Objective: To investigate the effect of S100A9 on hepatocellular carcinoma cell HepG2 and the relevant mechanism.Methods: CCK-8 assay and cell migration assay were used to study HepG2 growth and migration mediated by Hepatitis B virus X (HBx) respectively. Transfected S100A9-siRNA into cells for silencing S100A9 expression, then the growth and migration of HepG2 infected with AdHBx were analyzed. Real-time PCR and Western blot were used to detect S100A9 mRNA levels and expression in HepG2 cells infected with AdHBx or AdGFP. After the treatment with or without NF-κB inhibitor BAY11-7082, S100A9 mRNA levels and expression in AdHBx-infected HepG2 cells were detected again.Result: HBx enhances the growth and migration of HepG2 cells. Silencing S100A9 expression partially blocked HBx-induced growth and migration of HepG2 cells. The mRNA level and protein expression of S100A9 were significantly higher in HepG2 cells infected with AdHBx than with AdGFP, and that suggests S100A9 expression can be modulated by HBx. NF-κB inhibitor treatment efficiently suppressed the increase of S100A9 levels caused by HBx.Conclusion: Expression of S100A9 is regulated by HBx-mediated NF-κB activation, and S100A9 plays an important role in HBx-induced growth and migration of hepatocellular carcinoma cell HepG2.
Objective: R2R3-MYB transcription factors regulate primary and secondary metabolism in plants.Methods: A R2R3-MYB encoding sequence, characterized and cloned from drought treated transcriptome of Caragana intermedia, was named as CiMYB15. The gene was transferred into Arabidopsis thaliana, and the total flavonoids contents of transgenic lines and wild type were measured by spectrophotometric method, and the expression of AtCHS in transgenic plants was analyzed by qRT-PCR. A 1 580bp fragment of CiMYB15 promoter was isolated by genome walking. The results revealed that: (1) the length of CiMYB15 gDNA was 1 960bp, it’s consisted of three exons (134, 131 and 521 bp) and two introns (281 and 893 bp). The open reading frame (ORF) encodes a polypeptide of 262 amino acids. (2)The main cis-elements of CiMYB15 promoter include abiotic stress responded elements (G-box, P-box, GT1-motif and MBS), biotic stress responded elements (BOX-W1 and EIER), and MYB binding sites of flavonoids synthase regulatory genes. (3) The expression of CiMYB15 was induced by UV-B. (4) Total flavonoids contents of CiMYB15 overexpression plants were higher than that of wild type Arabidopsis. (5) Furthermore, the expression level of AtCHS was increased in transgenic Arabidopsis. In brief, CiMYB15 positively regulated the flavonoids metabolism.
The antifungal lipopeptides produced by Bacillus amyloliquefaciens TF28 have been isolated and identified and their antifungal activity have been studied. The extract of antifungal lipopeptides was prepared by acid precipitation, ethyl acetate and methanol extraction. After two rounds of HPLC separation, eight antifungal lipopeptides were obtained within 32~42 min of retention time, and they have been identified as fengycins by MALDI-TOF-MS, which show strong antifungal activity against Fusarium oxysporum and Fusarium graminearum. These results will contribute to the directional genetic manipulation of the strain TF28 to improve the yield of antifungal lipopeptides.
Chitinase encoding gene of Trichoderma reesei was optimized, synthesized and secretorily expressed in Pichia pastoris. The protein concentration of the expressed chitinase reached 0.17mg/ml. The optimum pH and temperature of the chitinase was 5.6 and 65℃, respectively, and enzymatic activity reached 0.52U/ml. The chitinase was continuously thermostable at 50℃. The low deacetylated chitosan was hydrolyzed by this enzyme and the composition and structure of these hydrolysates were analyzed. Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS) results showed that these hydrolysates contained at least 41 different kinds of chitooligosaccharides with degree of polymerization of 2~18 and different degree of deacetylation. Nuclear magnetic resonance (NMR) results indicated that, the reducing end of these oligosaccharide fractions were mainly composed of N-Acetylglucosamine while both glucosamine and N-Acetylglucosamine were found in the non-reducing end. These results would provide a reference for study of the relationship between the structure and function of chitooligosaccharides.
Astrocyte elevated gene-1 (AEG-1) was overexpressed in a diverse array of cancers and played an important role in the development and progression of cancer. The study aimed to construct the AEG-1-knockout U251 cell line by CRISPR/Cas9 technology and to explore the effect of AEG-1 on the metastasis in U251 Cells. Firstly, the designed sgRNA targeted to AEG-1 was synthesized, and was cloned into the pX459 plasmid to obtain the AEG-1-pX459 recombinant vector. The recombinant vector was transfected into human glioma U251 cells, and the activity of sgRNA was identified by TA cloning sequencing. Then, the U251 cells transferred with the recombinant vector were screened by puromycin to get the AEG-1-knockout cell line. The efficiency of gene knockout was detected by Western blot assay. Finally, the migration ability of the AEG-1-knockout cell line was evaluated by the methods of Transwell and Scratch experiment. The results showed that the AEG-1-pX459 recombinant vector was successfully constructed, and the sgRNA activity was confirmed by TA cloning sequencing. The AEG-1-knockout U251 cell line was successfully established. Western blot assay analysis showed that the knockout efficiency high to 98%. The Transwell and Scratch experiment results illustrated that the migration ability of the AEG-1-knockout cell line reduced obviously.
A high efficient in vitro regeneration system was developed from leaf explants of ‘Guichang’ kiwifruit (Actinidia chinensis) and the multiplication coefficient and rooting rate of adventitious buds were also optimized. Results revealed that the adventitious buds developing directly from leaf explants were noticed after 30d of culture. The maximum regeneration frequency of adventitious buds is 95.8% and 15.7 shoots was observed in each leaf explants when MS medium was supplemented with 4.0mg/L 6-BA+0.4mg/L NAA. The optimal culture medium for bud proliferation is MS+3.0mg/L 6-BA+0.3mg/L NAA+0.2mg/L GA3 and the proliferation coefficient reached 8.15 in every subculture during 1 to 6 subcultures. On the rooting medium with 1/2 MS+1.0mg/L IBA for 15d, the adventitious plantlets were successively transferred into agar power and matrix perlite supplied with 1/2 MS liquid medium for 15d and 95.83% of them rooted and the roots were also heavy and strong very much. 49 out of 50 plantlets (99%) survived acclimatization in the greenhouse after transplanting them to the Mixed matrix of pearlite and soil(1∶4)for two weeks. In conclusion, a highly efficient regeneration protocol via leaf explants of ‘Guichang’ kiwifruit was successfully established and it may provide theoretical and technical guidance for micropropagation and genetic transformation studies of ‘Guichang’ kiwifruit.
This study aimed to achieve the constitutive expression of human goose-type lysozyme 2 (hLysG2) in Pichia pastoris using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and to establish an efficient strategy for the production of recombinant hLysG2 (rhLysG2) on a bench scale. The hLysG2 gene was synthesized according to the codon usage preference of P. pastoris and cloned into pGAPZαA vector. The resulting pGAPZαA-hLysG2 plasmid was linearized and transformed into competent P. pastoris GS115, followed by Geneticin screening. The transformants with higher Geneticin resistance were selected to investigate the constitutive expression of rhLysG2 in P. pastoris. The lytic activity of rhLysG2 in the fermentation broth reached its peak after 60h of cultivation. SDS-PAGE and Western blot analysis showed that rhLysG2 was successfully secreted into the fermentation broth. There were a 23.8% increase in the total lytic activity and a 48h reduction in the cultivation time in comparison with those of the P. pastoris strain integrated with the pPIC9K-hLysG2 plasmid. Using chitin affinity and size-exclusion chromatography, rhLysG2 was purified with a yield of 187.4mg/L of fermentation supernatant, above 99.9% purity and a specific activity of 13 500U/mg under the condition of pH 5.6, 0.1mol/L of Na +, 30℃. In conclusion, rhLysG2 was expressed at high level in P. pastoris by codon optimization and had in vitro bactericidal activity against some pathogenic bacteria, which has laid a solid foundation for its possible future pharmaceutical applications.
Telomeres are specialized structures at the ends of the chromosomes, which have protective effects on the ends of chromosomes, and are associated with aging and many diseases. Long non-coding RNAs are RNAs that are greater than 200bp in length and generally do not produce a protein product. TERRA (telomeric repeat-containing RNA) is a type of long non-coding RNA that transcribed from telomere repeats. Emerging studies have shown that TERRA has functions of regulating telomere length, promoting the formation of heterochromosomes, also protecting the ends of chromosomes, and the expression levels of TERRA are related to diseases and aging. Since TERRA plays an important role in telomeres, the studies of TERRA has become a spotlight in researches related to telomere. At present, the transcriptional regulation and biological functions of TERRA have been well studied. Reviewing the biological characteristics and functions of TERRA and its relationship with disease and aging provides reference for the subsequent study of TERRA, which may be act as a target for the treatment of disease treatment and aging.
Purification of recombinant proteins has always been a bottleneck limiting its production. The traditional purification process is mostly batch based mode, which is characterized by high cost, low treatment efficiency and low recovery. Recently, with the development and application of continuous centrifugation, filtration, chromatography, and technologies, they have effectively overcome the defects of traditional processes. Based on downstream purification processes, the characteristics and applications of continuous technologies involved in large-scale industrial purification are summarized and analyzed. The core technology in the purification process - chromatography,and the prospects for the future technology of recombinant proteins purification are prospected, which is aimed at providing new theoretical evidences and guidance for optimization of biopharmaceutical production process.
Monoclonal antibodies have shown great value in the field of biology and medical research. They are new reagents in immunoassays and are the guided weapons for biological therapy. As a diagnostic reagent in vitro, monoclonal antibody can give full play to its advantages, such as good specificity, high sensitivity, and more convenient for quality control, which is conducive to standardization .Traditionally,mouse ascites were used to prepare monoclonal antibodies. Now monoclonal antibodies are also being developed in large-scale cultures of hybridoma cells in vitro. In particular, the needs of monoclonal antibodies in the diagnosis and treatment of diseases have further promoted the development of invitro culture and production techniques for hybridoma cells. Due to the semi-adherent nature of the hybridoma cells, both in suspension and adherent culture, large-scale invitro culture of hybridoma cells can be performed. This article reviews the invitro culture techniques of hybridoma cells, including hollow fiber cell culture system and bioreactor cell culture sysytem, as well as the optimization of different culture methods.
Objective: To analyze the development status and trend of immune system cell therapies in the sense of product manufacturing.Methods: Based on the Cortellis database of clarivate analytics,this paper analyzed the searching results with utilizing both quantitative and comparative analysis methods. Results: Currently,two immune system cell therapy drugs have been launched into markets,another one drug is at Pre-registration phase and four are at phase Ⅲ clinical trial phase. In addition, business deals related to immune system cell therapies are increasing in recent years. This paper analyzed the Top 10 valued deals related to immune system cell therapy drugs,indicating that the main agreement type was the drug development,commercial license.Conclusion: Although immune system cell therapy market still at its preliminary stage,with continuous development and improvement of future technologies,more drugs will be launched in the future,providing new options for the treatment of cancer and other diseases.
