Objective To prepare recombinant plasminogen activator(Pla) protein in E.coli BL21 cells that can be used in studying interactions between Yersinia pestis proteins and immunologic diagnosis of plague.Methods The pla gene was amplified by PCR and cloned into the pET28a expression vector.E.coli BL21 competent cells were transformed with the recombinant vectors, and isopropyl-β-D-thiogalactopyranoside ( IPTG) was added to induce expression of Pla protein. The expressed protein was detected by SDS-PAGE electrophoresis.The inclusion bodies of Pla protein were denatured in 8 mol/L urea, and then refolded using gradient urea solutions.The purified protein was identified by SDS-PAGE electrophoresis and Western blot.Results and Conclusion The constructed expression vector was demonstrated to be correct through agarose gel electrophoresis and sequencing.The recombinant Pla protein was accumulated as an inclusion body in E.coli, and the overexpression product was mainly a target protein, the yield of which was very high.SDS-PAGE purity of the bioactive Pla protein was obtained by denaturing and refolding the inclusion bodies.This study provides a simple and quick method for highly efficient preparation of biologically active Pla protein.

Objective To establish a simple and quick loop-mediated isothermal amplification (LAMP)method for detection of Yersinia pestis.Methods LAMP Primers were designed based on the specific sequence 3a in Y.pestis chromosome.LAMP reaction results were detected using turbidity meter or visual method.The specificity of the constructed method was evaluated by detecting Y.pestis and its closely-related bacteria.The different dilution DNA template was detected with LAMP and PCR to evaluate the sensitivity of the method.Results Thirty strains of bacteria closely related to Y.pestis were detected by the constructed LAMP,and all the results were negative,indicating that the method had a very high specificity.The detection sensitivity of this LAMP assay was 20 pg of DNA per reaction,which was ten-fold that of the regular PCR.The detection reaction was completed in 25 min.Conclusion This LAMP method is quick,sensitive, specific and simple,which is expecked to become an effective method for rapid detection of Y.pestis on the scene.

ObjectiveTo explore the Apolipoprotein-E （ApoE） gene polymorphism in patients with Alzheimer's disease （AD）, vascular dementia (VD) or mild cognitive impairment (MCI). MethodsPeripheral blood was taken from patient with AD, VD or MCI to determine the ApoE genotypes. ResultsThe most of the patients were ε3/ε3 genotype, while the ε2/ε2 and ε4/ε4 could not be detected. ε3/ε4 genotype （P=0.001） and ApoE ε4 allele （P=0.013） was more frequent in AD than in MCI. ApoE ε4 was more frequent in VD than in MCI （P=0.044）. ConclusionApoE ε4 allele is a risk factor in AD, and may be associated with VD and MCI.

Objective:To study the protective effect of alendronate combined with Lactobacillus rhamnosus on bone loss in ovariectomized mice.Methods:Fifty female C57BL/6 mice were divided into 5 equal groups ( n=10). Ovariotomy was performed in groups A, B, C and D while a sham operation was performed in group E. Group A was subjected to combined administration of alendronate and Lactobacillus rhamnosus, group B to administration of alendronate, group C to administration of Lactobacillus rhamnosus, and groups D and E to administration of physiological saline only. At 3 months after operation, all the mice were sacrificed to harvest their femurs. Micro CT scanning was performed to detect the bone mineral density (BMD), trabecular relative volume, bone surface area/bone volume, and trabecular thickness and number of trabecular bone. Three-point bending test was used to detect the maximum load, stiffness, ultimate load, Young＇s modulus, and fracture energy. Osteocalcin and alkaline phosphatase levels were measured using blood samples from the mice eyeballs. The 2 groups were compared in terms of all the above indexes. Results:The BMD [(669.87±67.87) mg/cm 3], maximum load [(14.35±0.75) N] and fracture energy [(1,497.43±38.29) J/m 2] in group A were significantly higher than those in group B [(520.07±9.01) mg/cm 3, (11.94±0.82) N and(1,277.61±35.12) J/m 2] and group C [(388.15±25.61) mg/cm 3, (11.10±0.93) N and (1,115.27±63.24) J/m 2] (all P<0.05). The osteocalcin level in group A [(22.25±1.78) ng/mL] was significantly higher than that in group B [(19.08±1.45) ng/mL] and group D [(19.33±1.66) ng/mL] (both P<0.05). The alkaline phosphatase level in group A [(83.21±9.69) ng/mL] was significantly lower than that in group C [(113.16±14.44) ng/mL] and group D [(137.96±14.01) g/mL] (both P<0.05). Conclusion:Alendronate combined with Lactobacillus rhamnosus may play a synergistic role in prevention of bone loss in ovariectomized mice, because combined administration of the two is more effective than administration of either of the two.

High cholesterol is one of the important factors inducing cardiovascular and cerebrovascular diseases. Drug therapy is the main method for reducing cholesterol, but has the disadvantages such as high cost and side effects. Studies have shown that intestinal bacteria play important roles in cholesterol metabolism. However, there are few reports on the screening and functional evaluation of cholesterol-lowering intestinal bacteria. In this study, 36 bile-tolerant bacteria were screened from healthy people stool through culturomics using bovine bile acid or artificial mixed bile acids as substrates. Taking Lactobacillus rhamnosus GG (LGG) as a positive control, three bile acid concentration groups (0 g/L, 0.3 g/L, 3 g/L) were set up to evaluate the cholesterol-lowering ability of bile-tolerant bacteria in vitro. Ten bacteria (including Proteus mirabilis, Providencia stuartii, Proteus vulgaris et al) were identified as the dominant cholesterol-lowering bacteria. Six of the above bacteria, Proteus mirabilis, Providencia stuartii, Proteus vulgaris, Proteus penneri, Wohlfahrtiimonas chitiniclastica, Providencia rettger, were evaluated for their ability to reduce triglycerides in vitro and tolerance to artificial gastric juice. Comparing with strain LGG, the six bacteria showed better triglyceride-lowering ability in vitro. With the decrease of pH value of artificial gastric juice and the increase of treatment time, the survival rate of six bacteria decreased. The above screening experiments and functional evaluation provide a basis for further development of potential cholesterol-lowering bacterial products.
Animals ; Cattle ; Cholesterol ; Gammaproteobacteria ; Humans ; Proteus mirabilis ; Providencia