Objective: To investigate the effect of MUC1 on proliferation, invasion and chemosensitivity of human colorectal cancer cell line HCT116. Methods: The HCT116 human colon cancer cells were transfected with lentiviral vectors with and without MUC1, then selected by puromycin. The expression of MUC1 was detected by SqRT-PCR and Western blot. There were two groups in our experiment: MUC1 lentivirus group and control lentivirus group. CCK method and soft agar colony formation method were used to detect cells proliferation, Transwell membrane assay to detect cells invasion, MTT method and flow cytometry to detect cells sensitivity to oxaliplatin and the colorimetric method to detect Caspase-3 activity. Results: HCT116 cell line with stable expression of the MUC1 was acquired. Compared with control lentivirus group, the number of soft agar colonies in MUC1 lentivirus group was increased significantly (P<0.05), the number of invasive cells was increased significantly (P<0.05). Chemosensitivity was decreased in the MUC1 lentivirus group(P<0.05). Caspase-3 activity was significantly low in the MUC1 lentivirus group(P<0.05). Conclusion: MUC1 is related to the anchorage-independent growth, invasion and chemotherapy of colorectal cancer.
Objective: Construct the expression vector p1301-YO-K2-YO based on the oil body expression technology which has two oleosins flanking the KGF-2,observe whether it can improve the expression level of KGF-2 and identify its biological activity. Methods: KGF-2 core gene and Arabidopsis oleosin gene were amplified by PCR, KGF-2-oleosin fused sequence was obtained by the fusion PCR,ligated with the blank vector also has a oleosin gene(p1301-oleosin),then named it p1301-YO-K2-YO,which can be tanslated into the oleosin-KGF-2-oleosin fusion protein. Agrobacterium-mediated transformation of Arabidopsis thaliana was conducted by floral dip.The total protein of the T2 seeds were extracted for SDS-PAGE,Western blot analysis,the proliferation activity of hair follicles was tested on C57BL /6 mice. Results: The plasmid which has two oleosins flanking the KGF-2 was successfully expressed in Arabidopsis thaliana. Western blot result shows this fusion expression strategy could be twice more higher than the single-oleosin configuration production in KGF-2 protein expression,and has a certain biological activity.
Reactive Oxygen Species was accumulated when the plant was under the abiotic stress, which damaged the cell structure and proteins. Carotenoids are non-enzymatic antioxidants, playing an important role to enhance the tolerance to oxidative stress in plants. β-carotene hydroxylase was a key enzyme in carotenoid biosynthesis pathway and coded by CHYB gene. In this study, the LcCHYB was overexpressed in transgenic Eustoma grandiflorum, the results shown When the plants were exposed to 10% H2O2, the Fv/Fm, ФPSⅡ, SOD and POD increased significantly, the content of total carotenoids, Zeaxanthin and Xanthophyll cycle were also enhanced significantly in transgenic line compare to the wild type. Overexpression of LcCHYB enhanced the tolerance to oxidative stress in Eustoma grandiflorum was proved.
Objective: This study was conducted to elucidate the response of genes in sunflower to salt-stress, isolate and identify the genes related to salt tolerance. Methods: The differentially expressed gene fragments produced after being treated with salt were analyzed by cDNA-AFLP. Results: 232 primer pairs obtaining differential expressed bands were acquired from 256 primers combinations. Using these primer pairs, a total of 845 differentially expressed up-regulated transcript-derived fragments(TDFs) were selectively amplified, 42 positive TDFs from them were identified after the second PCR amplification and reverse Northern blot. 12 TDFs of which were cloned and sequenced, and 10 nucleotide sequences were obtained. By Blastx analysis, 10 TDFs were tightly related to salt tolerence of sunflower, being involved in signal transduction related proteins, abiotic stress related functional proteins, senescene associated proteins and proteins associated with protein interaction. Conclusion: A number of genes that respond to salt stress were identified using cDNA-AFLP, the results will lay a foundation for determining potential molecular mechanisms of salt tolerance and the practice of molecular breeding of sunflower.
The genomic DNA were extracted from leaf tissue collected three cultivars of Pinellia, different phenotypes,collected from the field samples in Jingzhou, Hubei Province, which its corresponding the internal transcribed spacer(ITS)sequences were obtained using Polymerase Chain Reaction(PCR)directly and sequenced. The sequence alignment and phylogenetic tree analysis based on NCBI GenBank database, combined with the morphological characteristics of plants, indicated that HBB-01 belonged to Pinellia ternata,which is the "Regulations" from China Pharmacopoeia of Pinellia herbs, and HBD-02 belonged to P. tripartita,which is adulterants medicinal of Pinellia ternata. Notably, the result of HBF-03 is complex that its ITS sequence homology is up to more than 80%, which with the Colocasia,Arisaema,Pinellia. In the evolutionary relationship, they also have some connection. It suggested that HBF-03 may belong to varieties from Araceae. different genera but close kinship plants. The deduction needs further argument. In this paper, through the analysis of germplasm origins and phylogenetic relationships about the three cultivars of Pinellia, identificated the main varieties, which is artificial cultivation in Jingzhou, Hubei Province. The result has guided significance for the local medicinal, who have planting to meet "Chinese Pharmacopoeia" specification of P. Ternata. In addition, it has provided a reference basis for protection and breeding of high quality Pinellia resources.
Objective: To establish methodology for specific sorting of cardiac telocytes using c-Kit+ Combining with DiI Micro-label and flow cytometry. Methods: The cardiac telocytes were isolated via a c-Kit antibody conjugated magnetic bead method. The cells which have unique telocyte morphology, with long "Telopode", were labeled with DiI microinjection. The labeling DiI+ cells were sorted and recovered in 96-well plate by single cell flow cytometry technique. The immunofluorescence and RT-PCR were applied to confirm the phenotype of collected cells. Results: The cardiac telocytes (c-Kit+) which have unique telocyte morphology were able to be labeled by DiI microinjection. The DiI labeled cells were able to be sorted and recovered in single cell level. The adherent cells of collected DiI+ cells were 14.9% under culture. The 5.6% of cultured DiI+ cells was able to proliferate and the 2.4% of cultured DiI+ cells was able to form cell-clone. In addition, the formed cell-clone was able to subculture. The immunofluorescence staining confirmed that the recovered DiI+ cells were c-Kit-and CD34-positive. RT-PCR results also showed that the recovered DiI+ cells were positive for c-Kit, CD34, Vimentin and PDGFR-β, which are the markers for telocytes.Conclusion: The methodology which was established in present study was able to isolate and sort cardiac telocytes in single cell level. The sorted cardiac telocytes were able to proliferate and maintain the unique phenotype.
To investigate the efficiency of siRNA using aptamer-conjugated liposome(Apt-LP),A10-3.2 was used as a PSMA-targeting ligand in the design of a liposome-based siRNA delivery system to prostate cancer cells. The sequence of the aptamer used in this study is modified with 3'- Cholesterol and with 2'- Fluor pyrimidines. The cholesterol tag immobilizes the aptamer on the liposome surface by inserting into the hydrophobic lipid membrane. Liposome was conjugated to aptamer and used as a vehicle for siRNA target delivery. GFP expression assays of pEGFP - N1 against LNCaP (PSMA+) and PC-3 (PSMA-) cells demonstrated that the transfection efficiency of the synthesized Apt-LP complex was higher than that of the liposome. In addition, gene knock down assay of Apt-LP-siRNA complex in LNCaP (PSMA+) and PC-3 (PSMA-) cells showed that Bcl2 gene silencing effect significantly higher than that of LP-siRNA complex and more effectively induced apoptosis of LNCaP cells. These results suggest that the Apt - LP is a kind of effective siRNA targeting delivery system, has potential clinical application.
An expression system with high efficiency is very important for recombinant proteins production in biopharmaceutical field. Saccharomyces cerevisiae is a food-graded eukayotic organism. The features of short generation time, simple culture condition and well-characterized manipulation techniques make yeast S. cerevisiae an attractive cell factory for production of heterologous protein. Here, for the purpose to improve the efficiency of pHR expression system which was constructed in our lab previously., the promoter of pHR expression vector (PTEF) and host strain Y16 were modified by the way of error-prone PCR and mutagenesis respectively. After several rounds of screening, a mutant PTEFv1 with higher efficiency than the mother promoter PTEF was obtained. Two modified yeast strains Y16-E14 and Y16-E19 were identified with higher productivity of heterologous protein than yeast Y16. Then PTEFv1 and yeast Y16-E14 were used to construct the novel pHR-N expression system. To evaluate the ability of pHR-N expression system, yeast green fluorescent protein (GFP) and human serum albumin (HSA) were chosed to be expressed intracellularly and extracellularly respectively. The results showed that pHR-N system had higher ability to produce either intracellular GFP or extracellular HSA than pHR system. The pHR-N yeast expression system provides a valuable resource for future application in recombinant protein production.
The EchDA was systematically overexpressed by genetic engineering in the heterologous hosts Streptomyces lividans TK24. Mature peptide coding fragement of EchDA was amplified from Actinoplanesutahensis NRRL 12052. The expression plasmids pNW-S11, pNW-S12 and pNW-S13 are constructed by inserting EchDA,PermE*promoter and PSau3A promoter into vector pNW-S1. As a result, The EchDA was secretion expressed after induction with IPTGand the enzyme activity can reach 125 U/L under the best conditions for industrialized production.With optimization of the reaction conditions with 15 g/Liter FR901379, the molar conversion can achieve 100% within 24 hours.The highest enzyme activity by 300 L fermentation tank can reach 80 U/L, and the molar conversion can achieve 100%with 10 g/L FR901379. EchDA in industry have been relying on fermentationof original producer,Actinoplanesutahensis, which have low yield and activity. The construction of recombinant EchDA producing strain will be must to improve the current production process of echinocandin antifungal drugs.
The expression of target genes can be inhibited by RNA interference (RNAi) specificity via post-transcriptional gene silencing (PTGS) effects. RNAi silencing mechanism with efficiency, specificity and stability allows it to become an important tool in biomedical field. The focus is on the characteristics of RNAi technology and recent development in RNAi-based therapeutics, in particular structural modifications and designs for multi-target siRNAs were reviewed. It is illustrated that multi-target based siRNA therapeutics opens up a new therapeutic strategy and may help to improve the effectiveness of treatment. However, there are still some problems and challenges needing further researches for its widely use in clinical treatments.
In recent years, biocatalysis has started to provide an important green study tool in chemistry、 biology、Bioengineering and so on, currently, the ideal of using multi-enzymatic systems for these fields becomes increasingly attractive, it overcomes this limitation that many situations where a single enzyme cannot completely catalyze reactions, meanwhile, in the cascade reactions, it can cause the enzyme to act under a higher substrate concentration and combine different enzyme catalytic properties, also can eliminate interference factors by using the co-immobilized enzymes, therefore the whole catalytic efficiency may be improved. The research progress in Multi-enzyme Co-immobilization reaction systems was reviewed in three aspects including the classification of multi-enzyme reaction systems、the characteristics and applications of co-immobilization technology. In addition, Multi-enzyme Co-immobilization reaction systems were also predicted.
With the development of industry and agriculture, heavy metal pollution in soil is increasingly aggravated, which seriously threatens food production as well as human health. Because of its low cost, eco-friendliness and advantages on large-scale in situ remediation, phytoremediation has become an effective technology for the remediation of heavy metal contaminated soil in recent years. In this paper, the research and application progress of phytoremediation both in China and abroad is reviewed, and the soil remediation using energy plants especially sweet sorghum (Sorghum bicolor (Linn.) Moench) is highlighted for its great potential. Based on our research in Hunan Province, China, the advantages and feasibility of sweet sorghum in remediation of heavy metal contaminated soils is further discussed, and also the possible measures adopted to improve remediation efficiency is suggested. The plantation of sweet sorghum in heavy metal contaminated soils not only combine soil remediation with bio-energy production, but also transfer the heavy metals from the food chain into the energy chain, which shows broad application prospects.
Chitin deacetylase(CDA,E.C.3.5.1.41)is an enzyme that catalyzes the hydrolysis of acetamine groups of N-glucosamine in chitin, promoting the conversion to chitosan, a glucosamine polymer. Because of its unique properties,chitosan is widely used in medicine, food, chemical, cosmetics and so on. The present researches on CDA are reviewed. It includes the source, the purification and characterization, the structure and catalytic mechanism,the expression of gene cloning and applications prospect of the CDA, with analyzing the main research direction in the future are the expression of gene cloning, substrate modification and structure and catalytic mechanism of CDA.
Hyaluronic acid is a mucopolysaccharide composed of disaccharides unit of N-acetyl glucosamine and glucuronic acid polymerization. Now it has been widely used in medicine, cosmetics and food additives. Microbial fermentation is the most effective way to the current production of hyaluronic acid. Hyaluronic acid synthesis methods were Leloir way in organisms. Hyaluronic acid synthesis operon composed by hyaluronan synthase gene, UDP-glucose dehydrogenase gene and UDP-glucose pyrophosphorylase gene, it's expression is regulated by the CovS/CovR and LuxS, etc contro systeml. With the rapid development of molecular biology technology and the deepening of the understanding of HA synthesis related genes, from improve safety, increase production and regulation HA weight three aspects, people through genetic engineering means to build a high yield, security, a range of molecular weight of hyaluronic acid producing strains. Hyaluronic acid biosynthesis pathway, related gene expression regulation and production strain molecular biology transformation strategy and the research progress were summarized and prospected.
Thanks to the advantage of the characteristics of biotech drugs itself, biotech drugs is developing quickly. In the broader market, sales from biotechnology products are expected to account for 27% of the world's pharmaceutical sales by 2020, versus the current share of 22% in 2013. Roche is expected to remain the biggest player in the biotechnology space with sales increasing by $14.5bn to $43.5bn in 2020, representing an annual growth of 6% per annum. In support of the government, China have formed industrial clusters based on the Chinese medicine group as the leading. While total market of Chinese biotechnology drugs accounted for the global market is only 2%, but future is larger. At present China has formed industrial clusters,the biggest player of which is Sinopharm. With the increased demand for disease treatment, Biotech drugs has huge developing potential, the market will further expand.
