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1.
Chinese Journal of Biotechnology ; (12): 4314-4328, 2021.
Article in Chinese | WPRIM | ID: wpr-921508

ABSTRACT

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.


Subject(s)
Aminolevulinic Acid/metabolism , Corynebacterium glutamicum/metabolism , Fermentation , Metabolic Engineering , Rhodobacter capsulatus/enzymology , Rhodopseudomonas/enzymology
2.
Chinese Journal of Biotechnology ; (12): 4254-4265, 2021.
Article in Chinese | WPRIM | ID: wpr-921503

ABSTRACT

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.


Subject(s)
Aminobutyrates , Escherichia coli/genetics , Leucine Dehydrogenase/genetics , Threonine Dehydratase
3.
Article in English | WPRIM | ID: wpr-886694

ABSTRACT

@#Finding stable expression sites on the chromosomes of Chinese hamster ovary (CHO) cells is an effective method to solve the problem of unstable expression of CHO cells in long-term culture. Our group used lentiviral transfection to integrate the tracer gene (Zsgreen1) into the chromosome of CHO cells and found multiple potential stable expression sites. This study verified the ability of one of the sites located in the 148052-148157 bp region on chromosome NW_003614241.1 to stably express exogenous proteins.The expression of Zsgreen1 gene was first observed, and CRISPR/Cas9 technology was then used to integrate the enhanced green fluorescent protein (EGFP) gene into this site. Three strains of EGFP gene integrated cells were obtained. After 60 generations of suspension culture, the fluorescence intensity of the cells had no significant changes, which proved that this site can stably express the EGFP gene. The same method was used to construct recombinant CHO cell lines expressing the human serum albumin (HSA) gene, and was verified by Western blot that this site could express and secrete HSA. It shows that the above-mentioned sites can be integrated and can stably express exogenous proteins.

4.
Chinese Journal of Biotechnology ; (12): 1348-1358, 2019.
Article in Chinese | WPRIM | ID: wpr-771794

ABSTRACT

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.


Subject(s)
Biocatalysis , Cloning, Molecular , Escherichia coli , Glucosyltransferases , Streptomyces coelicolor , Trehalose
5.
Article in Chinese | WPRIM | ID: wpr-615026

ABSTRACT

This study was focus on investigating the anti-liver fibrosis effects of insulin-like growth factor-1 (IGF-1) in vitro.The effects of IGF-1 on human liver L-02 cell viability and cell cycle were observed.CC14-induced L-02 cell injury was set up to detect the anti-apoptotic activity of insulin-like growth factor-1 (IGF-1).Transforming growth factor β1 (TGF-β1) induced hepatic stellate cell line (HSC-T6) were used as a liver fibrosis model in vitro to analyze the effects of IGF-1 on the expression of liver fibrosis proteins and intracellular TGF-β1/Smad signaling pathway in HSC-T6 cells.The results showed that IGF-1 could relieve the growth inhibition effects of TGF-β1 on L-02 cells,increase the viability of L-02 cells injured by CCl4,decrease the expression of liver fibrosis proteins,and inhibit the TGF-β1/Smad signaling pathway by inhibiting the phosphorylation of Smad3.Our study suggested that IGF-1 exerted anti-liver fibrosis effects by stimulating L-02 cells proliferation,reducing cell damage and inhibiting ECM accumulation via interfering TGF-β1/Smad signaling pathway.

6.
Article in Chinese | WPRIM | ID: wpr-270024

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the changes of water level and the distribution of snails in Anhui province before and after runs of the Three Gorges Reservoir Project, and to determine the relationship between the two factors and schistosomiasis transmission.</p><p><b>METHODS</b>The hydrologic data of Datong hydrologic station and the data of snail status and schistosomiasis morbidity in Anhui Province were collected. The data from 1991 to 2002 and 2003 to 2012 were considered as before and after the impoundment of the Three Gorges Reservoir Project. Based on the prevalence of schistosomiasis, the cases of people and cattle were speculated, and the average infection rate of people and cattle were calculated. The t-test was used to compare the difference of snail area and the density of living snails before and after the impoundment of the Three Gorges Project. The pearson method was used to analyze the relationship between water level and snail area. The spearman method was used to analyze the relationship between the water level and the distribution of snails.</p><p><b>RESULTS</b>From 1991 to 2012, the range of the highest water level, the lowest water level, the difference between the highest and lowest water level, the mean in the abundant water seasons, the mean in the dry water seasons, and the difference between the abundant water seasons and the dry water seasons was 11.40-16.30, 3.68-5.20, 6.70-12.12, 9.92-14.40, 4.77-7.64 and 4.13-8.93 m, respectively. The snail areas was (28 613 ± 362) hm² and (29 477 ± 918) hm² (t = -3.00, P = 0.007), the density of living snails was 1.51 (1.15-2.43) and 0.43 (0.29-1.10) snails/0.11 m² (H = 4.28, P < 0.001) before and after the impoundment of the Three Gorges Project, respectively. The average infection rate of people and cattle was 1.68% (99 482/5 935 147) and 4.62% (13 923/3 011 33), and the average number of acute schistosomiasis cases was 328, before the impoundment of the Three Gorges Project; 0.60% (39 747/6 649 380), 1.65% (1 291/783 224) and 71 after the impoundment of the Three Gorges Reservoir Project, respectively. The snail areas had negative correlation with the highest water level, the difference between the highest and lowest water level, the mean in the abundant water seasons (r value was -0.514, -0.509 and -0.477; P value was 0.014, 0.015 and 0.025, respectively). The infection rate of people had positive correlation with the highest water level, the difference between the highest and lowest water level, the mean in the abundant water seasons (r value was 0.532, 0.587 and 0.446; P value was 0.011, 0.004 and 0.038, respectively). The infection rate of cattle had positive correlation with the highest water level, the difference between the highest and lowest water level (r value was 0.507 and 0.553; P value was 0.016 and 0.008, respectively). The number of acute schistosomiasis cases had positive correlation with the highest water level, the difference between the highest and lowest water level (r value was 0.481 and 0.486; P value was 0.023 and 0.022, respectively).</p><p><b>CONCLUSION</b>Following the runs of the Three Gorges Reservoir Project, the change of water level in the section of Anhui Province affected the distribution of snails and the infection of people and cattle to some extent. The snail areas showed an upward trend, and the density of living snails, the infection rate of people and cattle showed a downward trend. The runs of Three Gorges Reservoir Project has certain role to reduce flood and helpful for schistosomiasis control.</p>


Subject(s)
Animals , Cattle , China , Floods , Humans , Lakes , Prevalence , Rain , Schistosomiasis , Seasons , Snails
7.
Article in Chinese | WPRIM | ID: wpr-481926

ABSTRACT

Haemophilia is caused by lack of coagulation factor VIII or IX in patients′blood with inadequate hemostasis.Currently recombinant coagulation factor VII(rFVII)produced in different cells is used against clini-cal bleeding of haemophilia patients.To enhance the production and activity of rFVII;some eukaryotic cells such as baby hamster kidney(BHK);Chinese hamster ovary(CHO);insect cell and fish embryo;were used to express rFVII.Meanwhile;the effect of functional gene on the activity of rFVII and the limitation of rFVII production caused by post-translational modification were investigated by different methods.The role of rFVII in hemostasis;synthesis of rFVII in different eukaryotic cells and impact on production of post-translational modification are reviewed in this article.

8.
Article in Chinese | WPRIM | ID: wpr-445067

ABSTRACT

Objective To investigate the effect of different levels of positive pressure (PEEP) during one-lung ventilation on blood gas and hemodynamics in patients with thoracoscopic lung bullae resection surgery.Methods Seventy-eight patients undergoing thoracoscopic lung bullae resection surgery were divided into three groups by random number table method,26 cases in each:group Ⅰ was only given one-lung intermittent positive pressure ventilation (IPPV) after two-lung ventilation,group Ⅱ was given one-lung IPPV and PEEP 5 cmH2O (1 cmH2O =0.098 kPa) after two-lung ventilation,group Ⅲ was given one-lung IPPV and PEEP 10 cmH2O after two-lung ventilation.Blood gas and hemodynamics were recorded and compared in the supine position and lateral position two-lung ventilation,one lung ventilation 10 and 30 min among three groups.Results Oxygen saturation was maintained at 0.99-1.00 in three groups.pH value,base excess,arterial carbondioxide partial pressure (PaCO2) and HCO3-at each time point in three groups had no statistical significance (P > 0.05).Arterial oxygen partial pressure (PaO2) in group Ⅱ and group Ⅲ at one-lung ventilation 10,30 min was significantly higher than that in group Ⅰ [(336.2 ± 113.2),(348.5 ± 109.7) mmHg (1 mmHg =0.133 kPa) vs.(285.0 ± 103.5) mmHg,(357.6 ± 104.0),(358.9 ±103.2) mmHg vs.(276.0 ± 107.2) mmHg] (P <0.05),but were within the normal range,there was no statistical difference between group Ⅱ and group Ⅲ (p > 0.05).Heart rate,mean arterial pressure,left ventricular ejection time,systemic vascular resistance at each time point in three groups had no statistical significance (P >0.05).Stroke volume,cardiac output in group Ⅱ and group Ⅲ at one-lung ventilation 10,30 min were lower than those in supine position and lateral position two-lung ventilation and the same period in group Ⅰ (P < 0.05),but were within the normal range,there were no statistical differences between group Ⅱ and group Ⅲ (p > 0.05).Conclusions Two-lung ventilation after one-lung IPPV and PEEP 5 cmH2O in thoracoscopic lung bullae resection surgery can maintain satisfactory PaO2 and PaCO2,hemodynamic change is not obvious; PEEP 5 cmH2O compares with only IPPV can further improve PaO2,but PEEP 10 cmH20 can't further improve PaO2.

9.
Article in Chinese | WPRIM | ID: wpr-480364

ABSTRACT

A double antibody sandwich ELISA for quantitative analysis of recombinant fusion protein IL-2-HSA was constructed using a polyclonal antibody to human IL-2 for capture and a monoclonal antibody to HSA with HRP-labeled conjugate for detection.The optimal concentration of the first coating antibody and detection antibody were 2 μg/mL and 0.5 μg/mL,respectively.Regression equation of the linear calibration curve was:y = 0.442 9 x-1.143 3 with a correlation coefficient of 0.996 6,and the linear detection ranged from 39.06 ng/mL to 1 250 ng/mL.Recovery from the supernatant of fermentation broth was 98.13% to 102.94%.The specificity assay indicated that it had little cross-reactions with IL-2 and HSA.The soundness analysis suggested that fermentation broth,mouse serum and dilution had no influence on the method.The present method can be used in the studies on fermentation,purification and clinical diagnosis.

10.
Article in Chinese | WPRIM | ID: wpr-415253

ABSTRACT

According to the requirement of the national assessment for achieving the infection control criteria, 42 villages (among them,25 villages belonged to the first stratum, and 17 villages belonged to the second stratum) in 14 counties from 5 provinces, including Hunnan, Hubei, Jiangxi, Anhui and Yunnan, were selected as sampling villages for the assessment.The results from the field assessment showed that 154 out of 9 067 people were found infected with Sckistosoma japonicum, with an average infection rate of 1.7% ranged from 0.31 % to 4.10% , and only Yongping Village from Weishan County and Tenglong Village from Eryuan County were not found any case. A total of 46 out of 3 323 head of cattle were infected with S. japonicum, with an average infection rate of 1.38% ranged from 0.26% to 3.79% , and no any infected individual detected in Nanling County. No outbreak occurred in those sampling villages. Therefore, it is indicated that the five sampling provinces have reached the national criteria on infection control of schistosomiasis.

11.
Chinese Journal of Biotechnology ; (12): 1407-1412, 2008.
Article in Chinese | WPRIM | ID: wpr-275370

ABSTRACT

The gene of beta-glucuronidase from Thermotoga maritima was cloned into the plasmid pHsh, and expressed in Escherichia coli JM109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 65.9 kD. The optimal activity of beta-glucuronidase was found at pH 5.0 and 80 degrees C. The purified enzyme was stable over a pH range from 5.8 to 8.2 and had a half life of 2 h at 80 degrees C. The kinetic experiments at 80 degrees C with p-nitrophenyl-beta- glucuronide as substrate gave a K(m) and V(max) of 0.18 mmol/L and 312 u per mg of protein. The purified enzyme could transform glycyrrhizin to glycyrrhetinic acid.


Subject(s)
Cloning, Molecular , Enzyme Stability , Escherichia coli , Genetics , Glucuronidase , Genetics , Metabolism , Glycyrrhetinic Acid , Metabolism , Glycyrrhizic Acid , Metabolism , Hot Temperature , Kinetics , Recombinant Proteins , Genetics , Metabolism , Thermotoga maritima , Genetics
12.
Article in Chinese | WPRIM | ID: wpr-548742

ABSTRACT

Objective To identify the problem in the diagnosis and report of different types of malaria in China and provide evidence for making malaria control and elimination strategy and measurement. Methods All data are obtained from Disease Reporting Information System of China CDC, and the percentages, diagnosis methods and onset-diagnosis duration are described and compared among different malaria types and various report institutions. Results The overall number of reported cases declined by 71.77% during 2005-2008, with a relatively high percent for type-unconfirmed malaria. The percentages of laboratory diagnosis for falciparum malaria and vivax malaria were 91.44% and 71.14%, respectively. 28.22% of vivax malaria were diagnosed by using clinical diagnosis method, and 22.45% of type-unconfirmed malaria by using laboratory diagnosis method. 37.54% of falciparum malaria and 71.79% of vivax malaria were reported by hospitals in villages and towns, and 33.41% of falciparum malaria by general hospitals. The onset-diagnosis durations for falciparum malaria, vivax malaria and type-unconfirmed malaria were 72 h, 96 h and 72 h, respectively. Conclusions The laboratory diagnosis level is not good enough, though it is the main method for malaria diagnosis in China. The ability of laboratory diagnosis for malaria should be further strengthened in gross root medical institutions to reach the goal of malaria elimination.

13.
China Pharmacy ; (12)2005.
Article in Chinese | WPRIM | ID: wpr-528346

ABSTRACT

OBJECTIVE: To evaluate the long-term toxicity of Anti-EB-virus oral liquid on beagle dogs, and to provide safety evidences for clinical experiment. METHODS: Of the total beagle dogs, three groups totaled 18 were assigned to receive Anti-EB-virus oral liquid at high (76.41 g/kg), medium (25.47 g/kg) and low dosage (8.49 g/kg), and 6 (blank control group) to receive placebo. The ratio of male to female in each group was 1∶1. The beagle dogs were administered intragastrically with drugs qd, 6 days in a week for up to 26 weeks. All the indicators were monitored and the recovery of the beagle dogs was observed. RESULTS: Dogs in high dose group vomited obviously. Gastrointestinal irritation was also noted in medium dose subgroup, but was less severe than the high dose subgroup. During the recovery stage, one dog in high dose group was strong positive in urinary protein test and one in medium dose group was strong positive in urinary occult blood test. No significant drug associated toxic reactions were noted from beagle dogs' body weight, temperature, appetite, ECG, hematology test, blood biochemical analysis, ophthalmology test, marrow test, routine urianlysis, histopathologic examination, etc. CONCLUSION: The non-toxic dose of Anti-EB-Virus Liquid on beagle dogs was 8.49 g/kg in crude drug.

14.
Article in Chinese | WPRIM | ID: wpr-572173

ABSTRACT

Object To study the inhibitory effect of Anti-EB Virus Liquor (AEVL) on EB (Epstein-Barr) virus antigen expression and its cytotoxicity. Methods The effect of AEVL on Raji cell early antigen (EA) expression and B 95-8 cell virus capsid antigen (VCA) expression was assayed by indirect fluorescent technique; the cytotoxicity on nasopharyngeal carcinoma CNE 2 cells was determined by MTT method. Results At the non-toxic concentration, AEVL had markedly inhibitory effect on Raji cell EB-virus, IC 50 was 0.667 mg/mL and showed powerfully inhibitory effect on B 95-8 cell EB-virus VCA expression, IC 50 was 0.89 mg/mL; and it had strongly inhibitory effect on B 95-8 cell EB virus VCA expression stimulated by sodium n-butyric acid, IC 50 was 1.4 mg/mL. The IC 50 of AEVL on human nasopharyngeal carcinoma CNE 2 cell was 7.57 mg/mL. Conclusion AEVL could inhibit EB virus antigen expression and have cytotoxicity on nasopharyngel carcinoma cells at high concentration.

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