Objective: The aimed is to address the question of how PLCE1 gene polymorphism or genotypes affect its expression by cloning human PLCE1 gene and constructing eukaryotic expression vector of PLCE1 rs2274223G-rs3765524T(PLCE1 Minor) and PLCE1 rs2274223A-rs3765524C(PLCE1 Major). Methods: Overlap extension PCR (SOE-PCR) and In-Fusion Technology were adopted to construct PLCE1 eukaryotic expression vector. The targeted gene was acquired by double point mutations based on homologous recombination. The expression of overexpressed PLCE1 by gene transfection was observed by real-time quantitative PCR and Western blotting. Results: The full-length human PLCE1 that contains 6 927 base pairs was acquired and confirmed by DNA sequencing. By comparing with the reference sequence (NM_016341) in NCBI database, it was found to exactly matched the reference sequence except a 18bp insertion in the region of 3 554~3 572bp. Additionally, the recombinant plasmids of two PLCE1 haplotypes, PLCE1 Minor and PLCE1 Major, were constructed respectively. By transfection, it was found that the expression levels of mRNA and protein of PLCE1 Minor were significantly higher than that of PLCE1 Major. Conclusion: A new PLCE1 transcript variant was discovered and the eukaryotic expression vectors of two haplotypes of human PLCE1 were successfully constructed. It was found that PLCE1 rs2274223G-rs3765524T promotes its mRNA and protein expression. These findings laid the foundation of revealing the correlation between PLCE1 single nucleotide polymorphisms (SNP) and cancer susceptibility.
Objective: To investigate the expression of miR-146a/b and related upstream regulatory mechanisms and downstream target proteins in human heptoma cells. Methods: The proliferation activities of the normal human hepatocyte cell line LO2 and human heptoma cell lines HepG2 and Hep3B were assayed by MTT. Total RNA and protein were extracted from the cells and the miR-146a/b levels were detected by RT-qPCR and protein levels of p-ERK, ERK, p-AKT, IκB, IRAK1, and TRAF6 were detected by Western blot. The activities of PI3K, ERK, AKT, and NF-κB signaling molecules were inhibited in Hep3B cells, respectively, and the corresponding expression levels of miR-146a/b and protein levels of IRAK1 and TRAF6 were detected. Results: The order of proliferation activities were Hep3B > HepG2 > LO2. Compared to LO2 and HepG2 cells, the expression levels of miR-146a and miR-146b increased significantly in Hep3B cells (P<0.01). Compared to LO2 cells, the expression levels of miR-146a decreased significantly in HepG2 cells (P<0.001). In Hep3B cells, the protein levels of p-ERK1, ERK1, p-AKT, IκB increased 10.87, 24.68, 6.67 and 1.92 times of LO2 cells, respectively; and the protein levels of IRAK1 and TRAF6 decreased 0.23 and 0.003 times of LO2 cells, respectively. In HepG2 cells, the protein levels of IκB and IRAK1 increased 4.46 and 2.69 times of LO2 cells, respectively. In Hep3B cells, inhibition of PI3K and AKT activity significantly reduced the expression levels of miR-146a/b at 12 h and 24 h (P<0.05). Inhibition of ERK and NF-κB activity had no effect on the expression levels of miR-146a/b at 12 h and 24 h. Inhibition of PI3K, AKT, ERK, and NF-κB signaling molecular activities elevated the TRAF6 and decreased the IRAK1 protein levels. Conclusions: PI3K/AKT signaling pathway down-regulates the protein levels of TRAF6 by up-regulating the expression levels of miR-146a/b in Hep3B cells. This mechanism provides valuable clue for researches on hepatocellular carcinogensis.
The synthetic recombinant human glucagon-like peptide-1(7~37) (rhGLP-1) analogue gene was cloned into prokaryotic expressing vector pGEX-4T-3 fused with glutathione-S-transferases (GST) to construct a recombinant plasmid pGEX-rhGLP-1 analogue, then transformed into E. coli BL21(DE3). The constructed E. coli BL21(DE3)/pGEX-rhGLP-1 analogue was induced by IPTG and proteins were expressed, and then broken to extract inclusion bodies by high pressure homogenizer. The inclusion bodies were collected through centrifugation. The rhGLP-1 analogue was purified by urea denaturation, Glutathione-Sepharose 4B affinity chromatography and digested by enterokinase. By further purification with SP-Sepharose FF chromatography and RP-C18 reverse phase chromatography, the target rhGLP-1 analogue was gained with purity more than 96% by HPLC analysis and molecular weight was consistent with theoretical value by MS analysis. The biological activity analysis showed that rhGLP-1 analogue stimulated cAMP production from HEK293 cells expressing GLP-1 receptors.
Objective: To clone the goldfish B cell activating factor (BAFF) gene and construct soluble BAFF expression vector, and investigate the effect of the expression product on goldfish lymphocytes B cells. Methods: The full-length cDNA of BAFF from the goldfish (designated CaBAFF) was cloned using RT-PCR techniques. Using Rapid amplification of cDNA ends (RACE) to obtain the full-length cDNA of CaBAFF including the 5' and 3' untranslated regions (UTRs), and its soluble BAFF gene (designated CasBAFF) was linked with SUMO, efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by Western blot analysis. Laser scanning confocal microscopy analysis showed that SUMO-CasBAFF could bind to the surfaces of goldfish lymphocytes B cells. In vitro, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) assay analysis if the purified Nus-His-CasBAFF protein could induce the survival/proliferation of goldfish splenic B cells. Results: The goldfish BAFF was successfully constructed and obtained a soluble Nus-His-CasBAFF protein. In vitro, the soluble Nus-His-CasBAFF protein could induce the survival/proliferation of goldfish splenic B cells. Conclusions: The purified soluble Nus-His-CasBAFF protein could induce the survival/proliferation of goldfish splenic B cells. These results about goldfish BAFF gene would provide a basis for understanding the characteristics of the immune system of the goldfish.
10-Hydroxy-2-decenoic acid (10-HDA),a major fatty acid component from royal jelly,is known to have various biologic activities in antibacteria, antitumour and lifespan-extending.However, it is unclear about the underlying molecular mechanism of its biosynthesis. A NADPH-cytochrome P450 reductase (EC 1.6.2.4, NADPH-cytochrome P450 reductase, CPR) gene from Apis mellifera was cloned and expressed in Escherichia coli. Subsequently, the enzymatic properties of CPR were analyzed. Results showed that the target protein was successfully expressed in E. coli after induced by IPTG, and its molecular weight was 86.29 kDa. The CPR protein was then purified by Ni-NTA protein affinity chromatography and its specific activity was 77.33 (EU of CPR)/μg.The purified CPR protein displayed the maximum activity at pH 8.0 and 40℃. Its activity was not significantly affected by several metal ions except Ni2+, Zn2+ and Co2+ which caused a strong inhibition. The Km and kcat, towards cytochrome C at 30℃, were 76μM and 268/min, respectively. It lay a foundation for researching the function of CPR in 10-HDA biosynthesis pathway.
To improve the thermotolerance of AoXyn11A, a glycoside hydrolase family 11 mesophilic xylanase from Aspergillus oryzae, the amino acid residue Tyr13(Y13) was replaced by Phe (F). Based on the multiple alignment of the primary structures of AoXyn11A and seven thermophilic xylanases, the homology modeling of AoXyn11A and molecular dynamics (MD) simulation on its three-dimensional structure, a mutant enzyme AoXyn11AY13F was designed. Using pPIC9K-Aoxyn11A as a template, a mutant xylanase-encoding gene Aoxyn11AY13F was constructed by mutating a Y13-encoding codon TAC of Aoxyn11A into a F-encoding TTC with PCR technique. Then, Aoxyn11A and Aoxyn11AY13F were expressed in Pichia pastoris GS115, respectively, and the thermotolerance of expressed recombinant products, AoXyn11A and AoXyn11AY13F, were analyzed. The results indicated that the temperature optimum (Topt) of AoXyn11AY13F was 55℃, higher than that (50℃) of AoXyn11A, and that the half-life at 50℃ (t1/250) was 95 min, which was 15 fold longer than that (t1/250=6 min) of AoXyn11A. The thermotolerance of wild-type AoXyn11A were significantly improved by site-directed mutagenesis of Y13F.
Nitrile hydratase is consisted of α- and β-subunit and regulated by the activator for its functional expression. To investigate the influence of different proportions of subunits and activator (17k) on recombinant expression, the nitrile hydratase (NHaseK) from Klebsiella oxytoca KCTC 1686 was studied and several recombinant strategies were constructed. Taking the advantage of two T7 promoters (T7 promoter-1 and T7 promoter-2) in pETDuet-1, the three gene segments of NHaseK were inserted into this plasmid in eight different arrangements. When all the three gene segments were expressed under T7 promoter-1, NHaseK showed a specific activity of 0.78 U/mg as α- and β-subunits equivalently expressed, and was 24% higher than at the ratio of α-and β-subunit as 5: 3. With this optimal condition, 17k was added following the T7 promoter-2, which enhanced the expression level of 17k by 2-folds and improved the activity to 0.82 U/mg. Noted that the activity reduced to 10% when subunits were separately in control of different promoters, indicating that the α-and β-subunits needed to be transcribed simultaneously. Further, the influence of plasmid copy number was investigated and the number of 15~20 was appropriate to express NHaseK in E. coli. In conclusion, the equivalent expression of subunits and the abundant expression of 17k were in favor of the functional expression of NHaseK.
Flavor compounds is one of the most factors for beer quality. In order to obtain the key factor which influences the formation of flavor from the metabolic level, metabonomics was used to analyze the connection between the lager yeast intracellular metabolites and flavor compounds. The content of flavor compounds and lager yeast intracellular metabolites were measured simultaneously during beer fermentation. Then using principal component analysis (PCA) and partial least squares (PLS) to process the high-throughput data. PCA results showed that phosphoric acid, trehalose, succinic acid, glutamine, aspartate and alanine contributed to the principal components significantly, which means that these metabolites varied obviously in different stages of fermentation. PLS results showed that the greatest impacting factor on flavor compounds was mainly intracellular amino acids, including serine, valine, threonine, lysine, alanine, leucine and asparagine. These results provide theoretical guidance for beer flavor control.
The esterase EST12-7 was identified from the deep sea of the South China Sea and was used to prepared (R)-ethyl 2-chloropropionate efficiently through asymmetric hydrolysis reactions. The effects of temperature, pH, substrate concentration, organic solvents and reaction time on the preparation of (R)-ethyl 2-chloropropinate catalyzed by EST12-7 were investigated. The results identified that the optimal working conditions for the preparation of (R)-ethyl 2-chloropropinate using deep-sea microbial esterase EST12-7 were that: 13.8 μg/ml esterase EST12-7, 50 mmol/L (±)-ethyl 2-chloropropinate, 2% n-decyl alcohol, pH 8.5, 30℃, 0.05 mol/L Tris-HCl, 60 min. Under optimal conditions, the conversion of (±)-ethyl 2-chloropropinate could reach 49% and the optical purity (e.e.s) of product (R)-ethyl 2-chloropropinate could reach 98%. After comparing the kinetic resolutions of racemic methyl 2-chloropropionate and racemic ethyl 2-chloropropinate by esterase EST12-7, the chain length of the 2-chloropropionate esters could greatly affect the kinetic resolutions catalyzed by esterase EST12-7.
Objective: To co-expression Ebola virus proteins GP and VP40 in mammalian cells and generate Ebola virus-like particles. Methods: According to amino acid sequences, the nucleotide sequences of Ebola-Zaire GP and VP40 genes were optimized based on the codon usage bias in mammalian cells. The synthesized genes were cloned into expression plasmids pcDNA3.1 or pBudCE4.1 which has two expression units, and then 293FT cells were transfected with the recombinant plasmids using lipofectamine2000. The expression of recombinant proteins was detected by Western blot, and the VLPs were observed by electron microscope. Results: Specific reactive bands recognized by anti-GP antibody were found in cells transfected with the recombinant plasmid carrying optimized GP gene. The expression of recombinant GP protein mediated by the plasmid pBudCE4.1/GP/VP40 which co-expressing GP and VP40 was significantly higher than that mediated by co-transfection with the plasmids pcDNA3.1/GP which expresses GP and pcDNA3.1/VP40 which expresses VP40. Besides, VP40 expression was detectable although the level was low. Classical Ebola virus-like particles were found under the observation with electron microscope. Conclusion: Ebola virus proteins GP and VP40 were successfully expressed and assembled into virus-like particles in 293FT cells, which laid an important foundation for further studies on the development of a vaccine or virus detection reagent.
The aim is to analyze the vitamin C one-step fermentation process, and construct one step fermentation system including three strains of K. vulgare, G. oxydans and B. endophyticus. It was found that B. endophyticus could promote the growth and 2-KGA production of K. vulgare. They could grow fast under D-sorbitol, and 10 hours later of the fermentation process, B. endophyticus could consume D-sorbitol constantly. In the 5L fermenter, when the temperature was 30℃, stir speed was 600r/min, and the ventilation was 1.5vvm, the conversion rate of one step fementation of K. vulgare, G. oxydans and B. endophyticus was higher. In the fed-batch fermentation process, the mass conversion rate reached 81.89%, and in the batch fermentation process, the mass conversion rate reached 87.90%, it means the production process was optimized futher.
Glycerokinase (GK) and glycerol-3-phosphate oxidase (GPO) were immobilized to develop glycerol enzyme membrane and enzyme electrode for determination of glycerol concentration. Results showed that the higher current output for glycerol was obtained using a 1: 1 (GK: GPO) ratio for the random immobilization. The efficient two-enzymatic membrane was prepared by immobilizing GK in nuclear pore film coupled with GPO on Biodyne B. Glycerol enzyme electrodes were realized by assembling the GK-GPO membranes previously prepared. In this way, the optimum pH was 7.0, and the fit range of temperature was 28~32℃. In the best experimental conditions, the linear scope was 0.05~9.00 g/L with excellent linearity and high stability. The recovery was 98.4%~102.4%, and its relative standard deviation (RSD) was <5%. There was no significant difference (P>0.05) in measurement results among glycerol enzyme electrode, Periodate oxidation method and HPLC.
In the field of synthetic biology, quick construction of target metabolic pathways and rapid screening of combinatorial libraries is of great significance. A combinatorial screening method by co-transforming Saccharomyces cerevisiae with three plasmids was established and XR-XDH pathway was constructed in Saccharomyces cerevisiae as an application of the method. The gene expression cassettes of XR,XDH and XK were constructed efficiently using the Yeast Golden Gate (yGG) method. 100 recombinant strains with different promoter combinations were obtained through the three plasmids co-transformation system. Then the colonies were screened through spot assay on 2% SX plate and 16 colonies were chosen. In order to make the property more stable, the separate three gene modules of the corresponding colonies were assembled together to the expression vector pRS426, and then transformed to BY4741 to obtain the new recombinant strains. Oxygen-limited fermentation was carried out to test these strains. Among the 16 strains, Sc-LQH35(TDH3p-XR-ACS2t-FBA1p-XDH-ENO2t-PDC1p-XK-ASC1t) showed the highest products yield and fastest xylose utilization speed. Under oxygen-limited condition, xylitol and ethanol could accumulate to 7.14 g/L and 5.92 g/L separately when the medium contained 20 g/L xylose. The strain Sc-LQH39(TDH3p-XR-ACS2t-FBA1p-XDH-ENO2t-ZEO1p-XK-ASC1t) showed strong ability of producing xylitol on the oxygen-limited fermentation and xylitol yield could reach as high as 0.71 g/g. Three-plasmid co-transformation combinatorial screening method realized the flexible construction and rapid screening of the xylose-utilizing strains. Strains with high fermenting performance were obtained, and it showed that the method has great potential application in construction and screening of the recombinant strains.
Recently, genomic engineering technology using programmable nucleases boomed. The CRISPR/Cas9 system that consists of endonuclease Cas9 and guide RNA (gRNA) is derived from the adaptive bacterial and archaea immune system. Cas9 endonuclease introduces the DNA double-stranded break with the guidance of gRNA, therefore it enables researchers to precisely and efficiently manipulate specific genomic locus. Meanwhile,this system can reveal the unknown roles of genes in disease processes, and has great potential for clinical therapeutic applications. Here current progresses in disease modeling and gene correction by CRISPR/Cas9 were reviewed.
The cytochrome P450(CYP450) play critical roles in the drug metabolism and are responsible for the metabolism of more than 80% of clinical drugs. Genetic polymorphism in different ethnic and population is the main cause for individual variation in drug metabolism. The new advancement between the genetic polymorphism and drug metabolism of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were reviewed, which can offer the scientific reference for clinical individual therapy and the development of new drug.
Improving plant quantity traits through transgenic technology has been increasingly concerned in modern genetic transformation field.With the rapid development of genetic engineering,single gene transformation technology is insufficient for the plant modifications.In order to get better plant traits by multigene regulation,more researchers have focused on the study of genetic transformation,making the genes involved in metabolic pathways coexpression in plants.There are four genome expression routes for genes selection and basically plant genetic transformation methods cover the traditional and improved transformation methods,emerging polygene fusion.The advantages and disadvantages of each method in plant metabolism regulation and application progress were analyzed comprehensively and the future development was discussed.
In recent years, the achievements of cell drug in the field of basic research have promoted the clinical application of cell drug. Currently there are many kinds of diseases that can be treated with cell drug, including nervous system diseases, autoimmune diseases, cardiovascular system diseases, blood system diseases, digestive system diseases, lower limb ischemia, plastic and cosmetic surgery, anti-aging and anti-tumor,etc. The involved cells mainly include different stem cells, cartilage cells, hepatic cells, DC and CIK cells. More and more domestic and foreign institutions and organizations have already carried out the clinical research and application of cell therapy.
