With the help of analysis of mass spectrometry and data analysis according to the preliminary laboratory basic research, Co-IP and GST pull-down methods was used to prove the interaction of HBV X protein with Tab1. In order to study the carcinogenic mechanism of HBx protein, Some experimental evidence for further study of the role of HBx in the carcinogenic mechanism of HBV chronic infection were provided. pGEX-2TK-GST-HBx plasmid was successfully constructed, GST-HBx fusion protein was induced and incubated with GST-beads. pcDNA3.1/myc-His (-) B-Tab1 plasmid was constructed,then was transfected 293T cells to express Myc-Tab1. Finally the interaction of GST-HBx and Myc-Tab1 was verified in GST pull-down test. Moreover, the eukaryotic expression plasmids including pcDNA3.1/myc-His (-) B-Tab1 and pcDNA3.1-3×flag-HBx were successfully constructed, then were co-transfected into human embryonic kideny 293T cells and HepG2 cells to express fusion protein, further Co-IP test was demonstrated that the anti-Myc antibody could precipitate HBx from the cell lysate, and confirmed their intracellular interaction in two human cell lines. In conclusion, all results show that HBx and Tab1 could interact in vitro and in vivo, also established foundation to reveal the function and mechanism of HBV X protein.
Objectives: To determine the role of SFRP5 in bone morphogenetic proteins 9 (BMP9)—mediated osteogenesis in human umbilical cord-derived mesenchymal stem cells(hUC-MSCs). Methods: The hUC-MSCs are divided into control group,BMP9 group,BMP9+SFRP5 group and SFRP5 group. Alkaline phosphatase (ALP) activity was carried out on 3, 5 and 7 days respectively; while ALP staining on 7 days. Aizarin red staining and oil red O staining were detected on 21 days. Different groups of cells were collected for subcutaneous injection in nude mice. After 4 weeks, the ectopic bone formation was analyzed by micro-CT scanning. The specimens were stained with H.E. staining, Masson staining, Alcian blue staining and oil red O staining. Osteogenesis differentiation related proteins expression of Runx2 and OPN were detected by Western blot. Results: Compared with control, the ALP activity and alizarin red staining were higher than BMP9 group; and BMP9+SFRP5 group was lower than that of BMP9 group. A small quantity of lipid droplets was detected in BMP9 group; and lipid droplets increased significantly in BMP9+SFRP5 group and SFRP5 group. Ectopic bone formation subcutaneously in nude mice was observed in BMP9 group and BMP9+SFRP5 group. The bone density of BMP9+SFRP5 group was lower than BMP9 group(P<0.05). The osteogenetic differentiation in BMP9 group was greater than that in BMP9+SFRP5 group by H.E. staining, Masson staining and Alcian blue staining, and more adipogenesis in BMP9+SFRP5 group by oil red O staining. SFRP5 inhibited the protein expression of Runx2 and OPN induced by BMP9. Conclusion: SFRP5 can inhibit the osteogenic differentiation of human umbilical cord mesenchymal stem cells induced by BMP9.
Objetive: The object is to produce recombinant human proliferating cell nuclear antigen (PCNA) protein using insect cell expression system and purify and identify its antibody binding characterization. Methods: Human PCNA gene was amplified from HeLa cells and cloned into the baculovirus vector AcMNPV. Using insect cells, the recombinant baculovirus containing the PCNA gene was obtained. The virus infected the cells to express the protein, which was reached high purity by combining the nickel column affinity chromatography and the ion exchange chromatography. ELISA method was set to identify its binding activity. Results: Full length recombinant human PCNA (rPCNA) was produced in a baculovirus expression system. The optimal multiplicity of infection (MOI) value and infected time were 0.05h and 144h respectively. The produced protein samples were subsequently purified by a two-step procedure, including Ni-NTA affinity chromatography and ion exchange chromatography. The yield of rPCNA was up to 110mg/L cell culture, with a purity > 95% by SDS-PAGE. Indirect ELISA results showed that antibody binding activity of rPCNA was much higher than that of PCNA expressed by E. coli and rPCNA took a sensitivity and specificity of 93.3% and 85.0%, respectively. Conclusions: An expression and purification procedure for rPCNA and the produced rPCNA presented high antibody binding characterization were established which would have great potential applications on diagnosis of PCNA-associated diseases in vitro.
The investigation on the genomic study of Haematococcus pluvialis would be significant to explore the origin and evolution of green algae and the stress responses in Haematococcus pluvialis;and promote the development of Haematococcus pluvialis industry. The low-coverage draft genome of Haematococcus pluvialis was constructed by the Illumina Hiseq 2500 platform. The predicted genome size was approximately 547Mb, with the GC content of 59.2% by calculating k-mer distribution. The draft genome contained 11 059 predicted protein-coding genes and the average gene size and CDS were 1 711bp and 681bp; every gene contained 3.2 exons and the size of exon was 353bp in average. The analysis of metabolic pathway indicated that the low-coverage genome contained whole glycolysis, tricarboxylic acid cycle, phosphopentose, purine and pyrimidine synthesis and other basic metabolism pathway.
Objective: The metabolic pathway engineering of Corynebacterium crenatum SYPA5-5/△proB/△argF (SYPO-1) has been performed to further enhance the pathway flux of L-ornithine biosynthesis. Firstly, the four genes encoding N-acetyl-L-ornithine deacetylase (NAOD) from different bacterial sources were screened, cloned and expressed in Escherichia coli BL21 (DE3). Then the recombinant NAODs were purified and characterized. The argE gene from Serratia marcescens Y213 was overexpressed in the L-ornithine producing strain C. crenatum SYPO-1 to increase the L-ornithine production. Methods: The genes from different sources were sub-cloned into the pDXW10 plasmid and expressed under the tacM promoter in E. coli BL21(DE3). Then the recombinant N-acetyl-L-ornithine deacetylation were purified and their characterization were studied. The optimal N-acetyl-L-ornithine deacetylase was expressed in recombinant C. crenatum. The parameters of the recombinant strains during fermentation were also investigated. Results: The recombinant argE coding NAOD enzyme from S. marcescens showed a very higher activity than the other NAOD enzymes from E. coli BL21(DE3), K. pneumoniae and B. subtilis, the activity was 798.98U / mg, the optimum pH and temperature were 7℃ and 37℃ respectively. SmNAOD was expressed in C. crenatum, and the activity was 128.4U/ml, which was significantly increasing intracellular acetyl cycle levels. At the end of fermentation, L-ornithine yield increased to 38.5g/L with the overall productivity of 0.401g/(L·h) in the recombinant SYPO-2, which was approximately 21.3% and 33.2% higher than that of SYPO-1 and SYPO-3, respectively. Conclusion: The N-acetyl-L-ornithine deacetylase from S. marcescens Y213 has been screened and overexpressed in the L-ornithine producing strain C. crenatum SYPO-1, which could promote the rapid consumption of L-ornithine precursors and achieve L-ornithine accumulation. A huge potential of C. crenatum to overproduce not only L-ornithine but also L-citrulline, L-arginine from renewable resources such as glucose were demonstrated.
Cell-penetrating peptides (CPPs) have been widely used in decades for its ability to carry many macromolecular drugs across cell-membrane to exert their effects. Midkine (MK) is a heparin-binding growth factor with a heparin-binding domain (HBD). The HBD in MK that is rich in basic amino acids (MK-S0) was fused with enhanced green fluorescence protein (EGFP) and then it was found that MK-S0 could deliver EGFP into cells, and its transportation capacity is much higher that classical CPPs (such as Tat). After the sequence optimization on MK-S0, MK-Δ4 whose trans-membrane ability was increased about 16-fold than MK-S0 was obtained. The trans-membrane ability of MK-Δ4 was also suitable for a variety of tumor cells. The further investigation of endocytic pathways on MK-Δ4 was shown that MK-Δ4 penetrates cell-membrane through interacting with heparin sulfate on the cell surface and then via macropinocytosis. The results of cell growth inhibition by MTT method showed that MK-Δ4 could enhance the inhibitory effect of a ribosome-inactivating protein-MAP30 about 5.8-fold in HeLa cells which is significantly enhance the anti-tumor activity of MAP30. It was suggested that MK-Δ4 optimized from heparin-binding domain MK is a novel human-derived CPP with high efficiency, and is a new drug vector for anti-tumor therapy.
Objective: Optimization of previous developed self-deleting lentiviral vector carrying human β-globin gene and promoter in terms of preparation titles and transgene expressing efficacy. Methods: Based on the comparison of the predictions of three on line promoter prediction softwares; three self-deleting lentiviral vectors carrying three different lengths of β-globin promoters with gene were constructed. The optimization was carried out in terms of preparation titles and transgene expressing efficacy. The optimized LVV was used to transduce murine -thalassemia iPSC;and resulted cells were used to generate chimeric mice. RT-PCR and Wright Giemsa staining were then carried out on the mice blood. Results: The viral particles prepared by the optimized LVV principally have no difference comparing with their parental virus, FUGW. The functional expression of normal spliced human β-globin gene was detected in the chimeric mice. And the morphologically normal of erythrocytes was observed. Conclusion: An optimized self-deletion lentiviral vector, FCB-P265, was made.
The GLP-1 mutant gene and Fc of IgG4 were fused into GLP-1-IgG4-Fc, and were inserted into a mammalian expression vector pXC17.4. CHO-K1 cell was stable transfected with linearlized plasmid.High expressing clone with yield of 1.5g/L was got through ClonePix 2. The target GLP-1-IgG4-Fc was gained with SDS-PAGE purity more than 95% after purification by Protein A and Source 30Q. The HPLC and CZE purity of the target protein were more than 80%, and the purity was more than 99% by SEC analysis. The molecular weight was consistent with the theoretical value by MS and the peptide map was the same as that of the control by peptide mapping analysis. The biological activity analysis showed that GLP-1-IgG4-Fc stimulated cAMP production in HEK293 cell expressing GLP-1 receptors, and the biological activity was highly similar to the control.
The 5'regulation sequence of the chicken ovalbumin (ovalbumin, OV) gene is the preferred regulatory element for the development of poultry oviduct bioreactor. Using EGFP as reporter gene,the eukaryotic expression vector containing the OV promoter is constructed and effective promoter is screened by analyzing the expression of GFP in the primary cell cultures of chicken oviduct and CHO cell, OV promoter of 1.1kb is identified which is used for further experiments. A recombinant vector named pOV1.1k-HA to express HA protein of H5N1 subtype influenza virus is constructed. It is subsequently transfected into CHO cells, PCR and RT-PCR analysis of HA gene suggested that the vector could be delivered into CHO cells and then get them transcribed. The immunoreactivity and hemagglutination activity of HA protein are determined by Western blot and HA test. The 4-week-old SPF chickens are vaccinated with purified HA protein and boosts were conducted with the same dosage after two weeks. The HI antibody level is 6.3log2 of three weeks after the boost. All chickens are challenged with 106 EID50 of H5N1 virus ((A/Goose/Guangdong/1/96). The survival rate of all vaccinated chickens is 100% and that of control group is 0, also no detoxification in the vaccine group. It is indicated that complete protection is provided. Results show that the screened 1.1kb OV promoter could effectively drive the expression of HA protein and the expression of HA protein immunizing SPF chicken provided complete protection against avian influenza virus attack. The basis for the expression of protective antigens and precious drug proteins in the chicken oviduct bioreactor will be founded.
SpyCatcher can form an irreversible covalent linkage to its partner SpyTag via a spontaneous isopeptide bond. To explore the potential application of SpyTag/SpyCatcher system in the self-assembly of multiple enzyme complex in Escherechia coli, SpyCatcher and SpyTag were fused with P450BM3m monooxygenase and glucose dehydrogenase(GDH), respectively. The fusion proteins co-expressed in vivo were expected to self-assemble into a double-enzyme complex with the ability of coenzyme regeneration and efficient biosynthesis of indigo. At first, the self-assembled multienzyme complex formed via SpyTag/SpyCatcher system in vivo was confirmed by SDS-PAGE and Nano-Liquid Chromatography. After that, the ability to synthesize indigo by multienzyme complex was systematically analyzed under different culture conditions. When induced at 16℃ with 0.5mmol/L IPTG, the engineered bacteria showed robust ability to catalyze indole (2mmol/L) and glucose (4mmol/L). The yield of indigo produced by self-assembled complex cells was up to 258mg/L, which was 1.9 and 2.4 times higher than that of cells with free enzymes and P450BM3m single enzyme system accordingly. The conversion rate was 52% when reaction was balanced after 70min. These findings suggest that the SpyTag/SpyCatcher system can successfully assemble multienzyme complex for efficient biosynthesis of chemicals, which provide a new idea for the design of intracellular multienzyme complex assembly.
The DPEase gene from Clostridium cellulolyticum H10 was studied on the enzyme production in the food grade expression system Bacillus subtilis. The final enzyme activity was 495U/ml by high-cell-density fermentation in the 3L fermentor. The recombinant cells were immobilized by diatomite-sodium alginate (adsorption-occlusion method); the optimized immobilized conditions were as follows: 2% sodium alginate, 50g/L cell concentration, 2% CaCl2 and 1% diatomite. Under the optimum condition, the recovery rate reached 64%. Compared with the free cells, the immobilized cells had the same optimal pH, the optimal temperature was increased by 5℃;and the thermal stability was significantly improved. The conversion rate was still 28% after 7 times repeated operations; it was also maintained 81% of the residual enzyme activity, which had a high industrial application value.
In recent years, the emergence of drug-resistant bacteria poses a serious threat to the antibiotic therapy. sRNAs are new regulatory factors of gene expression, which regulate the physiological function of cell in response to various environmental changes by pairing target mRNA or protein. Studies have shown that sRNA can play an important role in the process of bacterial resistance, such as blocking the pathways of antibiotics into cells and releasing the drugs in the cells. The sRNAs regulates the expression of genes in related with bacterial resistance, which is helpful for elucidation of resistance mechanisms and discovery of new drug targets were reviewed.
The CRISPR/Cas9 system is not only a revolutionary tool for gene editing but also regulates gene transcription in various prokaryotic and eukaryotic organisms. In recent years,the system of CRISPR-dCas9 derived from CRISPR/Cas9 has been used in many fields such as gene imaging, high-throughput screening, gene regulation, investigating essential gene function and epigenetic regulation.The recent advances of CRISPR-dCas9 for activating or inhibiting gene transcription, reducing off-targeting efficiency and combing the intrinsic relationship between sgRNA and transcriptional regulation, application in the life science and further upgrading were described.
Microalgae have received growing interest as a potential biofuel feedstock, which has been regarded as a promising alternative source for next-generation renewable fuels. However, the commercial use of microalgae for sustainable biofuel faces some challenges due to low productivity and high cost. For this reason, two-stage cultivation and co-cultivation strategies were developed to improve the lipid yield. Besides changing the cultivation modes, more simple approach, addition of chemical additives or plant growth regulator are emerging as the potential lipid enhancing strategies. The principle and method of various novel technologies for improving microalgal lipid production were described and discussed.
