[EMA-pCR detection of enterohemorrhagic Eschrichia coli O157:H7].

OBJECTIVE: A rapid and effective method with ethidium monoazide bromide (EMA) in combination with PCR (EMA-PCR) was established to detect live Enterohemorrhagic Eschrichia Coli O157:H7. METHODS: The rfbE gene was used as the target gene for PCR detection of Eschrichia Coli O157:H7 by utilizing its pure isolates after the treatment of EMA as the template. The EMA concentration and reaction time was optimized. RESULTS: The use of 10 microg/mL or less EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The PCR amplification of DNA derived from 2 x 10(7) CFU/mL dead cells can be inhibited by 0.5 microg/mL EMA. The sensitivity of the method was 2 x 10(4) CFU/mL. The results demonstrated that it could detect 1% live bacteria from a mixed bacterial population. CONCLUSION: EMA-PCR can effectively detect live bacteria of O157:H7, it could be a potential rapid detection method applied in public health emergent events.

Construction and secretory expression of beta-galactosidase gene from Lactobacillus bulgaricus in Lactococcus lactis.

OBJECTIVE: This study is to examine the secretion effects of beta-galactosidase in Lactococcus lactis. METHODS: The usp45 and beta-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ. This recombinant plasmid was transformed into both Escherichia coli DH5alpha and L. lactis MG1363. The enzyme activity, gene sequencing, SDS-PAGE and hereditary stability were assessed and studied. RESULTS: The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence, and SDS-PAGE revealed an evident idio-strap at 116 KDa between L. lactis MG1363/pMG36e-usp-lacZ in both supernatant and cell samples. Beta-Galactosidase activity measured 0.225 U/mL in L. lactis pMG36e-usp-lacZ transformants, and its secretion rate was 10%. The plasmid pMG36e-usp-lacZ appeared more stable in MG1363. CONCLUSION: The authors concluded that these new recombinant bacteria well expressed and secreted beta-galactosidase, indicating that the beta-galactosidase expression system was successfully constructed, and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.

[Investigate the effects of compound radix notoginseng on renal interstitial fibrosis and kidney-targeting treatment].

OBJECTIVE: Investigate the effects of compound Radix Notoginseng on renal interstitial fibrosis and kidney-targeting treatment. METHODS: 100 healthy Sprague-Dawley rats were randomly divided into 5 groups: Unilateral ureteral obstruction (UUO) group, sham-operation (SOR) group, Radix Notoginseng (RN) group, compound Radix Notoginseng (CRN) group and Losartan (ARB) group. After operation, RN, CRN and ARB groups were intragastric administrated with RN (3 mL/d), CRN (3 mL/d) and ARB [20 mg/(kg x d)] respectively. Each group randomly included 18 rats for statistical analysis. The histological changes of renal interstitial tissues were observed by HE, Masson and PAS staining. Total kidney collagen content was determined by measuring the amount of hydroxyproline. The mRNA of alpha-SMA, collagen I and fibronectin were reverse transcribed and quantified by real-time PCR. The expression of alpha-SMA protein was assessed by immunohistochemistry and Western blot analysis. RESULTS: In UUO model, the obstructed kidney showed typical features of renal tubulointerstitial fibrosis, such as severe tubular loss, dilation, atrophy, infiltration of inflammatory cells, interstitial matrix deposition (P < 0.05). Partial correlation assay showed that the expression of alpha-SMA was related to the renal tubular injury (r = 0.55; P < 0.05). Administration of RN, CRN and ARB improved tubulointerstitial damage and collagen matrix accumulation induced by UUO in different degree. The expression of the alpha-SMA at mRNA and protein levels were significantly increased in the UUO group (P < 0.05), which was also suppressed by treatment with RN, CRN and ARB in different degree. Moreover, more effective role in preventing fibrosis was observed in CRN group than when compared with that of RN group. CONCLUSION: RN and CRN can inhibit UUO-induced renal interstitial fibrosis in rats, and CRN treatment is more effective than RN in reducing interstitial fibrosis.

Analysis of ß-galactosidase production and their genes of two strains of Lactobacillus bulgaricus.

A bacterial ß-galactosidase delivery system is a potential therapy for lactose intolerance. Currently, two Lactobacillus bulgaricus strains with different biological characteristics are under consideration as potential sources. However, differences in these ß-galactosidase genes and their resulting production levels are poorly characterized. The ß-galactosidase ORF of L. bulgaricus yogurt isolate had high variability and was terminated at site 1924 due to a stop codon. However, the full 114 kDa ß-galactosidase band was still resolved by SDS-PAGE, which may indicate that the interrupted ORF was translated into more than one peptide, and they together were folded into the complete enzyme protein that showed much higher ß-galactosidase activity (6.2 U/mg protein) than the enzyme generated from L. bulgaricus reference strain (2.5 U/mg protein).

[Hemagglutinin gene of influenza A (H3N2) virus from human samples in the 2009 influenza epidemic in Chengdu].

OBJECTIVE: To study the antigenic and genetic characteristics of influenza A (H3N2) virus in the 2009 influenza epidemics in Chengdu. METHODS: The influenza virus strains were isolated with MDCK cells from 4869 samples taken from the sentinel surveillance in 2009 in Chengdu. Hemagglutination inhibition (HI) and RT-PCR reaction tests were performed to guide the extraction of viral RNA from the culture fluid of the influenza A (H3N2) virus. The hemagglutinin gene was obtained by RT-PCR and sequenced. RESULTS: The separation rates of swine influenza H1N1, H3N2, H1N1, and B were 25.2%, 7.2%, 4.5%, and 1.5% respectively. The epidemic peaked in summer and autumn. Four amino acids changed in A, B, and D antigenic and receptor binding sites: site160N>K, site174K>R/N, site189K>Q, site277R>Q. Glycosylation sites were inserted to sitel60 or absent at site181 in some isolated strains. CONCLUSION: Swine influenza H1N1 viruses dominated the 2009 Chengdu epidemic, with H3N2, H1N1, and B strains coexisting. The influenza A(H3N2) viruses had gene variations due to antigenic drift.

Ginsenoside Rg1 modulation on thrombospondin-1 and vascular endothelial growth factor expression in early renal fibrogenesis in unilateral obstruction.

Renal interstitial fibrosis is the major histopathological change seen in a variety of renal disorders and is closely related to renal dysfunction. Progressive interstitial fibrosis accompanied by the loss of renal tubules and interstitial capillaries typifies all progressive renal disease. Thrombospondin-1 (TSP-1) is a major angiogenic inhibitor. It is demonstrated that TSP-1 levels were correlated with the loss of glomerular and peritubular capillaries and TSP-1 could promote renal scarring by effects on the endothelium. It has been reported that ginsenoside Rg1 inhibited renal interstitial fibrosis in rats via suppressing the expression of TSP-1. The present study was designed to examine whether ginsenoside Rg1 could modulate the integrity of the microvasculature and hence affect the progression of renal fibrosis in a rat unilateral ureteral obstruction (UUO) model. In UUO control kidneys, associated with interstitial fibrosis, lower peritubular capillary densities were prominent. These changes were all improved by ginsenoside Rg1 treatment. Interestingly, ginsenoside Rg1 decreased the expression of TSP-1 and enhanced vascular endothelial growth factor (VEGF) expression. The results show for the first time that ginsenoside Rg1 can evidently inhibit renal interstitial fibrosis in rats with UUO. The mechanism might be related to suppression of the expression of TSP-1 and to repair of the peritubular capillary.

LSKL, a peptide antagonist of thrombospondin-1, attenuates renal interstitial fibrosis in rats with unilateral ureteral obstruction.

The effects of LSKL, the peptide antagonist of thrombospondin-1 (TSP-1), on renal interstitial fibrosis in rats subjected to unilateral ureteral obstruction (UUO) were investigated. Rats were divided randomly into three groups (n = 20 each): UUO group, sham-operation group and UUO plus LSKL treatment group. Collagen deposition was studied using histopathology and reverse transcription polymerase chain reaction analysis (RT-PCR). TSP-1, transforming growth factor beta 1 (TGF-beta1), phosphorylated Smad2 (pSsmad2) and alpha-smooth muscle actin (alpha-SMA) in the kidney were measured using immunocytochemistry, western blotting analysis, RT-PCR and enzyme-linked immunosorbent assay. Biochemical analyses in the serum and urine were made. Histopathology showed severe tubular dilatation and atrophy, interstitial inflammation and collagen accumulation after surgery and LSKL significantly inhibited interstitial fibrosis including tubular injury as well as collagen deposition. The protein and mRNA levels of TSP-1 increased notably at different time point and significantly decreased in the presence of LSKL. The expression of TGF-beta1 and pSmad2 were upregulated in the obstructed kidney and substantially suppressed by LSKL treatment. Myofibroblast accumulation could be alleviated after administration of LSKL. Biochemical parameters did not show differences among the three groups. As TSP-1 is the major activator of TGF-beta1, we demonstrate that LSKL can attenuate renal interstitial fibrosis in vivo by preventing TSP-1-mediated TGF-beta1 activation.

[Carrying rate of Neissria meningitides in a healthy population in the Mianzhu post-earthquake residential area].

OBJECTIVE: To predict the possibility of epidemic outbreak of meningitis by testing Neissria Meningitides in a healthy population in the Mianzhu post-earthquake residential area. METHODS: A simple random sampling strategy was adopted to collect 887 throat swabs from a healthy population in the Mainzhu post-earthquake residential area. The TaqMan assay were performed to detect Neissria Meningitides. RESULTS: Three positive samples were identified. CONCLUSION: The carrying rate of Neissria Meningitides is not high enough to bring about an epidemic outbreak of Meningitis. However, efforts to maintain a hygienic environment in the post-earthquake residential area should be continued.

[Development of a multiplex PCR-suspension array for simultaneous detection of five bioterrorism bacteria].

OBJECTIVE: To develop a rapid, high-throughput screening method of gene suspension array technique to simultaneously detect five bioterrorism bacteria: Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp. and Burkholderia pseudomallei. METHODS: Highly validated specific primers were used to amplify diagnostic regions unique to each pathogen. Biotin labelled PCR products were hybridized to corresponding probes coupling on the unique sets of fluorescent beads. The hybridized beads were processed through the Bio-plex, which identified the presence of PCR products. RESULTS: Multiplex PCR-suspension array can detect five bioterrorism bacteria correctly with high specificity and high sensitivity, the results suggest the utility of suspension array system for high-throughput screening of bioterrorism samples. CONCLUSION: A multiplex PCR-suspension array for rapid detection of five bioterrorism bacteria was established.

Ginsenoside Rb1, a panoxadiol saponin against oxidative damage and renal interstitial fibrosis in rats with unilateral ureteral obstruction.

OBJECTIVE: To investigate the possible protective effect and mechanism of ginsenoside Rb1 against oxidative damage and renal interstitial fibrosis on rats with unilateral ureteral obstruction (UUO). METHODS: In total, 80 male rats were randomly divided into 4 groups, 20 in each group: the sham operated group (SOR), UUO group, UUO with ginsenoside Rb1 treatment group (treated with intraperitoneal injection of 50 mg/ kg daily) and UUO with Losartan treatment group (as the positive control, treated with 20 mg/kg by gastrogavage per day). The rats were randomly sacrificed on day 3, 7 and 14 after surgery, respectively. The histopathologic changes of renal interstitial tissues were observed with Masson staining. The mRNA of transforming growth factor beta 1 (TGF-beta 1), collagen I and fibronectin were reversed transcribed and quantified by Real-time PCR. Enzyme-linked immunosorbent assay was used to quantitatively detect TGF-beta 1 and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels. P47phox protein expression was assessed by immunohistochemistry and Western blot analysis. RESULTS: In the UUO model, the obstructed kidney showed typical features of progressive renal tubulointerstitial fibrosis, and the levels of TGF-beta1, collagen I and fibronectin increased (P<0.05). As compared with the UUO group, ginsennoside Rb1 significantly inhibited the interstitial fibrosis including tubular injury and collagen deposition, and decreased the levels of TGF-beta1 (P<0.05). Ginsenoside Rb1 also inhibited the heme oxygenase (HO-1) and 8-OHdG, two markers of oxidative stress (P<0.05). Moreover, ginsenoside Rb1 suppressed the expression of p47phox, a subunit of nicotinamide adeninedinucleotide phosphate (NADPH) oxidase (P<0.05). CONCLUSION: Ginsenoside Rb1 can obviously inhibit renal interstitial fibrosis in rats with UUO, its mechanism possibly via against the oxidative damage and suppressing TGF-beta1 expression.

[Construction and property study of recombinant Lactococcus lactis with non-fusion expressing of beta-galactosidase].

OBJECTIVE: To construct recombinant Lactococcus lactis strains exhibiting high beta-galactosidase activity in non-fusion way, and study their enzyme activities and enzyme secretion rates. METHODS: The recombinant plasmids pMG36e-lacZ 1.1480 and pMG36e-lacZ wch9901 which could express beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus in non-fusion way in Escherichia coli were obtained and transformed into Lactococcus lactis subsp. lactis MG1363. The beta-galactosidase activity of resulting recombinant L. lactis in different incubation periods and lactose concentrations, and their enzyme secretion rates in different culture conditions were examined. RESULTS: Recombinant L. lactis carrying pMG36e-lacZ wch9901 (MG1363/pMG36e-lacZ wch9901) exhibited the highest beta-galactosidase activity. Its enzyme activity was (16.95 +/- 0.09) U/mg pro, which was 2.75 folds of that of the native counterpart; recombinant L. lactis reached its enzyme producing peak after grown for 24 h; decreased enzyme activity of recombinant L. lactis were observed when incubated in medium containing lactose; the beta-galactosidase expressed by recombinant strains could be secreted into the culture medium, and the highest secretion rate (27.09 +/- 0.05)% was observed when the culture medium contained 20 g/L of lactose and without erythromycin. CONCLUSION: High level expression of non-fusion beta-galactosidase with secretion in recombinant L. lactis strains was obtained. This will be very helpful for the further developing of live delivery bacteria of beta-galactosidase.

[Effects of ginsenoside Rb1 on TGF-beta1 induced p47phox expression and extracellular matrix accumulation in rat renal tubular epethelial cells].

OBJECTIVE: To investigate the effects of Ginsenoside Rb1 (G-Rb1) on the oxidative damage and extracellular matrix accumulation in rat renal tubular epethelial cells induced by transforming growth factor-beta1 (TGF-beta1). METHODS: Cultured normal rat renal tubular epethelial cells (NRK-52E) were divided into control group, 10 ng/mL TGF-beta1-induced group, G-Rb1 treated groups in which rat renal tubular epethelial cells were treated with different concentration of G-Rb1 (10 ng/mL, 20 ng/mL, 40 ng/mL) after TGF-beta1 induction, G-Rb1 40 ng/mL group and 100 nmol/L DPI(diphenyleneiodonium, an inhibitor of NADPH oxidase) group. Intracellular reactive oxidative species (ROS) level was measured by flowcytometry. p47phox protein expression was assessed by immunohistochemistry and western blotting method. The expressions of collagen I (Col-I) and fibronectin(FN) gene were measured by real-time PCR analysis. The protein level of Col-I and FN were quantitatively detected by enzyme-linked immunosorbent assay. RESULTS: TGF-beta1 at 10 ng/mL significantly increased the intercellular ROS production and p47phox expression (P < 0.05). The levels of Col-I and FN were also significantly up-regulated with the stimulation of 10 ng/mL TGF-beta1 (P < 0.05). Compared to TGF-beta1-induced group, G-Rb1 and DPI depressed TGF-beta1-induced ROS production and p47phox overexpression. Meanhile, G-Rb1 and DPI decreased the levels of Col-I and FN. CONCLUSION: G-Rb1 could inhibit TGF-beta1 induced ROS production and decrease the levels of Col-I and FN in a dose-dependent manner. The mechanism might be partly related to the suppression of p47phox expression.

[Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria].

OBJECTIVE: To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. METHODS: 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. RESULTS: After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. CONCLUSION: The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

Ginsenoside Rg1, a major active component isolated from Panax notoginseng, restrains tubular epithelial to myofibroblast transition in vitro.

The medicinal herb, Panax notoginseng, has been used for thousands of years in traditional Chinese medicine and possesses anti-fibrosis properties. Epithelial-myofibroblast transition (EMT) plays an important role in renal tubulointerstitial fibrosis. The present study was designed to examine whether ginsenoside Rg1, a major active component isolated from Panax notoginseng, has an ability to block this phenotypic transition in rat renal tubular epithelial cells (NRK-52E) induced by transforming growth factor-beta1 (TGF-beta1). The morphology of tubular epithelial-myofibroblast transition was observed through light microscope and transmission electron microscopy. alpha-SMA and E-cadherin are two markers of tubular epithelial-myofibroblast transition, their protein expressions were assessed by immunohistochemistry and western blot analysis. Gene expression of alpha-SMA as well as the two major extracellular matrix components collagen I and fibronectin was measured by real-time PCR analysis. Enzyme-linked immunosorbent assay was used to quantitatively detect collagen I and fibronectin in the supernatant. Our results revealed that ginsenoside Rg1 obviously blocked morphologic transformation in NRK-52E induced by TGF-beta1. Meanwhile, ginsenoside Rg1 inhibited the expression of alpha-SMA and the loss of E-cadherin, subsequently decreased the levels of collagen I and fibronectin in a dose-dependent manner. In addition, western blot analysis indicated that ginsenoside Rg1 inhibited the expression of P-ERK1/2 in NRK-52E induced by TGF-beta1. These results suggest that ginsenoside Rg1 can restrain the process of EMT maybe via suppressing the expression of P-ERK1/2 in vitro.

[Ginsenoside R(g1) inhibit transdifferentiation in rat renal tubular epethelial cells induced by TGF-beta1].

OBJECTIVE: To investigate the effects of ginsenoside R(g1) on the transdifferentiation of rat renal tubular epethelial cells induced by transforming growth factor-beta1, (TGF-beta1). METHOD: Cultured normal rat renal tubular epethelial cells (NRK-52E) were divided into control group, TGF-beta1-induced group and treated with ginsenoside R(g1) at different concentration (10, 20, 40 mg x L(-1)) group. The morphology of tubular epithelial-myofibroblast transdifferentiation induced by TGF-beta1 was observed through light microscope. alpha-SMA and E-cadherin protein expression were assessed by immunohistochemistry and western blot analyses. alpha-SMA, collagen I and and fibronectin gene expression were assessed by real-time quantitative chain reaction. Enzyme-linked immunosorbent assay was used to quantitatively detect collagen I and fibronectin in the supernatant. RESULT: 10 mg x L(-1) TGF-beta1 could induce the transdifferentiation of tubular epithelial myofibroblast, showing fibroblast-like in morphology, with significantly enhanced expression of alpha-SMA, depressed expression of E-cadherin and increased secretion of fibronectin and collagen I (P < 0.05). Compared to TGF-beta1-induced group, ginsenoside R(g1) partly abrogated the alpha-SMA expression and E-cadherin depression triggered by TGF-beta1 in tubular epithelial cells in a dose-dependent manner (P < 0.05). Meanhile, ginsenoside R(g1) blocked morphologic transformation of tubular epithelial cells and decreased levels of collagen I and fibronectin (P < 0.05). CONCLUSION: Ginsenoside R(g1) could inhibit TGF-beta1 induced the tubular epithelial-myofibroblast transdifferentiation and decreased levels of collagen I and fibronectin in NRK52E.

[Beta-galactosidase gene from Lactobacillus delbrueckii subsp. bulgaricus gets non-fusion expression in Escherichia coli].

OBJECTIVE: To construct the food-grade recombinant probiotic strain with high activity beta-galactosidase, the beta-galactosidase gene (lacZ)from Lactobacillus delbrueckii subsp. bulgaricus was in non-fusion expressed in Escherichia coli. METHODS: From Lb. delbrueckii subsp. bulgaricus lacZ gene, the DNA sequence containing Shine-Dalgarno (SD) and ATGA sequences between upstream 18 bp and downstream 1 bp at start codon ATG was selected as upstream primer for PCR amplifying lacZ gene. Then lacZ cDNA was inserted into expression plasmid pMG36e to construct recombinant expression plasmid. Recombinant plasmids were introduced into E. coli, and positive clones were screened. To identify the gene recombination, the recombinant plasmid was cut by restriction enzyme and sequenced. To identify the protein expression, the beta-galactosidase activities of recombinant strains were determined. RESULTS: The restriction maps of recombinant plasmids were acceptable. The gene inserted into the recombinant plasmid had more than 99% homology with the lacZ gene of Lb. delbrueckii subsp. bulgaricus. The enzyme activity and enzyme activity ratio of E. coli DH5 alpha carrying pMG36e-lacZ 1.1480 were 3.074 U/mL and 6.939 U/mg pro respectively. The enzyme activity and enzyme activity ratio of E. coli DH5a carrying pMG36e-lacZ wch9901 were 4.755 U/mL and 8.537 U/mg pro respectively. CONCLUSION: lacZ from Lb. delbrueckii subsp. bulgaricus have gotten non-fusion expression in E. coli. The SD and ATGA sequences we selected can introduce lacZ non-fusion expression in E. coli.

[Development of a molecular beacon real-time PCR for the rapid detection of Mycobacterium tuberculosis].

OBJECTIVE: To develop a molecular beacon real-time PCR for rapid detection of Mycobacterium tuberculosis. METHOD: One set of primers was selected from the IS6110 gene in GenBank and the corresponding molecular beacon probe was designed. The specificity and sensitivity of the developed method were evaluated by tested with 10 different bacteria species. The developed assay were also applied to the diagnosis of tuberculosis. RESULTS: Only Mycobacterium tuberculosis strains possessing IS6110 gene generated fluorescent signals, and no cross reaction was observed with other 9 bacteria. The detection limit was 4 copies/PCR reaction. 100 Mycobacterium tuberculosis strains were positive tested by Real-time PCR. CONCLUSION: The established molecular beacon real-time PCR is a rapid, specific and sensitive method, and is a beneficial supplement of traditional methods for the tuberculosis diagnosis.

[The effect of ginsenoside Rg1 on the renal interstitial fibrosis of UUO rat].

OBJECTIVE: To study the effect of ginsenoside Rg1 on the renal interstitial fibrosis caused by unilateral ureteral obstruction (UUO) of rats. METHODS: 80 healthy Sprague-Dawley rats were randomly divided into 4 groups: the UUO group (UUO), sham-operation group (SOR), ginsenoside Rg1 group (Rg1) and losartan group (ARB). From the first day after initial UUO, ARB group was intragastrically administrated with losartan 20 mg/(kg x d). UUO, Rg1 and SOR groups were intragastrically administrated with identical volume of normal saline. Rg1 group was intraperitoneally injected with ginsenoside Rg1 50 mg/(kg x d). UUO, ARB and SOR groups were intraperitoneally injected with identical volume of normal saline. At day 3, 7, 14 after UUO, 6 rats selected randomly from each group were killed. The dynamic histological changes of renal interstitial tissues were observed by HE, Masson and PAS staining. The mRNA of transforming growth factor-beta1 (TGF-beta1) was quantified by real-time PCR. The protein levels of TGF-beta1 expression were assessed by Western blot and immunohistochemical method respectively. RESULTS: In UUO kidneys, the interstitial fibrosis including tubular atrophy, loss and dilation, inflammatory cell infiltration and interstitial matrix deposition was prominent. However, these morphological changes were notably reduced in Rg1 and ARB groups, and there was no significant difference between the two groups (P > 0.05). TGF-beta1 mRNA and protein expression were increased dramatically for UUO group at postoperative day 7 and 14 (P < 0.05). TGF-beta1 expression in Rg1 and ARB groups were significantly lower than that in UUO group (P < 0.05). CONCLUSION: Ginsenoside Rg1 can evidently inhibit UUO-induced renal interstitial fibrosis in rat, which may be related to the down regulation of TGF-beta1 expression.

[Phylogeny analysis and identification of two bacterial strains sourcing from human intestine and having resistance to acid and bile].

OBJECTIVE: To screen bacteria for the engineering bacteria expressing and secreting high activity beta-galactosidase, and two bacterial strains called as R92-2 and R111 with acid and bile resistances would be isolated from health human intestine to strain identification and phylogenetic analysis. METHODS: These two strains were first been identified with phenotype characteristic analysis. Then the 16S rDNAs of these two bacterial strains were amplified and sequenced with the primers designed by the conserve sequences. DNA sequences were blasted against GenBank, and the phylogenetic trees were constructed. RESULTS: Phenotype characteristic analysis showed that both of the bacterial strains belonged to lactic acid bacteria. Results of Blastn showed that 16S rDNAs of R92-2 and Weissella cibaria had 100% homology; 16S rDNAs of R111 and Enterococcus faecium had 100% homology too. CONCLUSION: R92-2 belongs to Weissella cibaria strain; R111 belongs to Enterococcus faecium strain. These two stains may be used to construct the engineering bacteria expressing and secreting high activity beta-galactosidase.

Non-fusion and fusion expression of beta-galactosidase from Lactobacillus bulgaricus in Lactococcus lactis.

OBJECTIVE: To construct four recombinant Lactococcus lactis strains exhibiting high beta-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. METHODS: The gene fragments encoding beta-galactosidase from two strains of Lactobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the beta-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the beta-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the beta-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5 alpha and Lactococcus lactis subsp. lactis MG1363 and confirmed by determining beta-galactosidase activities. RESULTS: The non-fusion expression plasmids showed a significantly higher beta-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the beta-galactosidase gene from Lactobacillus bulgaricus wch9901. The beta-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, beta-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate (27.1%) was observed when the culture medium contained 20 g/L of lactose. CONCLUSION: Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus beta-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.