Many studies have showed epidermal growth factor receptors (EGFR) were overexpressed in various types of cancer cells. The S3, an EGFR-binding domain derived from vaccinia virus growth factor (VGF), when fused to HE, a heparin binding domain derived from heparin-binding epidermal growth factor (HB-EGF),could enhance its tumor-homing ability. The incubation experiment of the fusion protein EGFP-S3-HE/EGFP-S3-HE-TATm (TAT is cell penetrating peptide) with normal/tumor cells proved that both the recombinant proteins had tumor cell specific targeting ability and significant penetrating effect.The fusion of S3-HE-TATmwith MAP30, a ribosome inactivation protein derived from Momordica charantia, significantly increased the inhibitory effect of MAP30 to tumor cells but still remained relatively low level effect to normal cells, Therefore, S3-HE-TATm is a new type of drug delivery vector for tumor targeting treatment.
Objective:To investigate the role of Runx1 gene on BMP9-incuced osteogenic differentiation of Mouse embryonic fibroblasts (MEFs). Methods:MEFs were infected with Ad-BMP9 and then the expression of endogenous Runx1 at the mRNA and protein level was determined by RT-PCR and Western blot.The recombinant adenovirus Ad-Runx1 was constructed and its expression was validated at the mRNA and protein level. MEFs cells were treated with Ad-Runx1 or/and BMP9, the ALP activity was detected by quantitative and staining assay, calcium deposition was detected by Alizarin Red S staining. Runx2, as a core osteogenic transcription factor, was detected by RT-PCR and Western blot at the mRNA and protein level. Result:The expression of Runx1 was up-regulated by BMP9 at the mRNA and protein level in MEFs cells; Ad-Runx1 could enhance the expression of Runx1 at the mRNA and protein level. Runx1 can increase ALP activity and calcium deposition of MEFs cells induced by BMP9,and promote then expressions of Runx2 at the mRNA and protein level. Conclusion:BMP9-induced osteogenic differentiation is partially improved by Runx1 in mesenchymal stem cell line MEFs.
Objective:To construct the prostate cancer cell line LncapC4-2 that overexpress GPRC6A gene, detect the cell migration and invasion abilities and the expression of EMT related genes, thus to establish the basis of exploring the role of EMT induced by GPRC6A in prostate cancer progression. Methods:The GPRC6A overexpression lentivirus vector pCDH-GPRC6A was constructed, The lentivirus were packaged using human embryonic kidney 293T cells and LncapC4-2 cell line was infected, and GPRC6A stably overexpressing cell line was screened using puromycin. RT-qPCR and Western blot methods were used to verify the overexpression of GPRC6A in the cell line, scratches and transwell cell migration assay were used to test the cell migration and invasion abilities and QPCR was used to detect the expressions of EMT-associated genes. Results:The lentivirus vector pCDH-GPRC6A was constructed successfully. In GPRC6A overexpression Lncap C4-2 cells, the GPRC6A expression was increased in mRNA and protein levels detected by using RT-PCR and Western blot methods. The cell migration and invasion abilities were higher in GPRC6A overexpression Lncap C4-2 cells than those in control cells. The expressions of EMT-associated gene E-cadherin and SNAIL were reduced and increased, respectively, in GPRC6A overexpression LncapC4-2 cells. Conclusion:The prostate cancer cell line LncapC4-2 that stably overexpress GPRC6A has been constructed successfully. GPRC6A maybe involves in the progression of prostate cancer by enhancing the abilities of cell migration and invasion, and promoting EMT in cancer cells.
Objective:To study the effect of PLCε regulated by miR-145 on EMT and metastasis in bladder cancer cell T24 and and the potential mechanism. Methods:①Using adenovirus infecting T24 cells,the migratory abilities of T24 cells were observed by wound healing and transwell chamber cell migration assay.The expression of EMT related molecules such as E-cadherin, n-cadherin、Vimentin were detected by RT-PCR and Western blot,at genetic and protein level respectively. In order to explore the molecule mechanism,western blot was used to test the protein level of p-GSK-3β and Snail. ② Using bioinformatic to calculate the possible microRNA regulating PLCε,referenced to the reported microRNA array, miR-145 was chosen for the target microRNA.Using miR-145 mimics transfecting T24,the expression of miR-145 and PLC ε were detected by quantitative Real-time-PCR, meantime, the PLCε protein expression was tested by Western blot. ③ After cells transfected with miR-145 mimics,the protein expression of EMT related molecule as well as p-GSK-3β and Snail were detected by Western blot. Wound healing and transwell chamber cell migration assay were adopted to detect he migratory abilities of T24. Results:① Wound healing and transwell chamber cell migration assay showed that the migratory ability was remarkably decreased in the Ad-shPLCε group,compared with the blank contol group and Ad-HK group(P<0.05).The expressions level of both mRNA and protein of N-cadherin, Vimentin were higher,while E-cadherin was lower in Ad-shPLCε group than that in blank contol group and Ad-HK group(P<0.05). Western blot result showed that the protein level of p-GSK-3β and Snail were significantly lower after treated with Ad-shPLCε(P<0.05). ② qPCR result showed that, transfection miR-145 mimics could restore miR-145 expression of bladder cancer cells T24,significantly higher than transfection negative control(P<0.01),by contrst,the PLCε mRNA expression was lower(P<0.05). Western blot result showed that the PLCε protein expression was markedly down-regulated (P<0.05),which was consistent with qPCR result. ③ After transfection miR-145 mimics, the migratory ability of bladder cancer T24 significantly lower than negative control(P<0.05). Western blot result indicated that,on the one hand, the expression of N-cadherin, Vimentin was lower than negative control, while E-cadherin was higher(P<0.05),on the other hand,overexpression of miR-145 could significantly inhibit the expression of p-GSK-3β and Snail(P<0.05).Conclusion:PLCε induce EMT and metastasis via GSK-3β/Snail pathway,overexpression of miR-145 can downregulate the role of PLCε on bladder cancer.
The clt-1 gene within Curvularia lunata genomic sequence was screened through Agrobacterium-mediated transformation (ATMT) method. Bioinformatic analysis showed that the molecular weight of the protein encoded by clt-1 was 81.984 8kDa, the oretical isoelectric point pI was 8.62, and the instability index was 55.08. CLT-1 was a hydrophilic protein, containing one transmembrane helix topologies. At subcellular level, CLT-1 protein located in the nucleus. More than one of the serine, threonine and tyrosine kinase phosphorylation sites in CLT-1 amino acids sequence were found. In the aspect of its function, CLT-1 protein contained a typical conserved BTB function domain, which may regulate the expression of some functional protein. The expression vector pC1300th-Pclt1-GFP was constructed for detecting its promoter functional activity by the fusion of clt-1 promoter fragment to the reporter GFP gene. Transformation of plasmid pC1300th-Pclt1-GFP was conducted by ATMT method of the spores of C. lunata wild-type CX-3. The putative transformants were obtained from selective regeneration medium and confirmed by PCR detection and green fluorescence analysis. The results indicated that GFP gene was integrated into the genome of CX-3 strain, and the clt-1 promoter could drive the GFP gene expression. Strong green fluorescence activity was detectable in the mycilia and spores of transformants under the confocal laser scanning microscope.
The integral Δ9-I desaturase gene from Mortierella alpina ATCC 32222 was expressed and purified from Pichia pastoris. The codon-optimized gene for theΔ9-I desaturase was appended to a cassette containing the human rhinovirus 3C protease cleavage site, the IgG-specific ZZ-tag, and an RGS-His10-tag. The highest expressing clone was determined by Western blotting and SDS-PAGE analysis. A membrane fraction was isolated by gradient centrifugation and detergents were used for efficient extraction of desaturase. Affinity purification of desaturases was conducted using the C-terminally fused IgG-binding ZZ-tag. The enzymatic activity was determined using cell lysate of Saccharomyces cerevisiae as substrate. The cytochrome b5 domain was characterized by wavelength scan and Na2S2O4 reduction experiment. Results showed that the Mortierella alpina Δ9-I desaturase was expressed successfully and transformant with high expression level was selected. The membrane fraction was collected at a centrifugation of 20 000g for 1h and only Fos-Choline-16 effectively extracted the desaturases from the membrane, when compared to the efficiency of SDS. The purified desaturase was intact and the cytochrome b5 domain was present. In the yeast lysate, the conversion rate of C16:0 and C18:0 substrate were (16.88±9.32)% and (20.61±7.55)%, respectively. Wavelength scan of the purified protein revealed an absorbance feature at 411nm and it shifted to 422nm in the presence of Na2S2O4. This result indicated that the cytochrome b5 domain can be reduced in vitro. Thus, the first successful purification of an integral desaturase that contained the fusion cytochrome b5 domain lays the foundation for the mechanism study of heme integral desaturase.
Background:Nodal is a member of the transforming growth factor beta (TGF-beta) super family, essential in maintaining the pluripotency of human embryonic stem cells (hESCs), which is not active in most adult tissues, its reexpression and signaling have been linked to multiple types of human cancer. Recent findings have revealed that Nodal is a critical regulator of melanoma growth, plasticity and tumorigenicity, and holds promise as a new tumor biomarker. Similar observations have been reported in multiple human cancers. To utilize Nodal as a useful biomarker for cancer detection, a double monoclonal antibody sandwich enzyme-linked immunosorbent assay (ELISA) may be a reliable, easy and cheap means. Methodology/principal findings:Recombinant Nodal was used as antigen to immunize BALB/c mice, by cell fusion between plasma cells and SP2/0 cells, 7 specific positive monoclonal antibodies were picked out, which were verified through indirect ELISA, Western blot and flow cytometry analyses. Therefore 15 pairs of antibodies can be used to set up a double monoclonal antibody sandwich ELISA, AF12-DG5 was the most appropriate antibody pairs. Combined with biotin-avidin systerm, DG5-biotin was used as detection antibody while the complex of streptavidin and HRP-biotin (mass ratio 4:1) as detection reagent. The best working concentration of antibodies were AF12 2μg/ml and DG5-biotin 2μg/ml. Linear range of this ELISA is 0~3 000pg/ml, regression equation is y=0.000 3x+0.150 5,R2=0.999, low limit of detection is 68pg/ml, average recovery is 99.6%. The result of serum detection indicates that more Nodal was expressed in 70% of gallbladder carcinoma patients, 92% of nasopharyngeal carcinoma patients and 97% of colorectal carcinoma patients, which were statistically significantly increased compare to normal human.Conclusions/significance:A sensitive and specific Nodal ELISA test has been developed. This test is high sensitive and easy to use and can discriminate cancer patients from normal person specifically. The achieved data suggest that the developed ELISA may be applicable as a research tool for detection of human cancer.
Objective:To construct virus-like particles(VLPs) presenting QPLGVGISGHPLLNKLDDTE epitopes of HPV 16 L1, and detect its antigenic specificity. Methods:The reported effective HPV 16L1 epitopes was selected and used to be presented by HBcAg VLPs. The oligonucleotides encoding for the peptide was inserted into plasmid pHBcAg. The recombinant plasmid was transformed into DH5α cells, and the expression of the chimeric proteins was induced with IPTG and identified by SDS-PAGE. The proteins was purified with a procedure consists of ammonium sulfate precipitation and sucrose density gradient centrifugation, and the presence of VLPs was detected with HPLC of size-exclusion chromatography and electron microscopy. Western blot showed the specific bands of the expressed recombinant protein. Mice were immunized with the mixed VLPs, and the serum to identify HPV 16L1 protein was measured by Western blot. Results:The constructed recombinant plasmid was proven to be correct by restriction enzyme digestion and DNA sequencing. The recombinant protein was expressed efficiently, and presented as VLPs. Western blot showed that the antiserum of VLPs immunizied mice could be recogniazed specifically by the recombinant yeast expression of L1 protein. Conclusion:HBcAg VLPs could present HPV 16L1 epitope and the recombinant HBcAg/16L1 VLPs could stimulate special immune response.
Bitespiramycin (BT) is a multi-component antibiotic consisted mainly of 4″-isovalerylspiramycin (isomycin) I, II and III. An isomycin I producing strain WSJ-2 has been obtained by inactivation of the 3-O-acyltransferase gene (sspA) in WSJ-1 which was responsible for the acylation of spiramycin I to II and III.However, the fermentation products of WSJ-2 contained plenty of spiramycin, and a low percentage of isomycin I. In order to improve the production of isomycin I through increasing the expression of 4″-isovaleryltransferase gene (ist), a recombinant plasmid pSET152-ia, carrying ist gene with positive regulatory gene acyB2 in Streptomyces thermotolerans, was constructed and introduced into Streptomyces spiramyceticus WSJ-2 by protoplast transformation. The recombinant plasmid pSET152-ia was integrated into the chromosome of WSJ-2 via Escherichia coli-Streptomyces shuttle vector pSET152, A new isomycin I producing strain, Streptomyces spiramyceticus WSJ-IA, was generated. The expression of ist in different culture period was detected by real-time quantitative PCR, which indicated that the ist expression of WSJ-IA was higher distinctly than that in WSJ-IA. The fermentation titer of high-yield WSJ-IA strains was up to (1160±108)μg/ml, increased by 314% compared to the original strain whose fermentation titer was only (280±20)μg/ml and the content ratio of isomycin I to spiramycin I in WSJ-IA was about 2.4 times of that in WSJ-2. These results demonstrated that the introduction of acyB2 gene can increase both the fermentation titer and isomycin production.
Steroids with unique biological activity are widely used as anti-inflammatory, diuretic, anti-androgenic, contraceptive and anti-cancer agents. In recent years, the role of biocatalysis and biotransformation in the synthesis of steroid drug intermediates is becoming more and more significant. In order to synthesize the potential steroidal compounds, biotransformation of androst-4-en-3,17-dione (androstenedione, AD) by Gibberella intermedia C2 was investigated. The two products was identified as 15α-OH-AD and 11α,15α-diOH-AD by structure identification. The results of the conversion mechanism research showed that androstenedione is first converted into 15α-OH-AD with the 15α-hydroxylation of AD, and subsequently, into 11α,15α-diOH-AD after the 11α-hydroxylation of 15α-OH-AD. G. intermedia C2 can selectively and sequentially perform two DHEA hydroxylation reactions. In addition, the optimum biotransformation conditions of androstenedione were obtained as follows:initial pH value 6.5, the strain incubated in 250ml shake flask with 30ml medium at 28℃, substrate concentration 6.0g/L, culture for 24h and biotransformation for 84h at 220r/min. The molar conversion reached 81.5% under above conditions.
HUMSCs is a stem cell population with high self-renewal capcity and multiple differention potential.They have secret special cytokine,induce apoptosis of tumor,migrate to tumor cells,adapt on gene editing and safety properties.Regarding their therapic value,many researchs use HUMSCs as cell-based treatment in tumor.The anti-tumor effect of HUMSCs is critically elaborated.
With the development of functional genomics, molecular marker technology is moving towards the direction of functional target marker genes. Functional marker is developed according to the specific region polymorphism motif of functional genes closely related to phenotype. Since these functional markers are directly derived from functional motifs within the gene, these markers do not require further validation to determine whether alleles are available in different genetic backgrounds.Gene-targeted and functional marker, conserved DNA and gene family based markers, transposable element based markers, resistance-gene based markers, RNA-based markers and targeted fingerprinting markers were discussed, which are aimed at providing a theoretical basis for the development and application of molecular markers.
Abiotic stresses, such as high salt, drought, low temperature and heavy metal pollution, have seriously affected the growth and reproduction of plants. Meanwhile, plants have evolved a series of various enzymes system against oxidative damage caused by abiotic and biotic stresses. Glutathione S-transferase,which comprise a large superfamily of multifunctional protein, can scavenge reactive oxygen species and protect plant cell membrane structure and protein activity when plants were subjected to high salt, drought and low temperature stresses. The role of GST in plant response to abiotic stress is reviewed, and this will provide valuable information for the plant genetic engineering in the future.
Single cell oil(SCO)is the most ideal material for biodiesel production. With the increasing depletion of fossil fuels, the production of SCO has been widely concerned. Yarrowia lipolytica is one of the best strain for the production of SCO. Many kinds of cheap substrates can be used as carbon source, so it has great application in industry. Its genetic background is clear, the whole genome sequencing has been completed, gene expression system has been constructed. On the basis of this, the oil accumulation ways were studied, and the strains with higher oil content were constructed. It has great significance to research the gene expression and lipid metabolism of Y. lipolytica for further study.
With the advantages of good targeting ability and less adverse reactions, monoclonal antibodies industry has grown rapidly in various therapeutic areas including tumor and autoimmune diseases. There are 55 kinds of antibody drugs in global market and the global sales have reached 91.63 billion dollar in 2015. Currently, China is in the stage of rapid development of antibody drugs. At present, 22 kinds of monoclonal antibodies were approved by CFDA, containing 10 kinds of domestic products and 12 kinds of imported products. The market scale of domestic monoclonal antibodies is 5.034 billion yuan in 2014. With the development of biotechnology, the market prospects of domestic monoclonal antibodies will become more and more extensive.
Molecular detect methods were widely used in diagnosis of infectious diseases, genetic diseases and prenatal prediction. Nowadays, with the more and more emerging infectious diseases and the development of precision medicine, automated molecular diagnostic system has attracted more and more attention as its rapid, accurate and convenient advantage. The automated molecular diagnostic systems and the company were summarized, and the development trend in this field was prospected.
With the rapid development of next generation sequencing technology and the researches on fermentation mechanism of food microorganism, data and knowledge of food microorganisms increased enormously, including genomic, metagenomics, metabolic and phylogenetic information. These data are distributed from different resources with various data formats. An integrated data platform is necessary for better understanding of biological knowledge from such growing heterogeneous data. As a result, we construct a food microorganism database using semantic web technology. We describe information of gene, genome sequences, gene ontology, protein sequences and structures, pathway and enzyme in the form of Resource Description Framework (RDF) from a wide range of open data resources. In this database, physiological information of microbes from culture collections could be linked to the genomic information and further linked to the metabolic information which allows flexible queries across different domains. User-friendly interfaces of the database provide the ability to answer a number of food microorganisms research related questions based on the linked data.
