Objective To display of heterologous proteins on the surface of E. coli . Methods The 1653 bp luciferase report gene was knocked in Ipp and ompA genes of chromosome by Red recombine system and selection-counter selection kan/sacB. Results The quantitative analysis results of exogenous lu-ciferase expression displayed that it could be expressed as fusion with the outer membrane proteins on the cell surface. The fusion protein of foreign protein and outer membrane protein Lpp-OmpA-Luc could be high-effi-ciently displayed on cell surface. Conclusion The stable expression of exogenous gene on the surface of E. coli had no effect on the bacterial growth and propagation.

Objective: To obtain phage-displayed ScFv library directly against prostate carcinoma cells, and select antibodies binding to prostate carcinoma cells specifically, so as to lay a foundation for developing diagnostic agents and clinical therapies of prostate carcinoma. Methods: Balb/c mice were immunized i. p . with purified membrane protein mixture of prostate carcinoma cells PC3, DU145. mRNA was isolated from the spleens of immunized mice, heavy and light chain genes ( VH and VL) of antibody were amplified separately by RT-PCR and assembled into ScFv gene with a specially constructed linker DNA. , the ScFv gene was ligated into the phagemid vector pCANTAB 5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to yield recombinant phage. After five rounds of panning with PC3 cells, the positive clones were selected with the ELISA from the enriched phages. Results: A ScFv library of 3. 5 ? 106 was obtained and one phage-ScFv which can bind specifically PC3 cells was found. Conclusions: A prostate carcinoma specific antibody was identified , which paves a way for study of prostate carcinoma.

OBJECTIVE: To observe and analyze the function and morphology of pharyngeal ostium of the eustachian tubes in secretory otitis media and chronic rhinosinusitis in children under direct vision,in order to provide an objective basis for clinical treatments. METHOD: Fifty cases of secretory otitis media,50 cases of chronic rhinosinusitis and a control group of 50 cases with hoarseness were examined under video laryngoscope to observe the pharyngeal ostium morphological changes of the eustachian tubes, and their functional statuses were tested by using acoustic impedance instrument. All the data were analyzed by statistical methods. RESULT: (1) In the secretory otitis group, the abnomal rate of the pharyngeal ostium of the eustachian tubes was 94% while the chronic rhinosinusitis group was 80%,and between them there was no significant differences (P > 0.05). But both of them had significant differences with the control group (P < 0.05). (2) In the secretory otitis group, the rate of the eustachian tube dysfunction was 70% while the chronic rhinosinusitis group was 26%, and between them there was significant differences (P < 0.05), and both of them have significant differences when compared with the control group (P < 0.05). CONCLUSION There are some abnormal points exist in the function and the morphology of the eustachian tube in secretory otitis media and chronic rhinosinusitis in children. Eustachian tube dysfunction played a dominant role in the pathogenesis of secretory otitis media in children rather than the morphological change did compared to the chronic rhinosinusitis in children.
Case-Control Studies ; Child ; Chronic Disease ; Eustachian Tube ; pathology ; physiopathology ; Humans ; Otitis Media with Effusion ; pathology ; physiopathology ; Rhinitis ; pathology ; physiopathology ; Sinusitis ; pathology ; physiopathology

To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.
Bacteriophage lambda ; genetics ; Chromosomes, Bacterial ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Knock-In Techniques ; methods ; Genes, Reporter ; genetics ; Lac Operon ; genetics ; Luciferases ; genetics ; Recombination, Genetic ; genetics

Objective To explore the clinical value of ureteroscopy and minimally invasive percutaneous nephrolithotomy ( MPCNL) in the treatment of ureteral calculi patients with acute renal dysfunction .Methods Clini-cal data of 124 ureteral calculi patients with acute renal dysfunction were retrospectively analyzed .86 cases were trea-ted with holmium-laser lithotripsy under ureteroscope .38 cases were treated with MPCNL under the guide of ultra-sound.Results Three months after operation ,the stone clearance rate was 100%,and no severe complications were observed.The renal function decreased to normal levels in 102 cases(82.3%).Conclusion The holmium laser lith-otripsy under ureteroscope and MPCNL can deal with double sites of ureteral calculi ,and offer advantages with less in-vasion,safety and efficiency ,which can be the first choice for the ureteral calculi combined with acute renal dysfunc -tion.

To identify the regulatory region that are responsible for the expression of mPC-1, we have isolated and characterized the mPC-1 gene promoter. Sequence analysis of the mPC-1 5' -flanking region and a series of truncated constructs were performed, which were transiently transfected into the prostate cancer cell lines and non-prostate cancer cell lines and analyzed through Dual-luciferase reporter assay system. The relative activity of mPC-1 gene promoter was by far higher than pGL3-control containing SV40 promoter and enhancer and p61-PSA containing hPSA 6 kb promoter in AR (androgen receptor, AR ) -positive prostate cancer cell lines. The region from 599 bp to 449 bp of mPC-1 promoter might contain a negative regulatory element. The expression of mPC-1 1.1 kb fragment is mainly restricted into prostate cancer cell lines. The relative activity of mPC-1 1.1 kb 5'-flanking region was regulated by androgen. The results demonstrated that the 1.1 kb fragment of mPC-1 5' -flanking region was relatively strong and prostate cancer cell specific promoter region.The 1.1 kb promoter of mPC-1 gene might be well suited to prostate cancer gene therapy if the promoter was properly modified.

Objective To evaluate the effect of spinal cord stimulation on the expression of neuronal nitric oxide synthase (nNOS) in the rats with diabetic neuropathic pain.Methods Forty-eight pathogen-free healthy male Sprague-Dawley rats,aged 2 nonths,weighing 250-300 g,were divided into 4 groups (n =12 each) using a random number table:control group (group C),diabetes mellitus group (group DM),sham operation group (group S) and spinal cord stimulation group (group SCS).Diabetic neuropathic pain was produced by intraperitoneal injection of 1％ streptozocin 75 mg/kg and confirmed by blood glucose level＞16.7 mmol/L on day 2 after streptozocin injection.Electrodes were implanted into the epidural space at 19 days after successful establishment of the model in S and SCS groups,and in addition spinal cord stimulation (frequency 50 Hz,pulse width 0.2 ms,intensity=2/3 of the lowest intensity) was performed for 30 min once a day at 26-28 days after successful establishment of the model in group SCS.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before establishment of the model (T1) and 2,7,14 and 28 days after successful establishment of the model (T2-5).The rats were sacrificed after the last measurenent of MWT at T5,and the lumbar segment of the spinal cord was removed for determination of nitric oxide (NO) content and nNOS expression in spinal cord tissues.Results Compared with group C,the MWT was significantly decreased at T4.5,and the NO content and nNOS expression in the spinal cord were increased in DM,S and SCS groups (P＜0.05).Compared with group DM,the MWT was significantly increased at Ts,and the NO content and nNOS expression in the spinal cord were decreased in group SCS (P＜0.05).Conclusion The mechanism by which spinal cord stimulation allenuales diabetie neuropalhic pain is related to inhibiting nNOS expression and reducing NO production in rals.

Objective To observe the effect of spinal cord stimulation on expression of spinal GLT-1 and GLAST in rats with diabetic neuropatbic pain.Methods Forty-eight healthy male SD rats,only 2 months old,weighting 250-300 g,using the random number table method,were divided into four groups (n=12):the control group (group C),diabetes neuralgia group (group D),false stimulation group (group N)and spinal cord stimulation group (group S).The model of diabetes was induced by the pedtoneal injec-tion of streptozocin (STZ),electrodes were placed into the epidural space 1 9 days after injection of STZ in groups N and S,in addition,group S was performed 26-28 days after injection of STZ.Mechanical contrac-tion leg threshold (MWT)were determined one day before STZ injection,2 d,7 d,14 d and 28 d after STZ injection.The rats were sacrificed,the lumbar spinal cord tissue were obtained for determination of GLT-1 and GLAST expression in spinal cord tissues on 28 d after measurement of MWT.Results Compared with group C,MWT was decreased 14 d and 28 d after STZ injection,and expression levels of GLT-1 and GLAST mRNA were increased on 14 d and 28 d (P<0.05);Compared with before STZ injection,MWT of group D,group N and group S was decreased on 14 d and 28 d (P<0.05);Compared with group D, MWT was increased,and expression levels of GLT-1 and GLAST mRNA were increased in group S on 28 d after STZ injection (P<0.05 ).Conclusion The mechanism of spinal cord stimulation reducing rats diabetes neuralgia may be related to elevating the expression of GLT-1 and GLAST.

Gsalpha gene mutation has been discovered in some human tumors. In our previous studies, three novel deletants of Gsalpha gene, Gsalpha L-1(500 bp), Gsalpha L-2(300 bp), and Gsalpha L-3(200 bp), and wild type Gsalpha-4(1 200 bp) were found in human leukemia cell lines and detected in leukemic cells from patients with acute leukemia. To investigate the construction, function and biological significance of the deletants, the plasmids of Gsalpha L-1, Gsalpha L-2 and wild Gsalpha-4 were transformed into E. coli DH5, amplified by PCR, and cloned in expression vector pET22b(+), and then transformed into E. coli, respectively. As a result, higher levels of expression of three recombinants were obtained in form of inclusion bodies. The results suggested that these Gsalpha isoforms have an open reading frame of gene and can be expressed in vitro. The data lay a foundation to study the relation of Gsalpha gene to leukemogenesis.