Microencapsulated recombinant cells technology is a novel approach to tumors therapy. It is great importance in encapsulated cells therapy to find a proper way for acquiring seed cells in large-scale in a short time for large scale preparation of biological microcapsule with high cell viability and recombinant protein production. The purpose of this experiment was to explore the way and technique to gain adequate seed cells for cell encapsulation with bioreactor culture system. The results showed that the recombinant CHO cells could expand rapidly in the bioreactor in three dimensions and the cell amount reached about 20 times of the initial amount after 10 days culture. The expanded cells could be encapsulated into the microcapsule and the microencapsulated cells could keep high viability and expand rapidly in the bioreactor. In comparison with the cells statically cultured in 24-well plates, a quicker growth and a higher endostatin production of microencapsulated cells were observed. B. Braun bioreactor provided the culture environment with relatively low shear force and good communication among cells in three-dimension, therefore recombinant CHO cells showed a strong ability to expanded in vitro. Large number of seed cells can be obtained rapidly with this culture system, which can meet the requirement of encapsulated cells therapy in both quantity and quality.
Recombinant expression vector pcDNA3-MCPCD59-DP containing human membrane complement regulatory proteins (hCRPs) MCP and CD59 cDNA was constructed successfully by using two independent promoters. After transfected into NIH3T3 cells with calcium phosphate-DNA precipitate method, NIH3T3 pcDNA3-MCPCD59-DP transfectants were obtained by G418 selection. Extraneous genes integration was identified by PCR. The co-expression of human CD59 and MCP at both mRNA and protein levels was confirmed by using RT-PCR and Western blot analysis. Human MCP and CD59 cDNA were integrated in NIH3T3 pcDNA3-MCPCD59-DP genomic DNA after continuous 30 times passages, indicating that NIH3T3 pcDNA3-MCPCD59-DP were stable cell lines. Human complement-mediated cytolysis assays showed that NIH3T3 cells transfected stably with pcDNA3-CD59, pcDNA3-MCP, and pcDNA3-MCPCD59-DP were protected from C-mediated damage and co-expressed human MCP and CD59 provided more excellent protection against C-mediated attack as compared with either CD59 or MCP expressed alone. These results suggest that the dicistronic vector represents an effective and efficacy strategy to overcome C-mediated damage and has potential therapeutic value for effectively controlling complement activation and finally for preventing hyperacute rejection in clinical gene therapy.
Abstract BMP2/7 heterodimer has stronger bioactivity than BMP2 homodimer, but the mechanism remains to be unclear. Treating the osteoblast MC3T3-E1 cells with BMP proteins, results of Alkalin phosphatase staining , Alizarin red staining , as well as p3GC2-LUX luciferase assay demonstrated that BMP2/7 heterodimers significantly promoted osteogenic differentiation to a higher extent than BMP2 homodimers. Overexpression of CIZ in MC3T3-E1 significantly inhibited the promotion of ALP and Osteocalcin expression by BMP2/7, and blocked activation of BMP/Smad. BMP protein may induce the expression of CIZ, but effects of BMP2/7 heterodimer is weaker than BMP2 homodimer. It suggested that the effects of BMP2/7 on CIZ expression is related to its bioactivity.
Objective To optimize the renaturation procedure of denatured LexA, prepare the repressor LexA from Pseudomonas aeruginosa (PA), which have the satisfactory biologic activity. Methods The LexA was renatured by the GSH/GSSG dilution method, and the renatured protein were purified by Ni2+ chelate affinity chromatography and gel filtration chromatography, following desalination by Sephadex G-25 gel column. The renaturation result were detected by the native polyacrylamide gel electrophoresis and RP-HPLC.The immunological activity of all LexA proteins, including the denatured , renatured protein and the renatured protein that was treated with the DTT, were determined by Western blot. Results The renatured LexA appears both monomer and multimer, which is confirmed by the native polyacrylamide gel electrophoresis analysis and RP-HPLC. Gel retardation experiments shows that the renatured LexA have satisfactory biologic activity.
Avian influenza, caused by avian influenza virus (AIV), is a highly contiguous disease. AIV can infect almost all wild and domestic poultries and cause severe economic losses for the poultry industry. The M2 protein of AIV is a transmembrane protein. It has highly conserved antigenic epitopes and is a potential candidate antigen for avian influenza vaccine with cross-protection. Marek's disease virus(MDV), avian herpesvirus have mostly been used as virus vectors to express a foreign gene for the protection of chickens against poultry. Among MD vaccine viruses, attenuated MDV1 strains such as CVI988 are more suitable for be vaccine vectors because they have the closest antigenic similarity to the very virulent(vv) MDV1. To develop a recombinant herpesvirus -based AI vaccine, the M2 gene of Avian Influenza virus (AIV) ligated with green fluorescent protein (EGFP) was inserted into the US gene, which located in the nonessential region of MDV1 (CVI988) genome and an EGFP marked recombinant plasmid was obtained. The M2 gene of Avian Influenza virus was expressed under the control of a CMV immediately early promoter. Through homologous recombination, this construct was transfected into cells infected with Marek's disease virus CVI 988/Rispens and the recombinant MDV1expressing AIV M2 gene was screened out. The PCR, dot-blot and western-blot results demonstrated that AIV M2 gene was not only cloned into the genome of MDV1 (CVI988) but also expressed. A group of one day-old chickens was injected with the recombinant virus and the specific antibody against M2 protein could be detected by ELISA after 21 days. When chickens were inoculated with commercial H9N2 subtype AI vaccine, the titers of antibody against AIV hemagglutinin from chickens inoculated with the recombinant MDV1were higher significantly (P<0.05) than those of H9N2 subtype AI vaccine alone. In addition, the excretive amount of live AIV from throat and cloacae of chickens inoculated with recombinant MDV1 were less significantly (P<0.01) after challenged with Avian influenza virus A/Chicken/Guangdong/00（H9N2）. All these results indicated that the recombinant MDV1expressing AIV M2 gene could be responsible for the prophylactic of avian influenza.
The Coat protein and Maturase gene of E. coli bacteriophage MS2 was amplified by PCR, then the gene was cloned into pET32a to construct the intermediate vector pET32a-CP. The conservative sequence of FMDV internal ribosome entry site (IRES) was cloned into the downstream of pET32a-CP bacteriophage gene to construct the prokaryotic expression vector pCPES. The recombinant plasmid pCPES transformed into E. coli strain BL21 (DE3) was induced to express with 1mmol/L IPTG. The expression products were purified by sucrose density gradient centrifugation. The expression products observed by TEM were circular virus-like particles, and the diameter of these particles was about 26 nm.. The stability of virus-like particles was detected, and the virus-like particles was identified by RT-PCR. The results showed that the virus-like particles contain the FMDV IRES RNA and have good stability. The study shows that the virus-like particles have great prospect as the standard and quality control in the area of RNA virus detection.
Poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx), a member of the polyhydroxyalkanoate family of biopolyesters, has superior mechanical properties and biocompatibilities that enable it to meet diverse biomedical requirements. One of the degradation product of PHBHHx is oligo-hydroxybutyrate (OPHB), whose derivatives and complexes are occurrent in organisms in a wide range. OPHB ramification is yielded by alcoholysis of Poly(hydroxybutyrate) (PHB). The effects of OPHB and its ramification treatment on human microvascular endothelial cells (HMECs) were investigated by MTT assay. OPHB ramification yielded showed cell toxicity, while OPHB promoted cell viability slightly, and more remarkable in semistarvation batch culture. This effect may underlie the good biocompatibility observed for PHBHHx.
With purpose of performing biotinylation of firefly luciferase in vivo, we fused a fragment of gene encoding C-terminal 87 amino acids of biotin carboxyl carrier protein (BCCP) of E.coli with luciferase cDNA from firefly Pyrocoelia pectoralis. Biotin was covalently linked to a specific lysine residue of BCCP via catalysis of biotin holoenzyme sythetase in E.coli, and firefly luciferase was biotinylated indirectly as a result of fusion with BCCP. Advantages of specific coupling between biotin and avidin or streptavidin allow firefly luciferase to be immobilized on solid support coated with avidin or streptavidin, so that more flexible and convenient methods of luminescence assay will be available in application. The details of cloning, expression and function of biotinylated luciferase will be discussed in this paper.
The optimization on liquid fermentation and purification of the fibrinolytic enzyme from Stenotrophomonas maltophilia（DR-929） was investigated in this paper.The results showed the best fermentation are amidulin 2.0%, soya flour 1.0%, yeast extract 0.5%, NaCl 1.0%, CaCl2 0.02%, MgSO4 0.05%, inoculum of 36 hours, fermental time 4d, initial pH 8.0 or 9.0, temperature 25℃, volume of media 30mL, volume of inoculum 5% or 6%.The purification process includes the following steps: removing cells by the centrifugation, 25%~70% saturation ammonium sulfate precipitation, HIC with Phenyl FF（high sub）, IEC with Q-Sepharose FF, gel filtration chromatography with Superdex 75. SDS-PAGE electrophoresis was used to examine the purification effect, and the results indicated that homogeneous strap in SDS-PAGE and has a molecular weight around 28.3KD.
Follow the vp2 gene of PPV was transformed into the competent cell of Pichia Pastoris, the vp2 protein of PPV was successfully expressed under the optimal expression conditions. The final concentration recombinant vp2 protein was 227.6μg/mL,using the recombinant protein ,an indirect ELISA assay for detection of PPV antibody was established . The optimal working circumstances for the antigen conditions(5.69μg/mL) and the serum dilution were 1：80 , determined chess titration. The positive circumstances of this ELISA assay is OD490 value of the sample serum ＞0.5，and the ratio of the tested serum OD490 to the negative serum is higher than 2.0.This ivp2-ELISA assay was compared with HA/HI and PPV ELISA kit of Cedi company ,result shows the agreement ratio is 97.2% and 91.2% .
Fermentation conditions for the production of spores of Bacillus mucilaginosus mutant 021120 were optimized through the single factor and orthogonal experiments. The results showed that the biomass and formation of spores were obviously affected by carbonate, followed by nitrogen. Addition of CaCO3 and enhancing air flux notably promoted the formation of spores. The optimal culture medium was composed of 2% starch, 0.4% Yeast, 0.1% K2HPO4, 0.1％ MgSO4·7H20 and 0.5 ％CaCO3, pH7.5. 6% of the inoculum prepared with two-stage extensive culture was inoculated into a 70L fermentor. The fermentation was carried out with 2.0-2.5 vvm of air flux at 32℃ for 38-42h, and the spores of 9.80×108 cfu ml-1 was obtained. Under these optimal fermentation conditions, the capsule was successfully controled and the formation of the spores was effectively improved, thus the satisfying bacteial product was obtained.
A fragment containing amino acid residues 561～578 of HSV-2 glycoprotein G(gG2) was obtained by PCR assembling technique, and doubly cloned into vector pET-KDO.The recombinant plasmid was transformed to BL21(DE3)plysS.Fusion protein,of molecular weight about 39,000D was highly expressed by induction of IPTG.Western-blot result showed the fusion protein had good antigenicity.After putification and digestion,the purity reached 95%.The digested purified protein was analysed by ELISA and showed good sensitivity and specificity. The recombinant protein should be useful for type-specific serodiagnosis of HSV-2.
Banana bunchy top disease is one of major diseases in banana, the pathogen of the disease is banana bunchy top virus. The virus genome consists of at least 6 components of circular ssDNA about 1.0~1.1kb. Each DNA component contains encoding region and non-coding one. On the basis of previous research, we want to understand further the promoter function of BBTV DNA components. According as the genomic sequence of BBTV Hainan isolate, a 540bp fragment（BV3.1） generated by PCR amplification, which is the promoter sequence of BBTV DNA3 component; and a 646bp splicing sequence（BV23）is consisted of non-coding regions of DNA2 and DNA3 components generated by overlapping PCR amplification. This two promoter sequences substituted the 35S promoter sequence in pBI121 vector and fuse with gus gene respectively, and then got the plant expression vector named for pBIBV3.1(540bp)、pBIVBV23(646bp) are constructed. By GUS histochemical staining, the very weak GUS activity was limited in the leave veins of pBIBV3.1 transgenic tobacco, it confirmed the promoter activity is associated with phloem specific expression. Whereas the faint GUS activities were detected in the mesophyll, the leaf margin of pBIVBV23 transgenic tobacco and in some leaf veins as well. It indicates that the transcriptional method of DNA2 component is different with one of DNA3 component.
Insects exhibit a particular resistance to infections. The activation of the innate immune response of insects is involved in the recognition of the infectious non-self and subsequent activation of cellular and humoral reactions. In humoral reactions, insect anti-bacterial peptides and lysozyme are very important in resistance to infections. Housefly (Musca domestica) is one of the most important kinds of insects and it has strong ability to adapt to the adverse circumstances. It is of momentous theoretical and practical significance to research the immunity system of housefly. In this research, the methods of inducement of housefly larvae were firstly studied. Then an antibacterial protein, whose molecular weight is 28kDa, was purified from housefly larvae, induced by 30% H2O2, through salt-out, Sephadex G-25 column, Sephadex G-75 column and CM-Sepharose Fast Flow column. This antibacterial peptide had activities against most of Gram positive and Gram negative bacteria.
Both pretreatment of lignocellulose and its saccharification process are treated with ultrasonic wave. The morphology, structure and crystal performance of the original and treated lignocellulose sample were characterized by SEM and FTIR. Moreover, the changes of raw materials caused by different pretreatment ways and the affect of saccharification rate brought about by ultrasonic wave were also studied. The result shows that the ultrasonic wave decreases the crystallinity of lignocellulose destroying the intermolecular hydrogen bonding effectively and improves the degradation rate of lignin and the saccharification rate of zymohydrolysis availably. The mechanism of activation of ultrasonic wave in zymohydrolysis process was discussed primarily.
Effect of Microbial Transglutaminase from Streptoverticillium SG215 on cutaneous wound healing in rats was evaluated using excision wound model. The wounds were visually observed, photographically documented and the wound area was measured at 5, 10, 15, 20th days. The volume of wound was measured by water injection method, the contents of protein, hydroxyproline (Hyp), hexuranic, hexosamines in the granulation tissues and regeneration of granulation tissues were investigated. The closure time and volume of the skin wounds in the experimental groups significantly decreased when compared to that in the control group (P<0.05). Wound contraction, dry(or wet) weight of granulation tissues and content of protein, Hydroxyproline, Hexuranic, Hexosamines in the wound tissues in the experimental groups were higher than that in the control group (P<0.05).These results clearly substantiate the beneficial effects of microbial transglutaminase in wound healing.
The yeast one hybrid system is the new type system deriving from the yeast two hybrid system to study the interaction between DNA and protein. This review briefly introduced the principle and the technical line of this system, and particularly summarized the progress of this system in plant osmotic stress-resist gene engineering field，such as cloning the transcription factor genes of osmosis resistance type; confirming the interaction between known DNA and protein; making sure the localization of the confirmed DNA binding domain with the interaction; also validating the transcription activation. Furthermore, we analyzed the problems existing in this system on the research of plant osmotic stress-resist gene engineering, as well as forecasting the development prospect according to our research.
Lipase is a kind of widely used hydrolase. Surface display is an efficient method of high-throughput screening for protein engineering of lipase. Besides, lipase displaying on surface of microorganism has many advantages, such as higher stability against high temperature and organic solvent, compared with free lipase, so the host strain displaying lipase can be used as whole-cell biocatalyst, which has some advantages compared with traditional immobilization of lipase. There are three kinds of host strain for displaying lipase: phage, bacteria and yeast. This article systematically describes surface display of lipase in the three display systems, and discusses their current uses and their possible trends in the future.
Cloning and expression of recombinant protein in Escherichia coli has been developed for a long time and it is widely used as a classical expression system now. Many advantages of this system have been confirmed, however, disadvantages also exist. The efficiency of expression is affected by many factors. This paper summarized strategies for high-level expression of recombinant protein in this system. These strategies mainly include selection of appropriate promoter, optimization of signal peptide, increasing the stability of mRNA and the efficiency of translation, preventing the use of low usage codons, reducing inclusion body formation and degradation of the target protein, using molecular chaperones and fusion protein, development of high-cell-density fermentation, and etc.
Abstract: Biotransformation of bioactive natural leading compounds is a kind of bioprocess in which the structure of the added bioactive natural leading compounds could be modified by biocatalysts (e.g., enzyme, microbial, plant and animal cells) in order to produce high efficient and low toxicity compounds. The biotransformation purpose of the known bioactive natural leading compounds is to improve its efficiency, or reduce its toxicity, or improve its solubility and bioavailability. The trace and high-valued bioactive natural leading compounds also could be produced by the biotransformation, and the biotransformation of bioactive natural leading compounds is still helpful to study the mechanism of drug metabolism. The current focus of the biotransformation of bioactive natural leading compounds is on the compounds of steroid, quinine, flavone and terpene, and some important biotransformation process has been successfully screened out. Fundamental research should be done in the following fields, such as the biotransformation mechanism of bioactive natural leading compounds, biotransformation process engineering, and the efficiency evaluation of bioproducts produced by biotransformation. The latest biotechnology (e.g., directed evolution of biocatalyst, combinatorial biotransformation, non-aqueous biotransformation, high throughput screening) should be introduced to the biotransformation of bioactive natural leading compounds, which will boost the fast development of the biotransformation of bioactive natural leading compounds.
In response to problems from the development of multi-drug-resistant pathogenic bacteria, it is urgent to find new antimicrobials. Antimicrobial peptides (AMPs) are a kind of ideal new antimicrobials with the advantages including their potential for broad-spectrum activity, rapid bactericidal activity and low propensity for resistance development, and have a bright future. Alpha-helical antimicrobial peptides are a main kind of AMPs. The following features was reviewed and elucidated including the structure and activity relationship (SAR) from different aspects including the degree of helicity, hydrophobic moment, hydrophobicity, net positive charges and so on, and the application of SAR on the molecular design and improvement of AMPs.
The value-increment utilization of marine shellfish proteins played a very important part in the marine bio-resourses sustainable utilization, enzymatic hydrolusis had becoming a important instrument in the value-increasing, recycling and ecologycal exploitation of marine shellfish proteins. In this paper, main types of comercial enzymes were proposed, including neutral protease, flavourzyme, papain, bromelain, pepsin et al, main parameters of shellfish enzymatic hydrolysis such as hydrolysis degree, physiological fators including antioxidative activity and radical scavenging capacity were also presented.The enzymatic products of shellfish proteins had been widely used as spices, functional food, aninal feed and medicines .The choice of enzymes, the flowsheet for enzymatic hydrolysis, and the applications of enzymatic hydrolysates of marine shellfish proteins were introduced in this paper. The research status and future of the utilization of marine shellfish proteins by enzymatic hydrolysis was reviewed. It presents a reference to the utiliztion of comercial enzymes in marine proteins especially shellfish proteins scientificly and industrially.
