence. Conclusion The successful construction of ES gene in pPIC9 expression vector using molecular biological method maybe helpful for the high expression of ES protein, which may lay the foundation for the treatment of malignant tumor through anti-angiogenesis appoach.
Human lymphocyte function-associated antigen 3 (hLFA3) has been identified as an important T cell accessory molecule. Rhesus monkeys (Macaca mulatta) have been widely used as animal models for human immune disorders. Due to the species-specificity of immune system, it is necessary to study M. mulatta LFA3 (mmLFA3). In this study, the gene encoding mmLFA3 CD2-binding domain (mmLFA3Sh) was amplified by polymerase chain reaction (PCR) and genetically fused to human IgG1 Fc fragment in pPIC9K to construct the expression plasmid pPIC9K-mmLFA3Sh-Ig. Approximately 3-4 mg mmLFA3Sh-Ig protein was recovered from 1 L of inductive media, and mmLFA3Sh-Ig produced by the P. pastoris can bind to the CD2 positive cells, and suppress the monkey and human lymphocytes proliferation induced by Con A and alloantigen in a dose-dependent manner. These results suggested that mmLFA3Sh-Ig might be used as a novel tool for pathogenesis and experimental immunotherapy of Rhesus monkey immune disorders.
Two vectors were used to construct the recombinant gene yeast cell that can be used to bioassay of the pollution of tetracycline antibiotics in the environment. In the expression vector, the GPD (glyceraldehyde-3-phosphate dehydrogenase) promoter was used to drive the gene expression of tetracycline repressor protein (TR) fused with V5 antigen epitope gene, while in the reporter vector, the tetracycline response element (TRE) was used to regulate Lac Z report gene expression. The specificity and the sensitivity of the recombinant gene yeast cell were evaluated respectively by different concentrations of tetracycline antibiotics and non-tetracycline antibiotics. The results showed that there were significant dose effect relationships between the tetracycline antibiotics and the yeast cells, while non-tetracycline antibiotics showed no dose effect relationships with this biosensor. It is illustrated that the recombinant yeast cells can be used to monitor the tetracycline antibiotic pollution on the environment.
ObjectiveTo explore the function of intercellular adhesion A (icaA), fibrinogen binding protein (fbe), and accumulation-associated protein (aap) genes in formation of Staphylococcus epidermidis-Candida albicans mixed species biofilms. MethodsThe experiment was divided into 3 groups:single culture of Staphylococcus epidermidis ATCC35984 (S. epidermidis group) or Candida albicans ATCC10231 (C. albicans group), and co-culture of two strains (mixed group) to build in vitro biofilm model. Biofilm mass was detected by crystal violet semi-quantitative adherence assay at 2, 4, 6, 8, 12, 24, 48, and 72 hours after incubation. XTT assay was performed to determine the growth kinetics in the same time. Scanning electron microscopy (SEM) was used to observe the ultrastructure of the biofilms after 24 and 72 hours of incubation. The expressions of icaA, fbe, and aap genes were analyzed by real-time fluorescent quantitative PCR. ResultsCrystal violet semi-quantitative adherence assay showed that the biofilms thickened at 12 hours in the S. epidermidis and mixed groups; after co-cultured for 72 hours the thickness of biofilm in mixed group was more than that in the S. epidermidis group, and there was significant difference between 2 groups at the other time (P<0.05) except at 72 hours (P>0.05). In C. albicans group, the biofilm started to grow at 12 hours of cultivation, but the thickness of the biofilm was significantly lower than that in the mixed group in all the time points (P<0.05). XTT assay showed that the overall growth speed in the mixed group was greater than that in the C. albicans group, and it was greater than that in the S. epidermidis group at 48 hours; there was no significant difference in the growth speed between the mixed groups and the S. epidermidis group in the other time points (P>0.05) except at 12 hours (P<0.05). The absorbance (A) value in the mixed group was lower than that in the S. epidermidis group at 2 and 4 hours, but no significant difference was shown (P>0.05); the A value of mixed group was significantly higher than that of the C. albicans group after 6 hours (P<0.05). SEM observation showed that mature biofilms with complex structure formed in all groups. The real-time fluorescent quantitative PCR showed the expressions of fbe, icaA, and aap genes in mixed group increased 1.93, 1.52, and 1.46 times respectively at 72 hours compared with the S. epidermidis group (P<0.05). ConclusionMixed species biofilms have more complex structure and are thicker than single species biofilms of Staphylococcus epidermidis or Candida albicans, which is related to increased expressions of the icaA, fbe, and aap genes of Staphylococcus epidermidis.