Abstract In order to study the optimal expression and purification condition of the GLP-1 derived polypeptide, by PCR technology synthetizing the gene of the GLP-1 derived polypeptide with preference codon of E.coli with the plasmid containing human wild-type GLP-1 gene. Expressed fusion proteins were purified and desalted with Ni-NTA column and C18 Sep-Pak column, respectively. After chemical cleavaged by formic acid hydroformicant, the hydrolysis products were purified with Ni-NTA column and HPLC. The target peptide was identified by mass spectrum. Experiment results showed in E.coli BL21 the optimal expression condition as follow：inducing temperature is 37℃, inducing time is 6h, and the concentration of the IPTG is 0.6mmol/L. The optimal chromatographic condition of getting HPLC as follow：mobile phase A (10% CNCH3∶90% H2O,0.1％TFA)，mobile phase B(100% CNCH3,0.1% TFA),flow rate is 1ml/min, 30 min of the linear gradient elution, B phase reaches 70% and the detection wave length is 280nm. The molecular mass of the GLP-1 derived polypeptide is 5.492 kDa,through mass spectrum identification. The yield of GLP-1 derived polypeptide prepared with the optimal expression and purification condition may reach 11.6mg/L fermentation product and its purity is equal or greater than 98%.
Abstract To investigate the inhibitory effects of siRNA expression vector on proto-oncogene Pokemon expression in SW480 cells and to provide experimental basis for further research about the biological function of Pokemon. siRNA expression vectors were constructed to express a short hairpin RNA to Pokemon. The recombinants were transfected into SW480 cells with liposome. Cellular morphology and transfection efficiency were observed. The expression of Pokemon were checked by real-time fluorescence quantitative PCR and Western blot. The transfection efficiency was about 36% and the cellular morphology changed greatly at 24h after transfection. siRNA expression vectors could specific reduce the expression of Pokemon mRNA and protein in SW480 cells. Compared with negative control, the inhibition ratio of Pokemon mRNA expression was 67.7% and the inhibition ratio of Pokemon protein was 73.6% respectively. siRNA expression vectors were successfully established that could effectively inhibit the expression of Pokemon in SW480 cells.
Objective This study was aimed to construct and identify the mammalian expression vector of pCAG-IRES-SHIP-GFP and to detect whether it could express in human acute leukemia cell line K562. Methods The cDNA fragment of SHIP obtained by RT-PCR was inserted into pCAG-IRES-GFP. The recombinant plasmid was confirmed by restriction enzyme digesiton, PCR and DNA sequecing. pCAG-IRES-SHIP-GFP was transfected into K562 cells with lipofectamine 2000. The expression of SHIP was determined by GFP fluorescence and Western blot analysis. FQ-PCR was used to quantitate SHIP mRNA. The expression of p-Akt、Akt were determined by Western blot. PI were tested by flow cytometry and MTT to verify whether exogenous SHIP could inhibit proliferation of K562 cells. Results The correct constrution of the recombinant plasmid pCAG-IRES-SHIP-GFP has been shown by restriction enzyme digestion, PCR and DNA sequencing. pCAG-IRES-SHIP-GFP could express SHIP protein in k562 cells. The K562 cells viability after transfected with SHIP gene droped down. Western blot analysis showed that phospha-Akt308 and Akt473 were reduced to 38.7% and 68% respectively. Conclusions The vector of pCAG-IRES-SHIP-GFP has been successfully constructed and it can be expressed in K562 cells. The expression of exogenous SHIP gene can lead to apoptosis of K562 cells by down-regulating the p-Akt expression. What found here might be one of the mechanisms involved in the pathogenesis of leukemia.
Human Interleukin-11(hIL-11) has no Cys residue in its natural form. By site-directed mutagenesis ,a Cys residue can be introduced to replace the 1st residue Gly and the rhIL11 was chemically modified by using 20 kDa mPEG-maleimide conjugated to this site. The mPEG-hIL-11 conjugate was purified and showed a single band on SDS-PAGE with an apparent molecular weight. The biological activity of purified mPEG-hIL-11 was determined using a dependent cell line 7TD1. The remaining biological activity of PEGylated-rhIL-11 was 30% of native rhIL-11, suggesting chemical modification of rhIL-11 by PEG is a promising approach for improving the pharmacological efficacy.
[ABSTRACT] Aim：to detect how Rb-deficiency will affect responses to TGF-β induced cell cycle arrest of hepatocyte. Methods: primary hepatocytes were isolated and cultured, and Rb-specific adenovirus siRNA was transformed into cells. Then TGF-β was added daily and cell growth and cell cycles variations. Western blot and FQ-RT-PCR were adopted to detect pRb, E2F, c-MYC and p16 expression changes. Results: Primary hepatocytes were isolated and infected withRb-specific siRNA adenovirus. In control cells, treatment with TGFβ prevented cells to enter S phase via decreased cMYC activity, activation of P16INK4A activity. In Rb-null hepatocytes, E2F and cMYC activity decreased slightly but P16INK4A was not activated and the great majority of cells continued cycling. Rb is therefore central to TGFβ-induced cell cycle arrest in hepatocytes. Conclusion: The present results show that otherwise genetically normal hepatocytes with disabled Rb genes respond less well to the antiproliferative effects of TGF-β.
Objective : To construct recombinant plasmid containing hp0525 gene of Helicobacter pylori ( H. pylori ) NCTC 11637 , and analysis of sequence nucleic acid, express it in E.coliBL21 and study the influence of the HP0525 protein on proliferation of SGC-7901 cells . Methods H. pylor hp0525 gene was amplified from the genome DNA by PCR,then operated T-A cloning and sequenced. The hp0525gene fragment was inserted directionally into vector pMD18-T to construct recombinant clones of hp0525 and was sequenced. The recombinant plasmid was transformed into E.coli BL21for expressing under induction of IPTG. Purify the expressed protein by Ni2＋-NTA column chromatography. Expressed product was analyzed by Western blot and MALDI-TOF. Added the purified protein into SGC-7901 cells, the effect of cell proliferation in SGC-7901 cells induced by the recombinant protein was observed by MTT. Results A 993 base pairs long hp0525 gene, which encodes a product of 330 amino acid, was obtained using PCR method and was cloned into pMD18-Tvector successfully. The sequence analysis for hp0525 showed that it shares 97%-99% homology with other strains in Gene bank. SDS-PAGE showed a protein band with a relative molecular weight of 36 000, which was consistent with the expectation. The expressed product reached a purity of 97% after Ni2＋-NTA column chromatography. The protein after dialyzed and annealed , was co-cultured with SGC-7901 cells, The protien of different concentration co-cultured with SGC-7901 cells for different times, found that the protein in low concentration stimulates proliferation of cells, to achieve some concentration, it inhibits proliferation of cells along with multiplication of the concentration of the protein; The protein inhibits proliferation of cells relay on the extension of time and concentration. Conclusion It is indicated that we have obtained the correct hp0525 gene and expressed in E.coli BL21. We obtained high purified protein by Ni2＋-NTA column chromatography, immunized serum was tested by indirect ELISA and the titers were up to 1：8000. the protein can inhibits proliferation of SGC-7901cells , and T-ATPase activity which posed a basis for further researching on its biological function.
Study on DC-SIGN protein expression and its biological activity measurement TANG Hui1 TANG Shao-hui1 YANG Dong-hua1 LV Zheng-bing2 YU Wei2 ZHANG Yao-zhou2 ZHOU Tian-hong3 XIAO Xin4 （1The First Affiliated Hospital，Jinan University,Guangzhou 510632,China）;（2（Institute of Biochemistry,Zhejiang Sci-Tech University,Hangzhou310018 ,China）;（3College of Life Science and Technology,Jinan University,Guangzhou 510632 ,China）;(4the Sixth Affiliated Hospital,Sun Yat-sen University,Guangzhou 510655,China ） [Abastract] Objective:To construct expression system of Bombyx mori baculovirus expressing DC-SIGN protein fragmen, and to measure the biological activity of it. Methods：Human DC-SIGN cDNA was amplied by RT-PCR from the differentiated DCs.The DC-SIGN gene was inserted into the bacubvirus transfer vector pBacPAK8,and the recombiant plasmid pBacPAK8-DC-SIGN were cotransfected into Bombyx mori cell lines with the linearized Bm-BacPAK6 virus genome DNA.After recombination in the cells and screening by the plaque assay, the recombinated virus Bm-BacPAK-DC-SIGN were obtained and again infected into Bombyx mori cell lines to express DC-SIGN protein fragmen which were detected by Western blot. The biological activity of the expressed DC-SIGN protein fragmen was measured by its combination with HIV-1 gp120.Results:Expression system of Bombyx mori baculovirus expressing human DC-SIGN protein fragment was constructed, Human DC-SIGN protein fragment was expressed and it can bind specifically with HIV-1 gp120.Conclusion: Human DC-SIGN protein fragment was successfully expressed in expression system of Bombyx mori baculovirus, which showed the natural DC-SIGN protein-like biological activity. It became the work basis of the antibody preparation against it and drug study on prevention and treatment of AIDS. [Key words]: Human immunodeficiency virus；DC-SIGN；Protein expression；biological activity measurement
Objective To clone and express TpN47、TpN17、TpN44.5 and TpN15 out membrane protein of Treponema pallidum, then develop an one-step double antigen sandwich enzyme-linked immunosorbent assay(ELISA) procedure on detecting Tp antibody. Methods The immuno-dominant epotope of TpN47、TpN17、TpN44.5 and TpN15 was amplified by PCR and subcloned into the expression pET-HT-JKM, the recombinant protein was expressed in BL21(DE3)plysS and purified with Ni-NTA affinity chromatography columns. Sandwich antigen ELISA was developed to detect the antibody of Tp in human sera. Results 30 control sera was tested by ELISA. Both the sensitivities and specificities were 100%. While detecting 385 T. pallidum human sera, the sensitivities of ELISA was 98.2% and the specificities was 99.5% compared with the results of the TPPA tests(P＜0.01). The concordance of results between the ELISA test and the TPPA test was 99%. Conclusion The TpN47、TpN17、TpN44.5 and TpN15 out membrane protein of Treponema pallidum were suitable for development of ELISA for serodiagnosis of syphilitics.
Abstract The biosynthesis cluster (ste) of a novel exololysaccharide called Ebosin producing by Streptomyces had been identified previously. The results showed that the gene products of ste15 and ste22 were glucosyltransferase and rhamnosyltransferase respectively. In this study, the ste22 gene was disrupted with a double crossover via homologous recombination in the mutant strain Streptomyces sp. 139 (ste15 -). The mutant strain Streptomyces sp.139 (ste15 –ste22 -) was identified by Southern blot and gene complementation also performed. Compared with Ebosin, the glucose and rhamnose of EPS15-22m produced by Streptomyces sp.139 (ste15 –ste22 -) were reduced obviously, it’s Mp and the antagonist activity for IL-1R decreased. Glucose, rhamnose and the antagonist activity for IL-1R were recovered in EPS15-22c producing by the gene complemented strain. This study elucidated that genes ste15 and ste22 play essential roles in the formation of repeating units of sugars during Ebosin biosynthesis. The activities of Ebosin new derivatives produced by the mutants have been studied further both in vitro and in vivo.
An Alternaria tenuissima cDNA yeast expression library was constructed The MAPK kinase PBS2 was isolated from an Alternaria tenuissima cDNA expression library, designated as AtPBS2. It has a size of 2,492 bases in length, encoded a protein of 683 amino acids. The AtPbs2p amino acid sequence shows 52%、52%、49% and 47% identities with AfPbs2p (XP_752961) of Aspergillus fumigatu、MGCH7 (XP_001522946) of Magnaporthe grisea and ScHog1p (EDN63254) of Saccharomyces cerevisiae, respectively. AtPBS2 cDNA sequences could complement the functions of S. cerevisiae ScPBS2 genes in sodium tolerance, suggesting a functional HOG pathway exists in A. tenuissima, AtPBS2 gene was involved in the stress adaptation regulation of A. tenuissima.
XYL1 gene, which encodes xylose reductase with dual coenzyme activity from Candida tropicalis, was transformed into Pichia pastoris X-33 by expression vector pGAPZB. The recombination strain was immobilized in Ca-alginate beads and fermentation characterization is studied using corn cob hydrolysates. Fermentation conditions were as follow: initial pH value 6.0, 30℃, initial cell concentration of 20%, the Liquid volume of 28%, rotation speed 130r/min.The average xylitol yield was 37.5% on the optimum condition. This result is expected to provide a new alternative method for producing xylitol on a large scale by bioconversion.
Pinellia ternate (Thunb.) Breit is widely used as a kind of traditional Chinese herb for more than two thousands years in China. In our research, endophytic fungus were isolated from Pinellia ternate (Thunb.) Breit. Total proteins were extracted by organic solvent from the fermentation products of the endophytic fungus. The agglutination activity of proteins extracted from the endophytic fungus were studied by erythrocyte of rabbit, and the result showed that the protein from strains gs1 exhibited high lectin activity. The lectin extracted from the strains gs1 was purified by Mannan-Sepharose 4B affinity column. It was indicated by SDS-PAGE that the molecular of the lectin was about 12 kDa. The saccharide binding specificities were studied. The lectin from the strains gs1 showed high affinity with mannose. It was measured by Brandford method that there was 9.58 mg lectin per 1000 ml cultures from the the strains gs1. The agglutination activity of the lectin from the strains gs1 was studied with various erythrocytes, and the result showed that the lectin from the strains gs1 could agglutinate erythrocytes of rabbit, rat and mouse, but could not agglutinate erythrocytes of human (A\B\AB\O type) and chick. The strain gs1 should belong to Aspergillus according to its taxonomic characteristics of the colony on PDA medium.
Abstract：Lactobacillus plantarum are one of probiotics. It can been broadly used as a kind of natural food preservative in the future. This paper determined flowing-alkaline was the better method of the high cell density culture than phosphate buffer.And then，flowing-alkaline culture was explored detailedly and completely.In order to raise cell concentration ,effects of fed-batch fermentation on lactobacillus plantarum Lp-2 was studied. The maximum yields of Lactobacillus plantarum Lp-2 with pH feedback feeding model was up to 13.56g/L more than 90.05% of batch culture
Yeast strains with improved ethanol yield are important for efficient bioconversion of lignocellulosic biomass for fuel ethanol. In this study, Candida shehatae CICC1766 was adapted to 4% (v/v) ethanol, and then subjected to UV mutagenesis. One respiration deficient mutant Rd-5 with improved xylose fermentation capability was selected. Protoplasts of Rd-5 were inactivated by UV treatment, followed by the PEG-mediated protoplast fusion with a Saccharomyces cerevisiae strain with good ethanol-fermenting capability. The xylose fermenting capability of the fusants was investigated, and the fusant F6 demonstrated good ethanol fermentation performance, producing 18.75 g•L-1 ethanol from 50 g•L-1 xylose with an ethanol yield of 0.375 or 73.4% of its theoretical value of 0.511. Comparing with its parent Candida shehatae strain, the ethanol yield of F6 was increased by 28%.
pHsh is a novel high level expression vector of Escherichia coli, in which the regulatory promoters are recognized by the 32-kD sigma factor ( 32). The concentration of 32 protein in E.coli cells without pHsh vector peaks at about 5 min after a temperature shift from 30℃ to 42℃, and then declines rapidly to a very low level because of its degradation and inactivation caused by interaction between three negative regulatory proteins-DnaK, DnaJ, GrpE and 32, the whole process is about 12 min. In E.coli cells harboring recombinant high-copy pHsh vectors, the heat-shock response can sustain 4～10 h, which indicates the concentration of 32 protein was increased compared to that in E.coli cells without pHsh vectors. The increasing of the concentration of 32 is most likely due to the decreasing of the concentration of DnaK, DnaJ and GrpE. In this paper, the changes of the three negative regulatory proteins in Escherichia coli proteome caused by the existence of the pHsh expression vector were assayed by two-dimensional electrophoresis. The genes encoding the three negative regulative proteins were cloned into pET vectors and overexpressed. After the resulting recombinant proteins were partially purified, they acted as molecular markers in order to identify their positions in 2-D gels. After thermal induction and referring to standard 2-D gel map in SWISS-2DPAGE database, the proteomes of E.coli cells harboring pHsh-xynIII vectors obtained from different periods were analyzed by two-dimensional gel electrophoresis and compared to those of E.coli cells without pHsh-xynIII, and the DnaK and GrpE were detected in 2-D gels, the data showed the DnaK protein in pHsh+ cells was obviously decreased compared to that in cells without pHsh, therefore the result verified the foresaid hypothesis.
Model on secondary metabolite phenazine-1-carboxylic acid (PCA) fermentation nutrition conditions of gacA inactivate mutant Pseudomonas sp. M18G was constructed by Plackett-Burman design (PB) and Response Surface Method (RSM). In PB design four key components selected from 12 different factors have shown to play an important role for promoting PCA production. Center Composite Design (CCD) was adopted to establish a fermentation model for the four nutrition components using RSM. The optimal concentration of the four components based on analysis of regression equation were determined that soybean meal 33.4g/L , glucose 12.7g/L, soy peptone10.9g/L, ethanol13.8g/L, and the highest PCA production could reached 1.89g/L after 60h fermentation and the yield increased to 6 fold over that before optimization. The contour graphs depicted interactions of the two nutrition components showed that soybean meal and ethanol played an even more crucial role for the highest production of PCA in fermentation.
Phaeodactylum tricornutum is a potential source of biodiesel for its abundance in fatty acids. Moreover as its fatty acid composition contains much eicosapentaenoic acid (EPA, 20:5D5,8,11,14,17), P. tricornutum represents an interesting alternative source for the industrial production. The absence of an adapted transformation system has been a serious limitation for its genetic manipulation. A novel transformation method was firstly established using micro-particle bombardment in P. tricornutum. The results showed that the GUS gene, a reporter gene, was successfully expressed. Transgenetic cells presented blue color under the microscope after stained. In addition, different factors which influenced transformation were optimized. The experiment indicated that the best parameters for P. tricornutu transformation: 60 µg plasmid DNA was coated onto the tungsten particles, 1500psi rupture-disc pressures was applied at a bombardment distances of 6 cm.. This newly method overcame the obstacle of P. tricornutum rigid cell walls. The adoption of endogenetic fcp promoter facilitated the expression of foreign gene. The P. tricornutum was tested for sensitivity to 5 antibiotics: kananycin( Km), ampicillin (Amp), streptomycin (Str), neomycin(Nm) and chloramphenicol(Cm). The result of sensitivity test showed that P. tricornutum were not sensitive to Km, Amp, Str and Nm. Otherwise, P. tricornutum is highly sensitive to Cm, the half lethal concentration of Cm in liquid culture was 60μg/ml. It suggests that Cm can be suitable to select regent for P. tricornutum genetic engineering. This method can be used as a potential tool in the research of P. tricornutu gene engineering and modification of its fatty acids synthesis pathway.
Objective To develop an effectually secrete expression in Pichia pastoris and purification system of recombinant human Arginase(Arg), and to test the activity of recombinant Arg. Methods Human arginase gene in the correct reading frame inserted into Pichia expression vector (pPIC9) after signal peptide, recombinant Pichia expression plasmid pPIC-Arg was transformed it into the host strain GS115. Results An recombinant expression plasmid pPIC-Arg was generated successfully, and was identified by DNA sequencing; The recombinant protein was expressed in GS115 with high level, the recombinant arginase gene was expressed highly and secrete effectually in Pichia pastoris, and the expressed product had the normal bioactivity. The target protein can be released into the media, the expression of protein in the supernatants accounted for more than 80%. AfterUltrafiltration and gel filtration, the purity of recombinant Arg reached 95%, and the specific activity is about 310 IU/mg. Conclusions A set of protocols for high efficient the Pichia expression and purification has been established, which is simple, efficient and applicable.
Sequence alignment analysis indicated that bovine, porcine and fish collagen type I contain differential sequences, which might be used as marker peptides for gelatin differentiation. In this research, bovine, porcine and fish gelatin were digested by trypsin. The peptides in the digest mixtures were identified by high performance liquid chromatography/mass spectrometry (HPLC/MS). Marker peptides specific for fish collagen type I were found in the digested fish gelatin. Actual sample analysis indicated that fish gelatin might be identified according to the marker peptides detected. The effect of hydroxylation of proline and the molecular weight range of gelatin on the relative abundance of marker peptides were also investigated. This research demonstrated that fish gelatin can be differentiated by detection of marker peptides in the tryptic digest of sample gelatin.
Seven promoter trapping lines, which GUS activity were detected in embryo, were found by screening 2227 lines from an indica promoter trapping pool. Of which one line accession NO.W9154 was further analyzed.A single copy T-DNA insertion was indicated in the W9154 line by Southern blotting and its T-DNA inserted locus was located in the third intron of an unknown protein gene in chromosome 3 in rice. Analysis on the upstream regulation sequence of the trapped gene revealed that it contained seed-specific promoter elements, such as RY motif、E-box、AACA and so on, besides general promoter elements TATA-box and CAAT-box. Further analysis was conducted on expression pattern of the candidate gene, and it was found that the candidate was only expressed in embryo and stem, but not other tissues in wild type rice. This result was completely consistent with that of the GUS expression in the trapping line w9154.Taken together,the analyses indicated that the trapped gene of the line w9154 might be an embryo development-related gene in rice.
DNA microarray technology emerged since 1990s allows a parallel analysis for thousands of genes in a single experiment. With the many strain's whole genome sequenceing, DNA microarray technology as a primary high-throughput tool has been widely applicated in monitoring of gene expression patterns, such as gene function prediction, detection of gene mutations, polymorphism analysis and so on. More attention has been taken on the application of gene chip technology in the research of Lactic Acid Bacteria（LAB）, as for several LAB strains genome sequencing completed and a lots of sequences of EST, 16S/16S-23S rDNA obtained. The basic principle of the gene chip and the application progress of DNA microarray in identification and expression analysis of LAB were introduced in this paper, which would provide reference for further application and exploitation of LAB DNA microarray.
This article explains the importance of proteomics in the coherent study of human reproduction and animal breeding from the study of proteomics technology, proteomics of human infertility, sperm-egg binding and mutual recognition of the mechanism. It illustrates that proteomics has become one of the main branches of life sciences in the future development. This provides the technological means and theoretical foundation for the individual dynamic changes in the protein. At the same time,it plays an important role in the drug development, the mechanism of life activities and in the field of livestock breeding.
As a rapid development biological technology, PCR has been widely used in many related fields such as sex identification for it is rapid, sensitive, convenient and specific. The PCR method used in sex identification varies from simple PCR, nested PCR to modified two-temperature gradient PCR. Besides the gene sequences such as Sry, which only locates in the Y Chromosome, gene sequences like zinc finger protein which present different gender characteristics between different genders were also detected. This has provided a brand-new primer designing thought and PCR method for sex identification. With the development of the study, the PCR technique is bound to become more mature on the application of sex identification and sex control. Much more progress will be made on promoting the application of PCR to other related fields at the same time.
Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to their cognate tRNAs, thereby ensuring the faithful translation of genetic code. In addition to their enzymatic function, these ancient and conserved enzymes have been discovered to have many additional functions in mammalian cells. As a result, several application aspects have been found. Aminoacyl-tRNA synthetases have attracted interest as essential and novel targets involved in bacterial protein synthesis, and they are now being pursued as targets for new antibiotic drugs. Engineered aminoacyl-tRNA synthetases are uesed for site-specifically incorporation of non-natural amino acids. The results with animals suggest that therapeutic applications for many human diseases such as tumor are possible with the cognate tRNA synthetases.
Laccases are able to oxidise both phenolic and nonphenolic compounds. They have broad ranges of subsrate specificity. Therefore, they are very useful for applications on pulp bleaching, textile dye decolouration, effluent detoxification, bioremediation and biosensors. However, the lack of a large number of and low cost laccases has become one of the most important obstacles to commercial application. And one of main solutions to this problem is heterologous expression of a laccase gene.This article reviews heterologous expression of fungal laccase in both yeast expression systems and filamentous fungi expressons syetem, and emphasizes on the factors of influencing heterologous expression of laccase as well as the strategies to enhance laccase production.
Xylane is the major component in hemicellulose, a recycling biological resource, and could be used widely in industry. Xylane degradation involved in many xylanolytic enzymes, including xlynase, β-D-xylosidase, acetylxylane esterase, arabinase, α-glucuronidase, ferulic acid esterase,p-coumaric acid esterase, and et al. The advances in xylanolytic genes expression control was reviewed in the paper, including the effect of XlnR, CreA, different substrates, pH, HAP-CAAT complexes and et al, and the research in future was discussed at the end of the paper.
Introduced policy support and research outputs and market demand of stem cell in the major countries. Research papers and Patent of stem cell have been analyzed by SCI-EXPANDED Database and Derwent databases in 1995-2008,so as to provide a useful reference for China.
