Heat shock protein 70 is essential for cellular recovery, survival and maintenance of cellular function. Heat shock protein 70 function as molecular chaperons in protein folding and it is a role of cardioprotection against various stresses. We have studied the different proteins expression of tissues from failing hearts using proteomics before. We found that HSP70 was highly expressed in the tissues and blood samples from heart failure patients compaired with non-failing hearts. For exploiting the use of HSP70 as a therapeutic marker, we sthudy the HSP70 expression using Neonatal rat ventricular cardiomyocytes(NRVM) by teating with TNF-a with various concentrations and time points in vitro. Primary cultures were prepared from neonatal rat ventricular myocytes. Cultured cardiomyocytes were incubated with TNF-a at doses of 40, 200 and 800U /ml for a dynamic time point as 1, 6, 12 and 24 hours. NRVM treated with heat shock(42℃) were used as positive control. The expression of myocyte HSP70 was quantified by Western blot analysis and immunohistochemistry using a specific monoclonal antibody. Immunohistochemical analysis revealed HSP70 were nearly not visible in control cells. However, at 12 h post-hypoxia/SD, HSP70-immunoreactivity (IR) was evident in the cells. These studies constitute the initial demonstration that TNF-alpha exerts concentration- and time-dependent effects on the expression of HSP 70 in NRVM. The expression of HSP70 influenced by TNF-a can indicate the possibility of a pharmacological approach to HSP70 induction and cardiac protection, which may ultimately be of clinical relevance.
Neurturin is a member of neurotrophic family, and the gene engineering product of the Neurturin can promote the survival of the dopaminergic neurons, which is a new approach to the therapy of Parkinson’s disease. However, a few researches have indicated that the wild type of Neurturin cannot be processed and secreted into the medium. Therefore, the study intends to rebuilt the gene of wtNeurturin and replaced the signal peptide of wtNeurturin by the counterpart of NGF. The hybrid Neurturin was inserted into the genome of adenovirus and packaged in AD-293 cells. The chimeric Neurturin could be expressed in cytoplasm of vero cells and be processed and secreted, which was detected by immunofluorescence and Western blotting. Finally, the culture of dorsal root ganglia of chick embryo test had indicated that Neurturin expressed by chimeric gene had good biological activity.
The radish phospholipid hydroperoxide glutathione peroxidase (RsPHGPx) fusion proteins expressed in Escherichia coli with high efficiency were purified and the antioxidant effects of RsPHGPx together with glutathione on hydroperoxide-mediated injury in mouse NIH3T3 fibroblasts were investigated. The cell viability measurement confirmed that 10μg/ml RsPHGPx, when added to the fibroblasts separately, can only slightly increase the cell viability. However, when combained with glutathione, the antioxidant effects increase dramatically. MDA assay suggests that RsPHGPx can scaveng the lipid peroxides, the results are accordance with the MTT results. ROS flourescence investigations and DAPI experiments show that RsPHGPx can increase the protection effect via inhibiting ROS genreation and this protection help maintain the normal nuclear and membrane conditions of NIH3T3 fibroblasts. This combain experiment of RsPHGPx and glutathione provides experimental evidences for the future production of RsPHGPx.
The focus is to evaluate the genetic stability and titer determination of the recombinant adenovirus (pAd-H5) expressing HA gene of H5N1 Avian Influenza virus (AIV). The recombinant adenovirus pAd-H5 had been serially passaged for 20 passages in 293 cell line. The passages of 5,10,15 and 20 were chosen to be identified by PCR and sequence; Using GFP rapid monitoring method to test the viral titer of pAd-H5, passage 20. Furthermore, the analysis supported that HA gene was amplified correctly, without any changes on the nucleotide in passages of 5, 10 and 15. While there was only one point mutation on HA gene (417 A→G) in passage 20, but this point mutation did not lead to amino acid changes (samesense mutation). The titer of pAdH5 in 293 cell line identified by GFP rapid monitoring method was 108.75pfu/0.1ml. All the results showed that the pAd-H5 has good genetic stability within 20 passages, and the titer was relativly high.
To construct an endophytic engineered bacterium, a strain of endophytic bacteria with resistance to banana Fusarium wilt and ability to degrade chitin was isolated from healthy banana plants. This strain was identified as Klebsiella based on its morphological, physiological and biochemical characteristics and 16S rDNA sequences analysis. The strain was named as KKWB-5. In order to obtain a secretion signal peptide, the chitinase II gene of KKWB-5 was cloned by PCR, and its signal peptide sequence was forecasted by Signal P3.0 Server. The chitinase gene was inserted into E.coli BL21(DE3) by pET22b to construct respectively the expression strain BL-chi1 and BL-chi2. The chitinase gene of BL-chi1 included the signal peptide sequence, while BL-chi2 removed it. After induced with IPTG, both BL-chi1 and BL-chi2 expressed a protein about 45KDa. The culture supernatant of BL-chi1 had a protein band about 45KDa too, while the culture supernatant of BL-chi2 and BL-pET22b control didn’t have. Biological activity determination showed that both concentrated liquid and purified liquid of the culture supernatant of BL-chi1 induced formed a clearing zone on the colloidal chitin plate. The results demonstrated the signal peptide sequence could be identified by E.coli BL21(DE3) and secrete the chitinase into culture supernatant, moreover, the secreted chitinase had biological activity. The isolation of KKWB-5 strain and cloning of signal peptide sequence of its chitinase served as a good foundation for further constructing engineered endophytic bacterium.
Cyanobacterial NADPH dehydrogenase (NDH-1) is an important photosynthetic membrane protein complex, and is essential to CO2 uptake, cyclic electrontransport around photosystem I and cellular respiration. The enzyme accepts electrons from NADPH and consists of at least 15 subunits, i.e., NdhA to NdhO.The NdhO is a newly identified subunit, and little is known regarding its roles in cyanobacteria. To obtain the ndhO gene inactivation mutant, the homologous recombination vector, pUC-ΔndhO, was constructed, and then this vector was transferred into wild type Synechocystis sp. strain PCC 6803 by using the natural transformation method. Further, after several subcultures, the transformations were examined by using the PCR and immunoblotting. The experimental results indicated that the kanamycin coding sequence had successfully inserted into the conservative region of ndhO gene, and completely inhibited the expression of ndhO gene. Therefore, an ndhO gene inactivation mutant, ΔndhO, has successfully been obtained, and it will further help us to reveal the roles of NdhO subunit in the NDH-1 complex and in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803.
In order to achieve efficient extracellular production of α-cyclodextrin glycosyltransferase (α-CGTase) , E.coli BL21 (DE3) carrying the α-cgt gene of Paenibacillus macerans JFB05-01 was investivated. By comparison of different induction conditions in complex and synthetic medium, the highest extracellular α-CGTase activity reached 113.0 U/mL (hydrolysis activity was 79100.0 IU/mL）at 30 h of culture in synthetic medium. The optimized condition is as follows: fed 0.8 g/L/h lactose as inducer in synthetic medium, which contains glycine. The enzyme production from this condition was as 2.3 times as that in complex medium.
The fermentation characteristics of recombinant E.coli JM001(△ppc)/pTrc99apck for succinate production was investigated. The results showed that its glucose consumption and succinate production rates were 4.2 and 15.3 folds as much as those of the parent strain E.coli JM001, respectively. The mass yield of succinate to glucose was 54%, besides the acetic acid concentration was rather low. It could be concluded that the reaction from phosphoenolpyruvate (PEP) to oxaloacetic acid (OAA) catalyzed by PEP carboxykinase (PCK) was more beneficial to the cell growth and succinate production compared to PEP carboxylase (PPC). Further investigation was carried out to optimize the fermentation conditions. The results showed that bacterial sludge was much better than 10% (v/v) broth for the inoculation from aerobic phase to the anaerobic one, succinate mass yield increased from 55.2% to 65.1% and the entire time to consume 20g/L glucose reduced to 48h. The results of glucose tolerance experiment showed that between 20g/L and 60g/L initial glucose, cell growth and succinate production were both satisfactory, succinate mass yield was from 64.18% to 69.10% with the increase of glucose concentration. But when the glucose was higher than 60g/L, cell growth and succinate production were clearly repressed. The higher the glucose concentration was, the more obvious the inhibition effects were. For 7L fermentor research, the initial glucose of the anaerobic phase was 40.25g/L, after 96 h it was completely consumed. The succinate mass yield was 67.75%. During the anaerobic phase, the glucose consumption rate was 0.42g/(L·h) and succinate productivity was 0.28g/(L·h).
Argininosuccinate lyase (EC 4.3.2.1; AL) genes from L-arginine producing mutant Corynebacterium crenatum SYPA were cloned and sequenced. Analysis of argH sequences revealed that only one ORF existed , which used ATG as the initiation codon and coded a peptide of 476 amino acids with acalculated molecular weight of 50.8 kDa. Only 10 nucleotide difference was found in the structure gene and the difference caused 3 change of amino acid by comparision of the gene sequences between C. crenatum SYPA and the Corynebacterium glutamicum ATCC 13032. The ORF sequence of argHs showed homologies of 99.4%. The argH gene from C. crenatum was expressed both in E.coli and C. crenatum SYPA. Then AL was purified by Ni-NTA affinity chromatography and the enzymatic characterization of it was determines. The expression vector pJC1-tac-argH was transducted to C. crenatum SYPA. The AL was expressed well and the activity was improved by 66.8%. The fermentive character of CCH1(pJC1-tac-argH) was also primary analysed. The result shows that the acid producing ability of recombinant strain is improved by 14.2%.
The liquid fermentation conditions for anthraquinone pigment by Fusarium oxysporum was optimized. Single factor experiment and orthogonal optimization experiment determined the optimal fermentation conditions for production as follows: the optimate composition of the medium:soluble starch 30%,(NH4)2SO4 3%, MgSO4 0.3%, KH2PO4 4%,and the optimal fermentation conditions: initial medium pH 6.0, medium volume 20%, inoculation amount 10%, Tween -80 1.0%, incubation temperature 28℃, rotation speed 200 rpm and the fermentation period was 120h. In the optimal conditions, the color value reached 8.184U/ml, and increased by about 1.8 folds compared with the unoptimized conditions. It is the first time to study the anthraquinone pigment produced by Fusarium oxysporum at domestic. And it is hoped to lay the foundation for its further application.
Control and Prevention, Academy of Military Medical Science, Beijing, 100071） Abstract: Construction of mutant strain is an essential method in pathogenesis researches. In the present study, the Brucella deletion mutant was constructed by using T cloning vector. At first, the homology arms of upstream and downstream of the target gene were fused with kanamycin resistance gene by fusion PCR. Then, the mutant cassette was ligated with T vector, resulted in the mutant plasmid. And the mutant plasmid was transformed into recombination competent cells and recombinants were selected by kanamycin resistance. As the results showed, the mutant construction was convenient and the mutant could be generated by only one round of selection with high efficiency. Therefore, this recombinational cloning strategy provides us a new method to construct the Brucella mutant and would accelerate gene function analysis of Brucella.
To screen anti-Nortestosterone monoclonal antibody (NT mAb) and develop heterologous ciELISA kit. Balb/C mice were immunized with artificial antigen of NT-17-BSA and a strain of hybridoma cell named NT1-B7 was produced, which can stably secret NT mAb. The isotype of mAb was IgG1 /k, ascites titer was 0.64×105, affinity constant (Ka) was 4.31×109 L/mol and IC50 value determined by ciELISA was 0.55 ng/mL. Of all the analogous competitors, 2.2% and 1.6% cross-reactivity (CR) were observed for trenbolone and estradiol, respectively, while no CR was obtained for other compounds. With coating antigen of NT-3-OVA, heterologous ciELISA kit was developed, which limits of detection in pork, swine urine, beef and cattle urine were 0.24, 0.23, 0.22 and 0.17 ng/mL, respectively. The recoveries of NT spiked in the four matrix were in the range of 85％-110％, while intra- and inter-relative standard deviation (RSD) were in the range of 4.4％-21.8％. when stored at 4 ℃, the NT-Kit could preserve 6 months or more. The pH value of validation detection in matrix ranged from 6.5 to 7.5, and the program could save 30-90 min after the incubation process was optimized. With high sensitivity and specificity, this ciELISA kit could be advantageously utilized for the primary screening of sample matrix for presence of 19-NT residue.
Single-factor and response surface test were used for optimization of Ganoderma lucidum medicinal solid fermentation medium. Optimized medium composition were as follows: brewer's spent grain as substrate, the ratio of material to water was 1∶1.4, Astragalus membranaceus 12.52%, glucose 4.78%, KH2PO40.21%, MgSO4·7H2O 0.25%, VB1 trace. Under the optimized conditions, GAPS concertration reached up 8.42mg/g, increased by 29.54% compared with unoptimization.
The viral macrophage inflammatory protein-II (vMIP-II) is encoded by Kaposi's sarcoma-associated herpesvirus and the residues 1~21 of the N-terminal of vMIPII(NT21MP) was shown to strongly and selectively bind CXCR4 to inhibit the chemotaxis of SK-BR-3 cells.The N-terminal of vMIP-ⅡcDNA was derived by chemical sythesis method. DNA fragment encoding the NT21MP peptide was inserted into pGEX-KG vector and the recombinant plasmid was expressed in E.coli BL21(DE3). NT21MP peptide, which was recombinated with a glutathione S-transferase (GST) at the N-terminal, was expressed in E.coli cells.SDS-PAGE and Western Blot analysis demonstrated that the recombinant protein existed mainly in the supernatant of E.coli lysates. The supernatant was further purified by affinity chromatography, ultrafiltration and fast protein liquid chromatography. The bioactivity of GSTNT21MP was determined by Transwell chemotaxis on CXCR4-expressing breast cancer cell line SK-BR-3 in vitro. The results indicate that The chemotaxis of SK-BR-3 cells toward stromal cell-derived factor-1(SDF-1) was reduced by GST-NT21MP and may serve as a potential target drug for the treatment of breast cancer metastasis.
Bacterial lac operon can regulate gene expression in mammalian. Modified lac operon alter the inducibility and affinity binding to lac operator, which will be better to adapt higher animals’ internal environment. Galactosidase has a positive regulation effect on the lac operon system. Lac operon regulate galactosidase inducible expression can improve transgenic animals’ ability of making full use of galactoside. Following from the structure and function of lac operon、the gene regulation properties, combined with galactosidase’s physiological functions and applied in transgenic animals are reviewed. Analysis prospect of the lac operon mediated galactosidase applied in transgenic animals.
Detailedly described several reliable methods for constructing amiRNA , which was based on WMD3 (Web MicroRNA Designer 3), including molecular designing and flexible synthesis strategy in vitro. When designed online, four steps were needed sequentially: inputting target sequence(s), screening candidate amiRNAs, gaining four oligonucleotides carrying amiRNA-related sequence and other proper sequences that flanked nature miRNA. For overlapping PCR synthesis strategy, amiRNA can be amplified using these four oligonucleotides and two primers designed based on plasmid. Uracil-excision based strategy could also be employed to generate amiRNA, while primers need altering. Specific primer-annealing based method was equally represented here, which may guarantee the synthesis of amiRNA in one-step-PCR. With appropriate restriction sites, the synthesized amiRNA expression cassette can be cloned into corresponding sites of destination vector. It is foreseeable that such scientificness of molecular designing principle and the accuracy of synthesis strategy would aid greatly in gene functional analysis, promising a profound impact on life science.
In flowering plants, MADS-box genes control diverse developmental processes. FRUITFUL (FUL) is a MADS-box gene which functions early in controlling flowering time, meristem identity and cauline leaf morphology and later in carpel and fruit development in Arabidopsis thaliana. Its homeotic genes also play important roles in regulating plant growth and development in other plants. This article reviews the research advances in the expressional patterns and functions of FUL gene and its homeotic genes. And the potential value in the breeding of crop and orchards is discussed.
The paper focus on recent research of cutinase, including the major source, the cloning and expression, as well as the fermentation of cutinase. It also elaborates the current application progress of cutinase in the following aspects, such as cotton fiber bioscouring, wool anti-felting finish, and biological modification of synthetic fibers, etc. In addition, the cutinase, as a key enzyme in promoting the textile cleaner production industry, was summaried briefly in the future textile application.
γ-Linolenic acid is an essential fatty acid of ω-6 series with a variety of special physiological function for human beings. It is available commercially in seed oils from black currant, evening primrose, borage, and so on. However, this means is limited by many factors, and can not meet the huge requirement of human beings. Therefore, it might be expected that potential commercial sources of γ-linolenic acid may be found among the microorganism. The present review outlines the status of our current understanding on γ-linolenic acid by microbial fermentation, including the strains and the metabolite pathways. Breeding strategies, including mutation breeding and molecular breeding, are especially introduced. In addition, this paper also summarized the effect of various factors on the fermentative production of γ-linolenic acid, such as carbon sources, nitrogen sources, cheap substances, trace minerals, temperature, morphology, and solid state fermentation. At last, the future research emphasis was prospected.
Dehydrogenases, which use nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+) as the coenzyme for their catalyzed reactions, are the most important group of oxidoreductases in the nature. In recent years, Biosensors based on NAD(P)+-dependent dehydrogenases have been developed rapidly. Two key technologies are quite important for the construction of these biosensors. One is the electrochemical regeneration of oxidated form of coenzyme, and another is the coenzyme immobilization in electrode surface. This paper reviews methods to aid the electrochemical regeneration of coenzyme. Common ways of coenzyme immobilization were also introduced. In addition, research progress of the two key technologies was reviewed.
It was totally twenty years now from Sep. 14, 1990 when the first successful case of gene therapy. The treatment was for adenosine deaminase (ADA) deficiency patient which born with severe combined immunodeficiency (SCID) and without functioning immune systems in NIH Clinical Center of USA. So far a lot of progress had been made in gene therapy. It was developed from pure gene recombinant technology for DNA transfering to both DNA and RNA molecular levels, and from deficient gene upregulating such as augmentation, correction, replacement, etc. to both gene expression up-regulating and down-regulating (gene inactivation) strategies. Gene therapy was the year news of Science in 2009. In China, the first gene therapy medicine was approved for the therapy, and a dozen reagents were on the study, as well several ones on clinical trails. Gene therapy has a potential for trestment the hereditary disease, becomes the promising biotechnolgy in life science.
