Abstract : Objective :To detect the dopaminergic neural cells differentiated pathway, the related gene in dopaminergic neural cells development process were investigated. The role of Human amnion epithelial conditional media (ACM) and SHH, FGF8 was focused on in cultivation process of human umbilical cord blood mesenchymal stem cells (CB-MSCs). Method: Preparation of the mononuclear cell fraction was performed by the Ficoll gradient technique according to the manufacturer’s instructions . ACM and SHH, FGF8 were combined to induce CB-MSCs, and there were four groups CON, ACM, SHH+FGF8, ACM+SHH+FGF8. After 48 hours of co-cultivation , Immunophenotypes of CB-MSCs were observed by immunocytochemistry and the related gene in dopaminergic neural cells development process En-1、Foxa2、Lmx1b、Gli-1、Pitx3，Nurr1 and Ngn2 were detected by RT-PCR. Results: After 48 hours of co-cultivation ，the positive cells numer of TH and DAT were higher in induction groups than in control group. By RT-PCR, En-1，Foxa2，Pitx3, Lmx1b were higher in ACM, SHH+FGF8, ACM+SHH+FGF8 groups than in the control group(p<0.01). Nurr1,Ngn2 were strongly higher in SHH+FGF8. Conclusion:The related gene in dopaminergic neural cells development process，En-1、Foxa2、Lmx1b、Gli-1、Pitx3，Nurr1 and Ngn2 play a role in the differentiated pathway form CB-MSCs to dopaminergic neural cells.
Engineered targeting fusion protein XE-TNFαm2 was prepared.In which,XE is the second excellular domain of CXCR4,a co-receptor of HIV/SIV;TNFαm2 is a mutation protein of TNFα.Its side effect was reduced 18 fold.It has been used in clinic for cancer therapy.Our previous study indicated that one of XE-TNFαm2 functions is to kill the cells infected by HIV/SIV,but, not to kill the normal cells without HIV/SIV. The purpose in this study is that to explore whether XE-TNFαm2 could lead cell to apoptosis,as its effect mechanism.The result shows that XE-TNFαm2 only can lead the apoptosis of cells infected by SIV,but can not lead this effect on normal cells without HIV/SIV.It indicates that XE-TNFαm2 is an accurate targeting fusion protein to specifically kill cells infected by HIV/SIV,but not be able to kill normal cells without HIV/SIV.It side effect is minimized。
Akt1 is a serine-threonine protein kinase that has been implicated in the control of cellular metabolism, survival and growth. Elevated expression of Akt1 has been noted in a significant percentage of human tumors, promoting cellular metastasis. Conversely, some studies have revealed hyperactivated Akt1 inhibited the invasiveness and metastasis of breast cancer cells. To clarify the definite effect of Akt1 on tumorigenesis and development, Akt1 was silenced by RNAi in the highly metastatic murine breast cancer 4T1 cells. Akt1 silencing didn't affect the proliferation of breast cancer cells in MTT assay, while reduced the migration in Transwell assay. Consistent with the above results, Akt1 silencing didn't change the primary tumor weight, but significantly suppressed lung metastasis of 4T1 cells. These observations indicated Akt1 plays an important role in murine breast cancer metastasis, and suggested that Akt1 might be a therapeutic target for breast cancer metastasis.
In this study, peptide vaccine was designed based on B cell and T cell epitope of IBV, and the multi-epitope based peptide vaccine was expressed in E.coli fused with glutathione S-transferase (GST). ELISA and Western blot analysis showed that purified fusion proteins (EpiA) had an excellent immune activity with chicken anti-IBV serum. The expressed fusion proteins were purified and emulsified with Freund adjuvant. Vaccination twice at an interval of 2 weeks with the emulsified the fusion proteins elicited anti-IBV specific antibodies and specific cellular responses and 73% of the vaccinated chickens were protected against mortality. We proposed use of a biosynthesis system instead of peptide synthesis, provided a new way to prepare multi-epitope based peptide vaccine
Objective: To obtain a purified protein fragment with ATP-dependent kinase activity of the kinase domain of histidine kinase YycG in Streptococcus pneumoniae by a prokaryotic expression system and make use of its activity to search for new bacterial inhibitors. Methods: The protein of YycG kinase domain was expressed by a prokaryotic expression, identified by SDS-PAGE and Western Blot, and then purified by His-tagged Ni affinity column, and finally the activity of this purified protein was determined by a Kinase-Glo Luminescent Kinase Assay; Effective inhibitors were screened out from 105 candidate compounds based on their inhibitory action on the kinase activity of YycG protein, and were testified for their antibacterial effects in vitro. Results: The interest protein expressed in protocaryon was about 35KDa, reaching a purity of 95%, with obvious kinase activity of hydrolyzing ATP. Several effective kinase inhibitors were filtered out, and a selective one was verified overt bactericidal effects in vitro assay. Conclusion: The kinase inhibitors screened out based on their action on YycG may be important cues for developing antibacterial drugs or disinfectant.
To obtain polyclonal antibody against human NANOGP8, the NANOGP8 gene was amplified by PCR from the template obtained in our previous work and identified by DNA sequence analysis. Then it was digested by EcoRⅠand SalⅠ, and ligated with pET-28a vector which was by the same treatment. Sequenced and blasted with the NCBI GenBank, the recombinant plasmid was named as pET-28a-NANOGP8. The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by 1mM IPTG at 16℃ and 37℃. SDS-PAGE showed that the fusion protein was expressed in the form of inclusion body in the sediment when induced at 37℃. After dissolution and affinity purification with Chelating Sepharose Fast Flow, a single protein band about 45 KD was identified in the SDS-PAGE and Western-blot analysis. One new zealand rabbit was immunized with purified NANOGP8 protein to prepare for the polyclonal antibody against NANOGP8. The NANOGP8 antiserum was obtained and characterized by Western-blot, ELISA, and compared with commercial goat anti-human Nanog antibody. Results showed that the antibody had high affinity and specificity, and higher titer compared with commercial anti-human Nanog antibody. The studies provide a favorable tool for further study on relation of tumorigenesis, development and NANOGP8 in the future.
Objective In order to express and purify the mono-phosphorylated PZR proteins. Methods The bi-- phosphorylated His-PZR recombined prokaryotic expressions plasmids was used as the template, then site-directed mutagenesis was used to amplified the mono- phosphorylated PZR a Tyr263(TAT) Phe263(TTT)and PZR b Tyr241(TAT) Phe241(TTT) mutants. The mutants’ and C-Src recombined prokaryotic expressions plasmids were co-transformed to the E.Coli. BL21. Through IPTG induction and Ni-NTA purification, we want to get the mono-phosphorylated PZR a and PZR b proteins. Results The mono- phosphorylated PZR a and PZR b recombined prokaryotic expressions plasmids have successfully been constructed; The mutants have successfully been induced to express by IPTG. They represent 8.5% of the total bacterial protein in E.Coli. BL21, and theirs relative molecule mass is 10 000. After Ni-NTA purification, the mono-phosphorylated PZR a and PZR b proteins have been obtained. Conclusion These results laid a foundation to future study the bio-function mechanism between different ITIM of PZR with protein tyrosine phosphatase in the signal transduction.
In order to express and purify the mono-phosphorylated PZR proteins, the bi-phosphorylated-site His-PZR recombined prokaryotic expressions plasmids was used as the template, then through the overlapping PCR the mono- phosphorylated-site PZR-Phe263(TTT) and PZR-Phe241(TTT) mutants were amplified. After recombined and purified, the mutants' and C-Src recombined prokaryotic expressions plasmids were co-transformed into the E.Coli. BL21. Through IPTG induction and Ni-NTA purification, the mono-phosphorylated PZR a and PZR b proteins were obtained successfully. These results laid a foundation for future study on the PZR bio-function mechanism.
The desulfation rate and immunoactivity of polysaccharide of Gracilaria lemaneiformis was investigated after using two desulfation method (Miller, et al and Nagasawa, et al ).The results showed that using the Miller method, the best desulfation effect was obtained when Oxalic acid was used as catalyst, and the desulfation rate achieved to 71.4%, but the polysaccharide recovery rate was only 36.4%. Using Nagasawa method, the desulfation rate was 72.9% with DMSO mixed with methanol 10%, which had a better desulfation effect than 2% pyridine or 2% pyridine+2% trimethylchlorosilane, and the polysaccharide recovery rate was 48.9%,. The immunostimulation of polysaccharide was decreased after desulfation.
Abstract [Objective] The purpose of this paper was to investigate the effects of mixing intensity on the alga growth. [Method] Spirulina platensis was cultured in the flat plate photo-bioreactors both indoors and outdoors, the alga growth areal yield and chlorophyll content under different mixing intensity were measured. [Result] Results showed that the alga growth yield increased with the mixing intensity increasing; the physiological characteristic was not influenced by the mixing intensity when the alga was cultured indoors, while it was influenced by the mixing under the outdoor condition. [Conclusion] To intensify the culture liquor mixing can effectively enhance the alga yield.
The effects of different carbon sources added to aerobic medium on cell growth, enzyme activity and metabolite distribution was investigated in dual-phase fermentations. The results showed that both cell density and enzyme activity were enhanced by adding 4mmol/L glucose or 12,54,80mmol/L sodium acetate to aerobic medium, respectively. Then centrifugated cells that had grown aerobically in different conditions were transferred to anaerobic fermentation, the enzyme activity and metabolite distribution changed. To analyze the anaerobic enzyme activity and metabolite distribution, it was concluded that during the anaerobic fermentation of Escherichia coli NZN111 overexpressed malic enzyme, PEP carboxykinase(PCK) was the key enzyme of succinate production, pyruvate kinase(PYK) was associated to the accumulation of byproduct pyruvate, and isocitrate lyase(ICL) have some influence on the concentration of succinate. The yield of succinate in anaerobic stage could reach 89.0%, which was 16.6% higher than that of the control by adding 80mmol/L sodium acetate as carbon source in aerobic medium.
The objective of the study is to remove murine embryonic stem cells (mESC) from the differentiating cell culture and purify the differentiated cells by Magnetic Activated Cell Sorting (MACS). Neural differentiation of mESC was induced by a 5-stage method. The specific cell surface marker, SSEA-1, was used to identify ES cells in the differentiating cells. The optimal dilutions of mouse anti mouse SSEA-1 IgM primary antibody and FITC conjugated goat anti mouse secondary antibody were determined before the flow cytometry test. The incubation time and incubation temperature of primary antibody were all optimized to make the cytometry test accurate. After the optimization, stage 4 cells were dissociated into single cell suspension, incubated with antibody of SSEA-1 and microbeads conjugated goat anti mouse IgM, and then sorted through the magnetic field. The rate of SSEA-1 positive cells in pre- and post- separation groups was assessed by flow cytometry, and the viability of cells was evaluated by trypan blue staining counting under light microscopy. The proportion of SSEA-1 positive cells in the separated cells can be reduced from (7.19±1.36) % to (1.34±0.80) %. The survival rate of sorted cells was more than 92%, similar to that of pre-separation cells. The MACS system we used can effectively remove mESC from the differentiated cells. The sorted cells will be well provided for the subsequent studies about transplantation therapy.
Investigators in gene (genome) manipulation frequently meet difficulty in amplification of GC-rich DNA sequences. This difficulty has not been well solved worldwide. Through systematic screening, 1,2-propanediol and ethylene glycol were found specifically efficient in enhancing the amplification of high GC content human genomic DNA. We randomly selected 104 GC-rich sequences (GC content from 60% to 80% and length from 700 to 800bp) from human genomic DNA to test the two organic solvents and all 104 amplicons were well amplified.
Gene trapping is a powerful method for gene analysis relying on mutation generated by random insertion. Various of new kinds trapping vectors have been endlessly designed and applied for trapping because of the local of conventional trapping vector. Based on plasmid PL-451,combinding with the site specific recombination target sites—Loxp,Frt and LoxP511A,LoxP511B which mutant one base of the LoxP,we constructed a conditional gene trapping vector.The trapping vector contains multicloning sites for cloning of the homology regions,which make it can realize the target trapping depending on homologous recombination. Furthermore, the exprssion of promoterless EGFP with splice acceptor（SA）and polyA in the vector can tagging the work of the trapping cassettes.Construction of this vector establish substantial foundation for conditional trapping of target gene.
Pinellia ternate and Pinellia pedatisecta lectins with biological functions could be obtained through procaryon expression. Their biological similarities and differences were analyzed and further experiments were conducted to discuss the agglutinative activity difference between polymer lectin and metamer lectin. The results showed that agglutinative activity of Pinellia pedatisecta was four times more than that of Pinellia ternate. The differences of the agglutinative activity and pharmacologic action were mainly because two site mutations of Pinellia pedatisecta in the third active site compared with the amino acids sequence in Pinellia ternate. In addition, the metamer lectin expressed by prokaryotic expression system did not agglutinate rabbit red blood cells. This research conduct further discussion about the difference between two lectins and the difference between polymer lectin and metamer lectin. It would be applicable for solving the problem of lacking Pinellia resources.
MiRNAs are an important class of gene regulators.And miRNAs play a pivotal role in the regulation of genes involved in diverse processes such as development,differentiation,and cellular growth contro1.But little is known about it's role and molecular mechanisms due to lack of efficient and effective target identification methods.Therefore,recent progress on the adjustment target of miRNA was reviewed.The adjustment target of miRNA is very helpful to reveal the pathogenetic of some diseases, discover novel molecular targets for treatment and establish foundation for gene therapy.
Reverse genetic technique, also named virus rescuing, was a new technique in molecular virology in last 90’s,which played a significant role in the study of rabies virus in these years.The reverse genetic technique for rabies virus rescuing and the research on pathogenesis,vaccine and vector of rabies virus with reverse genetics were reviewed.
Entomopathogenic fungi are the main biological factors of controlling pest population in nature, but its application was limited because of their poor performance in field. With the rapid development of fungal molecular biology and gene engineering, large progress has been made in improving the strain’s virulence and creating highly efficient and stable strain. The latest advance of entomopathogenic fungi in genetic engineering was reviewed in this paper, mainly including selective markers, some transformation methods, and the application of improved strains.
Salinity is the main limitation factor for plant growth and crop production. To improve salinity tolerance of plants, many approaches by genetic means towards to manipulating expression of functionally related classes of genes such as signaling pathways, ion channels and compatible solutes in the stabilization of biological structures under salinity stress are been developed. This review focuses on recent progress in molecular engineering to improve salt tolerance in plants and the possible problems in research.
Lycopene, one of the most important dietary carotenoids, plays an important role in reducing the risk of chronic diseases (e.g. certain cancers, cardiovascular disease). Blakeslea trispora is one of the microorganisms which produce lycopene. The present review is focused on the physicochemical properties of lycopene, biological characteristic of Blakeslea trispora, isolation of lycopene mutants of Blakeslea trispora,the medium optimization , extration of lycopene and the market condition and its developmental future.
The excessive use of fossil fuels will lead to the depletion of natural energy resources and greenhouse gas (CO2) emission increase thus is considered unsustainable. To achieve the harmonious development of economy and environment, foil fuels need to be replaced by renewable energy resources. Use of renewable energy will not emit greenhouse gas. Bio-diesel is one of the ideal renewable energy resources and can satisfy the demands for energy requirement and environment protection. So bio-diesel has been fast developed in recent years worldwide. Microalgae absorbs carbon dioxide in the air mainly by using solar energy to generate the microalgae lipids needed for bio-diesel production. And this bio-diesel could be the ideal substitute for foil fuels. This review provides a brief overview of some features of oleaginous microalgae species and their lipid synthesis, microalgae autotrophy and heterotrophy, bioreactor and engineering microalgae latest research progress. Perspectives of microalgae cultivation for bio-diesel production are also discussed.
Butyric acid can be used to produce cellulose butyrate fiber and ester derivatives, and to be applied in foodstuffs and perfume industries. Recent researches have found that butyric acid is a preferred carbon source for colonic epithelial cells, and can inhibit histone deacetylase, showing great anticancer potentials. With more and more functions of butyric acid being found and applied in bio-related fields, and with consumer’s growing preference to bioproducts, biotechnological production of butyric acid will receive more competence than petroleumbased chemical synthesis. Low product concentration and poor selectivity are presently the main restricting factors. Workers have made considerable progress on more cheep carbon sources, optimization of fermentation process, simplifying downstream processing, and genetic engineering of producing strains. Any achievement on these aspects in the future can contribute to put fermentation of butyric acid into industry.
