Objective: Try to improve the production and quality of TNFR-Fc fusion protein in CHO cells by inhibiting protein degradation. Methods: The stability and degradation pathways of TNFR-Fc in CHO cells were investigated by measuring the cell density and survival rate with additional lysosome inhibitor (Leupeptin) and ubiquitin-proteasome inhibitor (MG-132), respectively. The qualities of the total and extracellular TNFR-Fc expression were detected by Western blot and ELISA. Extracellular TNRF-Fc was purified by Protein A Affinity Chromato- graphy. The protein purity was analyzed by High Performance Liquid Chromatography (HPLC), while the enzyme activity of TNFR-Fc was assessed by measuring the inhibition level toward the cytotoxicity of L929 cell induced by TNFα. Results: TNFR-Fc was determined to be degraded via the ubiquitin-proteasome pathway;Adding 50 μmol/L MG-132 when cell culture, the production of total TNFR-Fc and dimer TNFR-Fc were improved 42.35% and 28.60%, respectively. Moreover, purified TNFR-Fc exhibited good bioactivity and the EC50 is 2.86ng/ml. Conclusion: MG-132 adding when cell culture may become a competent strategy to improve the recombinant protein production of CHO cells expression system.
Aim:To observe the adjuvant efficacy of murine zona pellucid 3(mZP3) DNA vaccine through intranasal vaccination with PolyI:C and IL-15, and chitosan as delivery vector. Methods: Mice were delivered with chitosan-loaded mZP3 DNA vaccine or added PolyI:C and IL-15 by intranasal route, and to detect specific IgG antibody level in serum, sIgA in vaginal fluid and lung fluid by indirect ELISA, to detect lymphocyte proliferation by MTT, to do histology analysis of mice ovary for morphology of ovary. Results: PolyI:C and IL-15 can promote IgG antibody level of immunized mZP3 mice, promote lymphocyte proliferation and ovarian abnormalities in mice. Conclusion: The results suggest that antagonist of TLR PolyI:C and IL-15 have a synergistic effect and may be enhance the immunity effect of mZP3 DNA vaccine and reduce murine fertility rate to some extent.
The nattokinase encoding gene (sNK) was synthesized by modifying its sequence based on the optimized codon usage in the plant. The first intron of tomato fruit-specific expression E8 gene was inserted in sNK gene to construct sNKi gene by overla Pextension PCR method. These two synthetic nattokinase gene were transiently expressed in tobacco NC89 leaves by agroinfiltration. Real-time quantitative reverse transcription PCR (RT-qPCR) result showed that higher expression level was detected in tobacco leaves which infiltrated with sNKi gene, compared with that infiltrated with sNK gene. Fibrinolytic activity was detected in transiently expressed samples of two synthetic genes by fibrin plate method, which indicated that the target genes can be normally translated and show thrombolytic activity in tobacco leaves. The translational expression level of sNKi gene was significantly higher than that of sNK gene. Those proved that intron can significantly improve the transient expression of sNK gene.
Objective: In an effort to understand the physiological functions of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and exploit ACO gene more profitably, an ACC oxidase gene (HaACO1) was cloned from sunflower and analyzed by bioinformatics method, and expression pattern of HaACO1 gene in sunflower was revealed under salt stress. Method: The full length cDNA sequence of ACO gene in sunflower was obtained by RT-PCR and 5'/3'RACE methods based on the sequence of 4-4-7TDF (accession No. KM823963), a gene fragment of ACC oxidase from Inner Mongolia Sunflower Hybrid No.4. The cDNA sequence and its encoded protein was analyzed by bioinformatics method. The genemic DNA (gDNA) of ACO sequence was cloned by PCR. The expression level of HaACO1 gene in roots, hypocotyl and leaves under NaCl stress was tested by quantitative Real-time PCR (qRT-PCR). Results: The full length sequence of HaACO1 gene in sunflower was 1 135bp, and its open reading frame (ORF) was 942bp, then the protein sequence analysis results showed that the HaACO1 gene encoded 313 amino acids with molecular weight 35.84kDa and pI 5.13. The sequence accession No. in GenBank was KP966508. The nucleotide sequence and the amino acid sequence of HaACO1 was highly homologous to other species (75%~83%, 77%~88% respectively). The full length of the coding region of gDNA was 1 018bp, comprised of two exons separated by one intron. The sequence has been submitted to GenBank (Accession No. KP988289). The results of qRT-PCR showed that the expression level of HaACO1 had specific expression differences in different organs. Different concentration of NaCl treatmet or different induction time with NaCl also lead to the specific expression of HaACO1 in sunflower. Conclusion: HaACO1 belongs to ACO gene family in plants, and this gene has distinctive expression pattern when plants response to salt stress.
The disruption of competitive metabolic pathways is conducive to the carbon flux converted into the purpose amino acid biosynthetic pathway. In the present study, based on the prediction of genome-scale metabolic network model, markless knock-out technology to construct separately recombinant strains C. crenatum MT-M4 △pta and C. crenatum MT-M4 △ack which led to interdict the acetic acid biosynthetic pathway were employed. As shake-flask fermentation results shown, L-arginine production of C. crenatum MT-M4 △pta was significantly increased by 25.60% higher than that of original strain, reached to 15.46g/L with glucose transformation rate increased by 29.41%. L-arginine production of C. crenatum MT-M4 △ack was 13.82g/L which was 12.81% higher than that of original strain, with glucose transformation rate increased by 26.02%. In addition, the growth of C. crenatum MT-M4 △pta and C. crenatum MT-M4 △ack was increased by 9.19% and 7.71%, respectively. Thus, pta and ack deletion was not only beneficial to the improvement of L-arginine production, but also conducive to the cell growth. However, pta deletion compared with ack deletion was more conducive to L-arginine accumulation.
Objective:To generate and identify the cell line for detecting the unknown flaviviruses. Methods: The defective replicon with a deletion of prM-E gene and the insertion of the red fluorescent protein mCherry reporter gene was constructed on the basis of the infectious clone of dengue virus type 4 (P4), which was named P4-△prME-mCherry. To select the packaging cell lines for detecting flaviviruses,The multi-fusion PCR method to construct the defective eukaryotic expression plasmid pCDNA3.1-P4-mCherry on the basis of P4-△prME-mCherry were used. BHK-21cells were transfected with thepCDNA3.1-P4-mCherry, then the G418-resistant BHK-21cellsexpressing the defective replicons were obtained, which were named BHK-Flavivirus.Results: The expression of mCherry reporter gene were detected after the BHK-Flavivirus cells were infected by Flaviviruses,such as Japanese encephalitis virus (JEV,P3) and Dengue virus(DENV,P4). By contrast, mCherry was silent when theBHK-Flavivirus were infected by three related plus-strand RNA viruses, including Sindbis virus (SINV,XJ-160, YN87448), Chikungunya virus (CHIKV,SD08Pan) and Getah virus (GETAV,HB0234),and Tahyna virus (TAHV,XJ0625). Conclusion:These result indicated that the BHK-Flavivirus were specific and effective to detect Flavivirus in cell culture, which is a simple, color-visible detection method could detect corresponding mosquito-borne viruses quickly and specifically without affecting the viral multiplication capacity, making it possible to be used in the detection for clinical examinations and the screening of viral biological warfare agents.
Due to its safety and excellent exocrine capacity, Bacillus subtilis is used as the heterologous expression system of lipase currently. How to improve the secretion level of lipase in Bacillus subtilis has become a research hotspot. The expression of LipS in Bacillus subtilis has the problems that the signal peptide of LipS can't secrete itself well and the expression level of LipS isn't very high. To solve these problems, the screening of different signal peptides with different structure in Sec pathway and Tat pathway maybe a good method. The results show the signal peptide phoD can secrete the LipS more well than the other signal peptides. Then the strategies such as changing the expression system with an inducible expression system is took, optimizing the signal peptide phoD and expression conditions, the secretion level of LipS increased significantly. The secreted lipase activity reached 62.07U/L, accounting for 62.30% of the total lipase activity. Compared with the extracellular activity of the original system, the new system increased about 13.7 times.
AKT, also known as protein kinase B, is a pivotal component of pathways associated with cell survival, metabolism, invasion and metastasis. Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of Janus protein kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway and may involve in the phosphorylation of AKT and tumorigenesis. The review is focused on the biological function of SOCS3 and the role of SOCS3 in AKT signal pathway, which may be beneficial to targeting AKT signal pathway in cancer therapy.
miRNA are a class of endogenous single-stranded small molecule RNA within eukaryotes, about 18~26 nucleotide, which can trigger the target mRNA degradation or translational repression of target mRNA through specific complementary base pairing. miRNA dysregulation can have profound cellular consequences. In cancer, the loss of tumour-suppressive miRNAs enhances the expression of target oncogenes, whereas increased expression of oncogenic miRNAs (known as oncomirs) can repress target tumour suppressor genes. This realization has resulted in a promotion to comprehend the feasibility of targeting oncogenic miRNAs and restoring tumour-suppressive miRNAs for cancer therapy. In conclusion, with the deepening of clinical studies in miRNA,new ideas and methods are provided for molecular diagnostics and cancer therapy.
Long noncoding RNA (lncRNA),longer than 200 nucleotides, can not encode protein, and directly plays a role in the form of RNA at transcription and post-transcription level as decoys, signals, guides, and scaffolds. Compared with the expression of encoding protein gene, the expression of lncRNA is lower. Genomewide studies have uncovered there are only a small amount of the encoding gene, while there are a large number of ncRNAs, which play regulatory role in plants, animals and human. In recent years, the study of ncRNA has been mainly on miRNA and siRNA, having achieved fruitful results. However the research of lncRNA has just begun, some researches have shown that lncRNA has extensive biological functions,such as chromosome modification, X chromosome silencing, transcription interference, transcription activation, and nuclear transport. Meanwhile the related research methods represented for RNA-seq, microarray, and FISH are also developing.
Alexa Fluor is a derivative of rhodamine or coumarin. As an organic fluorescent dye, Alexa Fluor has some obvious advantages such as narrow excitation spectrum, wide emission spectrum, high quantum yield, good light stability and less temperature and pH impact. Recently, though Alexa Fluor dye gets a wide range of applications in the field of cytology and molecular biology research, there are few articles about Fluor Alexa dyes. elaborate Alexa Fluor's application and make prospects for its development in cytology and molecular biology science based on the characteristics are elaborated.
Inhibition of p53 by abnormaloverexpression of Mdm2 (murine double minute 2, also known as Hdm2) and MdmX (murine double minute X, also known as Hdm4) is relevant to nearly half of cancers. Thereby it is clinically profound that the removal of the inhibition of Mdm2/MdmX to p53 by designing Mdm2/MdmX-specific inhibitors. Structurally Mdm2 and MdmX are similar to each other, but the discovery of Mdm2 inhibitor has so far made advanced progress compared to the design of MdmX inhibitors. The progress on the design of MdmX inhibitors based on the structure-activity relationships of nutlins (Mdm2 inhibitors), structural biology and combinatorial chemistry were reviewed. The potential application of natural compounds is discussed for searching novel scaffolds of Mdm2/MdmX inhibitors.
Phytase is a kind of enzymes that stepwise hydrolyse phytic acid to release phosphate radicals and inositol derivatives. As a new type of feed additives, it has great application potential in the areas of animal nutrition and environmental protection. Here the classification, sources, production technique, and downstream processing of phytase, as well as the strategies employed for improving phytase production and the trends for further research and development in the future are summarized.
Heavy metals are a class of pollutants that may produce certainly toxic actions towards plants, plants have evolved mechanisms of heavy metal stress tolerance in long-term evolution. Based on plant heavy metal tolerance, making a brief overview that the study of tolerance mechanisms of plant response to heavy metal stress at home and abroad in recent years. The molecular mechanism that stress effects of heavy metals on plants, plant antioxidant systems, osmotic adjustment substance including proline, soluble sugar or protein, and different types of gene families under heavy metal are mainly discussed. In order to improve the ability of plant resistance to heavy metals and help phytoremediation.
Streptomyces are well known as a particularly abundant source of secondary metabolites, including antibiotics, immunosuppressants, anti-cancer agents and many other bioactive compounds. Many of them have important application in the field of clinical medicine and aquaculture, etc. Nevertheless, the synthesis of secondary metabolites is often closely related with environmental and nutritional factors. Herein,the influence of inorganic phosphate and the molecular mechanism of the two-component PhoR-PhoP signal transduction system on the synthesis of secondary metabolites are reviewed. Streptomycetes sense and respond to phosphate via the two-component PhoR-PhoP signal transduction system.When the concentration of phosphate in the environments decreases below a threshold level. PhoP plays a major role in the repression of the central and secondary metabolic pathways to slow down the consumption of phosphate, and the activation of scavenging uptake and transport systems that allow the cell to recover inorganic phosphate from external sources, which ultimately affect the secondary metabolites production and morphological differentiation.
Microbial lipases, being major sources of commercial ones, which have been widely utilized in many industrial fields, such as foods, beverages, lipids, detergents, feeds, textiles, leathers, advanced materials, fine chemicals, medicines, cosmetics, papermaking, pollution treatment, and bioenergy, etc. Compared with other microbial lipases, bacterial lipases have more types of reactions and exhibit higher activity and better stability in many reactions. Amongst bacterial lipases, the most excellent ones are those being from the genus Pseudomonas. As one of the most excellent and the most widely used lipases, the study on Pseudomonas lipases has been a hot topic in the field of lipases. Research advances in molecular biology of Pseudomonas lipases, including mining and cloning of genetic resources, molecular mechanisms of gene expression regulation and protein secretion, strategies for efficient overexpression, protein crystallization and 3D structure analysis, and protein engineering, were summarized and reviewed. Furthermore, the future research directions of Pseudomonas lipases were prospected so as to provide a useful reference for the follow-up studies.
Cytidine is good intermediates of anti-tumor and antiviral, also can be used as health food ingredients.The metabolic regulatory mechanism of cytidine biosynthesis in Bacillus amyloliquefaciens was discussed and the breeding strategy of constructing cytidine high-yielding strain was summarized. The focuses were on elaborating the six breeding strategies, such as improving the pyrimidine operon transcriptional level of Bacillus amyloliquefaciens, enhancing cytidine anabolic pathways and central metabolic pathways to cytidine synthesis, blocking degradation pathway of cytidine, increasing the pentose phosphate pathway to the sub-flow of cytidine synthesis pathway, reducing the bypass pathway and accelerating cytidine secretion.Thereby, the strategies of breeding high yield strain were proposed, which could provide some ideas for breeding cytidine high-yielding strains, and solve the existing problems in the processing of cytidine industrial production.
