Objective: To express tumor targeting GP-CDD-iRGD fusion protein with specific fibroblast activation protein (FAPα) restricting sites in prokaryotic cells,purify the expressed product and determine its toxicity to FAPα positive and negative tumor cell lines, evaluate the feasibility to remove the fusion tag in vivo. Methods: Design and synthesis the gene of GP-CDD-iRGD, insert it into pGEX-4T3 vector. The recombinant plasmid was transformed into E. coli BL21 cells for expression under induction of IPTG. The expressed recombinant fusion protein was analyzed for expression level and form by SDS-PAGE, then purified by GST affinity tag protein purification kit, evaluate the anti-tumor activity and FAPα targeted cleavage effect through in vitro toxicity tests. Results: Restriction analysis and sequencing proved that recombinant plasmid pGEX-GP-CDD-iRGD was constructed correctly. Recombinant GP-CDD-iRGD fusion protein, with a relative molecular mass of about 36000, was solublely expressed and reached a purity of about 90% after purification.ED50 of the protein is about 18.5μmol/L. Conclusion: It showed significant toxicity to FAPα-positive 4T1 cells while FAPα-negative cell lines with no effect which established a foundation of further reasearch of its antitumor activity in vivo.
Human Astrocyte elevated gene-1 (AEG-1) encodes 582 amino acid residues and locates at chromosome 8q22. AEG-1 involves in multiple signal pathways and represents an important genetic determinant regulating multiple events in tumorigenesis and development. In order to further explore its role in the development of malignant tumors, recombinant AEG-1 protein was used to immunize the BALB/c mice. At the end of immunization, the mouse spleen cells were fused with SP/20 cells to develop hybridoma cells, and after ELISA screening with AEG-1 protein coated plate, an anti-human AEG-1 monoclonal antibody secreting hybridoma cell line was obtained and named as 1E3. The results of Western blot and Immunocellchemistry/Immunohistochemistry detecting showed that this AEG-1 monoclonal antibody could special reacted with AEG-1 protein in cancer cells. Then, the variable region fragment genes of anti-AEG-1 antibody from 1E3 cells were amplified by RT-PCR method, and both the mRNA and protein sequences were analyzed and compared in BLAST, which indentified them as mouse-derived IgG variable heavy and light chains(VH and VL). Moreover, structural analysis of these VH and VL genes were also did by using Kabat online analysis system, and confirmed that both VH (encoding 117 amino acids) and VL (encoding 119 amino acids) genes had complete FWRs and CDRs regions. All these results have perhaps laid a foundation for further study of AEG-1 function in the development of malignant tumors and the potential use of this bio-marker in the clinical diagnosis of malignant tumors.
Objective: Examine the anti-fibrotic effect of a novel truncated transforming growth factor-β typeⅡ receptor (tTGF-βRⅡ) in rat hepatic fibrosis model.Methods: The recombinant truncated type TGF-betaⅡ receptor proteins was obtained from the Escherichia coli BL21 strain containing recombinant truncated TGF-betaⅡ receptor vector, by cultivating, inducing, and purification. The rat model of CCL4-induced hepatic fibrosis was established to assess the effect of the tTGF-βRⅡ protein on the treatment of fibrosis. SDS-PAGE detect the purification of truncated TGF-betaⅡ receptor proteins. The mRNA expression levels of α-SMA,Col1 and Col4 were detected by real-time PCR analysis in hepatic tissue. The content of serum ALT and AST were checked with automated biochemistry analyzer. The expression of α-SMA,Col1 and Col4 protein in hepatic tissue was detected by immunofluorescence. Liver fibrosis of the rats was evaluated by three histological methods: HE staining,Masson staining and PAS staining. Results: SDS-PAGE detection results show that the purified recombinant truncated TGF-beta Ⅱ receptor proteins was obtained. Histological examination revealed that the tTGF-βRⅡ protein prevented progression of hepatic fibrosis. The tTGF-βRⅡ protein reduced activities of serum ALT and AST. The tTGF-βRⅡ protein reduced both mRNA and protein expression of α-SMA,Col1 and Col4 in the liver tissue. The results of three histopathological staining shows that the treatment group significantly reduce the degree of liver fibrosis.Conclusion: The tTGF-βRⅡ protein prevented progression of hepatic fibrosis in rat and provided a potential treatment for liver fibrosis and other fibrosis diseases.
Objective: Helicobacter pylori (Hp) urease is an important colonization factor as well as a pathogenic factor, and the enzymatic activity sites of Hp urease locate in the Hp urease B subunit (UreB). Research and development of Hp vaccine based on urease is a promising strategy for the prevention of Hp infection. The aims are to obtain recombinant Hp urease with high purity, and study its immunological properties. Methods:The UreB gene was obtained from Helicobacter pylori standard strain SS1 (Hp SS1), and the expression vector pET-rUreB and the recombinant strain BL21(DE3)/pET-rUreB were also constructed by gene cloning technology. After protein expression and optimization, the recombinant protein rUreB was purified by Ni2+-charged column chromatography and anion-exchange chromatography using DEAE Sepharose FF. Then, the immunological properties of rUreB protein was investigated by intraperitoneal immunization experiments in BALB/c mice with Freund's adjuvant. Results:Hp UreB gene was obtained successfully from Hp SS1 through PCR, and the expression vector pET-rUreB and recombinant strain BL21(DE3)/pET-rUreB were constructed successfully by gene cloning technology. After protein expression optimization and purification, about 69 mg of pure target protein was obtained from 1 L of fermentation broth and the purity of rUreB was 96.5%. Mice immunized with rUreB using Freund's adjuvant could induce high level of antibodies specific for both natural Hp urease and UreB by ELISA, which can significantly inhibit the activity of Hp urease. Conclusion:The recombinant UreB was expressed at a medium level in E. coli and had good immunological specificity. Besides, the antibodies induced by rUreB can effectively inhibit the activity of Hp urease. This will provide an experimental basis for the development of Hp vaccine based on urease.
Objective: To express and purify the Kunitz type serine protease inhibitor IsKuI-1 (GenBank NO. AAY66517) from E. coli, and analyze its anticoagulation and anti-protease activity. Methods: The prokaryotic expression plasmid pET32a-sumo/IsKuI-1 was constructed and then transferred into E. coli BL21 (DE3). The fusion protein Trx-SUMO-IsKuI-1 was expressed after inducing with IPTG and purified by Ni-NTA resin affinity chromatography. The fusion tag was cleaved with SUMO protease on the resin bed. Prothrombin time and activated partial thromboplastin time assay was used to detect the anticoagulant activity and a single stage chromogenic assay was used to determine the inhibitory activity against serine proteases. Results: rIsKuI-1 was obtained successfully using the prokaryotic expression system and showed no prolongation of clot time. rIsKuI-1 has potential inhibitory activity against human neutrophil elastase (IC50 =1.83 μmol/L), but no inhibition of human cathepsin G, human pancreatic chymotrypsin, human pancreatic trypsin, human chymase, bovine pancreatic α-chymotrypsin and porcine pancreatic trypsin. Conclusion: IsKuI-1 is an inhibitor of human neutrophil elastase. So it laid a foundation for the further investigation of the biological function and application of IsKuI-1.
Thermophilic Esterase 2 (EST2) from Alicyclobacillus acidocaldarius belongs to the HSL group of the esterase/lipase superfamily. It has been reported to be the most active hyperthermophilic esterase with excellent thermostability, which makes it a good choice for many industrial applications, such as food industry, fine chemical industry, pulp and paper industry and environmental bio-mediation. However, the biomass yield and the basal enzyme expression level are remarkably low in the cultivation of hyperthermophiles, which results in the high cost for the large-scale production of EST2 in its natural host. Heterogenous production of EST2 in a proper host organism holds great potential to solve this problem. E. coli and Pichia pastoris are the most widely used hosts for the production of heterologous proteins. In order to establish the high-level expression system, the gene encoding EST2 was cloned and expressed in Pichia pastoris and E.coli, and the enzymatic properities of the recombinant proteins were systematically characterized and compared. The recombinant EST2 expressed in E.coli and Pichia pastoris showed very similar properties: the optimal temperatures were 75℃ and 77.5℃, the optimal pH were both 8.0, the specific activities were 4656.6 U/mg and 4078.3 U/mg, and the enzymes were stable up after incubation at 70℃ for 4.5h. After the optimized cultivation conditions in the shake flake, the expression of EST2 in different hosts was conducted in a 5 L fermentor by high cell-density fermentation. In Pichia pastoris, the recombinant EST2 was predominantly expressed extracellularly with activity of about 960 U/ml and a cell dry weight reached 68g/L after induction with methanol for 90h. On the other hand, the EST2 activity increased to 14825.6U/ml after 25h of fermentation in E.coli, with a cell dry weight of 60g/L. The expression level and productivity in E.coli are 14.4-fold and 73.2-fold higher than that of Pichia pastoris, respectively. The results illustrated that E.coli is more suited for the high-level expression of EST2 than Pichia pastoris, which will further promote wider application of EST2 in biotech industries.
A recombinant plasmid pET-DsbA/PPFE-I was constructed and used as template to amplify fibrinolytic enzyme gene PPFE-I. This was then used in Pichia pastoris expression vector pPICZαA / PPFE-I; pPICZαA / PPFE-I and integrated into the genome of Pichia pastoris SMD1168 by electroporation. Results showed that after methanol induction for 72h, enzyme activity was 286 IU / ml. When compared to the wild type, the enzyme activity had improved 2.6 fold. SDS-PAGE electrophoresis analysis showed that the recombinant fibrinolytic enzyme (rPPFE-I) was expressed. In human fibrin degradation test rPPFE-I was used to firstly degrade the α chain human fibrinogen, followed by β chain, while the γ chain degradation was slowest. Endogenous Paenibacillus polymyxa fibrinolytic enzyme gene expression in Pichia pastoris was achieved, this would provide a new way to develop thrombolytic drug from endophyte sources.
Scenedesmus acuminatus, a new isolated freshwater green microalga, could be potentially used as feedstock for biodiesel production. The effects of nitrate concentration (3.6 mmol/L, 6 mmol/L, 9 mmol/L, and 18 mmol/L) on the growth and photosynthetic physiology of the algae was investigated with column photobioreactors. The results showed that nitrogen concentration exerted considerable influence on the growth of S. acuminatus, and the maximum biomass of 5.19 g/L was obtained under 6 mmol/L nitrate experimental group. The content of chlorophyll a, b, and total carotenoids of S. acuminatus were positively correlated with nitrogen concentration. The total lipid content of S. acuminatus increased remarkable during the whole culture period, and achieved its peak value of 54% of dry weight, which was 17% higher than that obtained under high nitrogen concentration (18 mmol/L). Meanwhile, the total carbohydrate and protein contents decreased significantly during the whole culture period. The maximum efficiency of light energy conversion of PSⅡ(Fv/Fm), relative electron transfer efficiency (ETR), and the actual energy conversion efficiency(Yield) decreased significantly as the nitrogen supply decreased. The photosynthetic oxygen release rate decreased, with a contrary increase of respiratory rate during the whole culture period. In conclusion, the growth and photosynthetic physiology of S. acuminatus were evidently influenced by the nitrogen concentration, and adjusted to their changing environment.
The HBsAg binding protein (SBP) was screened out from human liver cDNA phage expression library with HBsAg as a probe. SBP has the ability to enhance the immune response of Hepatitis B vaccine. It is proved to be a potential efficient immune adjuvant. The secretory SBP-expressing Pichia pastoris strains with high protein expression quantity has successfully constructed, then was fermented in the 18L fermentor. The results showed that SBP could achieve the maximum expression after 38h in methanol induction according to real time detecting; meanwhile hybrid proteins were the least so that it's the most appropriate time to collect bacteria liquid.15L supernatant could be obtained at low temperature centrifugation. Through the ultrafiltration membrane package whose interception was 5kDa, the supernatant was concentrated to about 2L. Then the concentrated fermentation supernatant was passed through sephadex S200 and the TDEAE column respectively. The attained 300ml SBP protein was with a purity of 98% and 1.125mg/ml as concentration. The SBP yield from fermented liquid was about 22.5mg/L.Finally, the lyophilized proteins were preserved at low temperature. The work on the fermentation process optimized the time, identified the purification process and lay a solid foundation for the mass production of SBP.
Objective: EH is a derivative of hirudin with three amino acids (EPR) at the amino terminal of hirudin. The expression of EH in yeast has long time of production and low efficiency of expression. To improve the production efficiency of EH, the soluble expression of EH in E. coli was studied. Method: Three recombinant prokaryotic expression engineering bacteria, BL21 (DE3)-pET-24-eh, BL21(DE3)-pET-22-eh, BL21(plySs)-pET-22-eh were constructed. The three engineering bacteria were cultured and EH expression was induced by IPTG. Results: The results of SDS-PAGE and Western blot showed that EH was expressed in intracellular soluble pattern in all three engineering bacteria, and the expression level is better in BL21(DE3)-pET-24-eh. Then in BL21(DE3)-pET-24-eh, the induction temperature, the induction time, the concentration of IPTG and the density of bacteria at induction time were optimized. The optimized condition is as follows: induction at 37 ℃ for 6 h, IPTG concentration is 0.4μmol/L, the bacteria density at induction time is OD600 = 1. Finally, the specific anticoagulant activity of purified EH protein (the purity is 96.93%) and the cleavage product of EH by FXa were determined. Conclusion: The results indicated that the intact EH has no anticoagulant activity, and after cleavage with FXa, EH released anticoagulant activity of hirudin. Therefore, the prokaryotic expression system of EH, and EH were constructed which can be expressed in an intracellular soluble pattern. These results will support the subsequent fermentation process and the ultimate industrialization of EH.
A suitable screening method to select thymidine phosphorylase high-product strains was quickly established, which was applied to the UV mutagenesis breeding. The results showed that adding 25mmol/L or less thymidine in the liquid medium did not affect the cell growth. The thymidine can be converted into thymine by the catalysis of thymidine phosphorylase in the bacterial and the OD290nm of product thymine is much higher than thymidine under the same concentration which can lead to significant increase of the fermentation broth. Based on the above principles, effective pre-screening method was established. Furtherly, the UV mutagenesis time was optimized as follows: initial mutagenesis time was 20s, after stopping 5min irradiate 10s, 15s or 20s to create mutant library by controlling the mortality rate about 80%. Through pre-screening and re-screening, the thymidine phosphorylase activity of EB27, EA42 could reach 1.025 U/mg wet cells and 0.916 U/mg wet cells, which was increased by 50% and 35% than that of the original strain. The high-product strains remained stable after transferred for 5 times.
To select algal strains with fast growth rate or high oil content, Isochrysis zhangjiangensis is mutated with atmospheric and room temperature plasmas (ARTP). The mutants are screened by well and flask at room temperature ordinary light conditions and stress conditions, such as high temperature ordinary light, high light ordinary temperature. An integrated screening method is set up. Through 600 ml bioreactor cultivated evaluation, the rate of the detection of mutants is 0.7% at room temperature ordinary light conditions, 1.1% at stress conditions, 0.6% through once mutagenesis and 1.2% through secondary mutagenesis. The results show that screening rate is high at secondary mutagenesis and stress conditions. It is easier to obtain mutants with character change.
Efficient utilization of xylose is an import microbial trait for fermentative production of bio-based products using renewable lignocellulosic feedstocks. Escherichia coli WL204 was previously engineered for fermentative production of optically pure L-lactic acid from xylose. E. coli WL204 was reengineered for D-lactic acid production by functionally replacing the l-lactate dehydrogenase gene (ldhL) with a D-lactate dehydrogenase (ldhA), resulting in strain E. coli LHY02. In 10% xylose fermentation test, LHY02 produced 84 g/L D-lactic acid with an optical purity of 99.5% and a volumetric productivity of 0.93 g/L/h.
2-keto-D-gluconic acid is an important precursor for D-isoascorbic acid and sodium D-isoascorbate, a significant antioxidant and food additive. Gluconobacter suboxydans has lots of oxidases in the periplasmic space and can be used to produce 2-keto-D-gluconic acid through two-step oxidation-reduction reactions: the oxidation from glucose to gluconic acid, catalyzed by the glucose dehydrogenase and the oxidation from gluconic acid to 2-keto-D-gluconic acid, catalyzed by the 2-gluconate dehydrogenas. To inactivate other by-product forming pathways and improving the production of 2-keto-D-gluconic acid, a genetically stable strain by integrating glucose dehydrogenase (gdh) and 2-gluconate dehydrogenase (ga-2-dh) genes into the chromosome of Gluconobacter suboxydans to replace glycerol dehydrogenase (gldh) and sorbitol dehydrogenase (sdh) gene respectively was constructed. By the verifying PCR, color reaction and HPLC, the characteristics of engineered strain indicated that gdh and ga-2-dh gene were strengthened and gldh and sdh gene were knocked out. Using compound medium with 10% glucose as carbon sources, the engineered strain could produce 72.3 g/L 2-keto-D-gluconic acid without by-products 5-keto- gluconic acid after culturing in shake flask for 72 h and its 2-keto-D-gluconic acid production increased 1.4 fold comparing to J12 wild type. Genetically stable strains constructed can metabolize glucose to produce2-keto-D-gluconic acid without the addition of antibiotics which is favorable for the industrial production.
With the completion of genome sequencing on more and more organisms, post-genomic era has begun. Gene trapping, as a powerful tool in the functional-genomic era, has been widespreadly applicated in many fields of biomedicine research because of its great merits in cloning, finding novel genes and unraveling their biological functions. The principles, classifications, manipulation procedures, advantages and disadvantages of gene trap technique and its application in developmental biology, novel gene identification, stem cells differentiation, cancer and reproductive medicine research are discussed, which will provide helpful references for relevant researchers.
Under the influence of energy crisis, environmental crisis and resources crisis, the development of green alternative energy attains more and more attention worldwide. Many studies have found that, compared to ethanol, isoamyl alcohol possesses higher energy density, lower vapor pressure, higher octane number and closer physical and chemical characteristics to gasoline.The technical barriers, process and key enzymes-KDCs about isoamyl alcohol production were reviewed. Finally, the future development tendency of the biosynthesis of isoamyl alcohol was also suggested.
The biobutanol industry is limited by yield, productivity and low proportion in acetone-butanol-ethanol fermentation. Strain improvement is considered as an important and basic strategy to solve the problems. In industry, many high-yield strains were obtained mainly by mutation breeding, in which compound mutation and rational screening are more efficient strategies. Genetic engineering breeding has achieved rapid development. The high-yield strains could be obtained by modification of the butanol pathway and other genes which inhibit butanol synthesis in Clostridium acetobutylicum, as well as construction and optimiztion of the butanol pathway in E. coli. However, the effects were still lower than those of mutation breeding. The most important strategy would be still to increase yield and proportion of butanol in the following strain improvement.
The marine has a largely source of novel compounds with potent anticancer activity and receives extensive attention from researchers. Recent studies in the field of marine anticancer drugs have revealed promising compounds with proven anticancer activity. Three examples are cytarabine, ecteinascidin-743 and eribulin mesylate, which represent the first three described marine anticancer drugs. In addition, there are a number of marine anticancer products in anticancer preclinical or clinical trials. The domestic studies of marine anticancer products play an important role in the international studies. But they are seriously out of line with the industrialization. The scientific advancements and development directions in marine anticancer drugs were examined. It analysis the industrialization problems in domestic marine anticancer drugs, such as insufficient development of marine anticancer resources, lack of intellectual property rights, insufficient capital investment, long period of clinical trials and so on. In order to solve the problems existing in industrial development and promote the healthy and orderly development of domestic marine anticancer drugs, highlights the viewpoint that the application rate of marine anticancer products can be increased greatly by the research system of autonomous intellectual property drug based on market demand and multidisciplinary technology in the "Industry-University-Institute" cooperation mode. The purpose is to put forward some useful suggestions for the industrialization of domestic marine anticancer drugs from the point of key technologies, market outlook and industrial policies.
