Objective:To evaluate immunological activity of chimeric protein carrying one PV2 antigenic epitope on rotavirus VP6. Methods:one chimeric protein was constructed by inserting an epitope derived from neutralizing antigenic sites 1 of PV2 in rotavirus VP6 as a vector through gene cloning and recombination; the chimeric protein was expressed in E.coli cells and detected by Western blot,immunofluorescence assay and neutralization test. Results:The chimeric protein was favorably constructed and expressed in E.coli cells efficiently. Antibodies induced in inoculated guinea pigs by the chimeric protein could both neutralize the infection of RV and PV2 in vitro. Conclusion:The constructed chimeric protein possesses dual immunogenicity and can elicit production of antibodies in immunized guinea pigs which can protect infection of RV in MA104 cells and PV in Vero cells. The results are valuable for development of new RV/PV2 bivalent vaccine.
An expression vector pET-28a-VEGFⅠ-M2-GRP plasmid containing the VEGF and GRP antigen epitopes was designed and constructed. After being expressed in recombined strains induced by lactose, the fusion protein was lysised by ultrasonic. The resulting inclusion body was washed,dissolved, and dialysised for renaturation and purified by ion-exchange chromatograph to get the target protein.VEGFⅡ/GRP was identified by Western blot. The C57BL/6J mice subcutaneous tumor model of prostate cancer cell line RM-1was established to receive treatments with VEGFⅡ/GRP vaccine.The tumor growth,the blood vessel densities and the concentration of anti-VEGF antibody were examined to assess the effect of anti-angiogenesis and anti-tumor. ELISA showed that the concentration of anti-VEGF antibody in the mice serum of VG group was significantly higher than that of NS group (P<0.05),the anti-angiogenesis effect of VG group was superior to that of NS group (P<0.05).Thus,the constructed fusion protein can inhibit angiogenesis.
Objective:Although BMP9 has a prominent effect on promoting osteogenic/odontogenic differentiation of stem cells from the apical papilla (iSCAP) in the previous experiment, the mechanism is not clear. Smad signaling pathway is one of the most canonical pathway in BMP signaling pathways. Thus,the aims are to investigate the effects of Smad signaling pathway on BMP9-induced osteogenic/odontogenic differentiation of iSCAP. Methods:First of all, BMP9 was introduced into the iSCAP by using recombinant adenoviruses method and phosphorylated forms of Smad1/5/8 protein was assayed by Western blot after 36 hours. Subsequently, taking the dominant negative mutant receptor ALK1 (dnALK1) recombinant adenoviruses and BMP9 conditioned medium together into the iSCAP. Then, activity of Smad1/5/8 was detected by Western blot. Chemoluminescence quantitative and staining assay were applied to measure the activity of alkaline phosphatase (ALP), Alizarin red S staining was used to examine calcium deposition. At last, expression of transcription factor RUNX2 and osteogenic/odontogenic target genes (such as OCN、OPN、DMP1) were measured by RT-PCR. Results:It was found that BMP9 stimulated the activation of Smad1/5/8 in iSCAP,and this phenomenon was decreased by dnALK1 in iSCAP. BMP9-induced osteogenic/odontogenic differentiation(early marker ALP,expression of OCN and OPN, late osteogenic/odontogenic marker calcium deposition) were significantly inhibited by dnALK1,which is a competitive inhibitor of BMP9. Furthermore, expression of transcription factor Runx2 and osteogenic/odontogenic target genes were reduced by dnALK1.Conclusion:Taken together, these results reveal that Smad1/5/8 was activated in BMP9-induced osteogenic differentiation of iSCAP.And inhibition of Smad1/5/8 activity results in reduction of BMP9-induced osteogenic/odontogenic differentiation of iSCAP, which implies that BMP9 can induce osteogenic/odontogenic differentiation of iSCAP through Smad signaling pathway. AlK1 is an important receptor of BMP9-Smad signaling pathway, dnALK1 can inhibit BMP9 effectively, however, whether can it inhibit the other signal pathways are remained to be studied. In addition, animal experiments should be performed to verify the inhibition of dnALK1 in BMP9-induced osteogenic/odontogenic differentiation of iSCAP.
LfcinB is a small piece of peptide derived from bovine lactoferrin. It was reported that antibacterial activity of LfcinB stands its leading place among all kinds of lactoferricin. Preliminary research showed that, activated LfcinB can be expressed through E.coli expression system and Pichia pastoris expression system, but with limited yield and difficulty in purification. So building a new LfcinB expression system is significative. Baciullus thuringiensis can form ICPs consisting of plenty of δ-endotoxins during spore formation. It was subjected to lysis after fermentation,releasing spore and crystal into culture medium. Based on the above advantages of Bt, a fusion protein of LfcinB and Cry60Ba about 35kDa was constructed. The fusion gene was constructed by fusing the gene encoding LfcinB to the 3' terminus of the intact cry60Ba gene. The recombinant vector containing the fusion gene was transformed into acrystalliferous Baciullus thuringiensis strain 4Q7 recombinant strain 4Q7/pPFT60Ba-LfcinB was obtained. After cultivated in GYS medium for 48 h, the recombinant strain produced parasporal crystal, which was collected and subjected to SDS-PAGE analysis. The result showed that the expected protein band about 38 kDa was obtained. The purified fusion protein was hydrolyzed with HCl and subjected to SDS-PAGE analysis. The result showed that 3kDa target protein was obtained, which was then excised and was subjected to LC-MS/MS analysis. The result showeds that Cry60Ba and LfcinB from fusion protein was separated after hydrolysis and single LfcinB was obtained.This indicated that expressing fusion protein of crystal protein and LfcinB in Baciullus thuringiensis may be a efficient expression system of LfcinB, which laid the foundation for massively producting active LfcinB through genetic engineering methods.
Terpenes are important secondary metabolites that play a vital role in plant growth and development, some of which can be induced in response to biotic and abiotic stresses. The Tianta 5(F1) and its parent lines were treated with 200 mmol/L NaCl when they were at three-leaf stage. The qRT-PCR results showed the expression level of terpene synthase 2(TPS2), terpene synthase 3(TPS3) and geranylgeranyl diphosphate synthase 4(GGPS4) genes were up-regulated then down-regulated of all lines under salt stress, and the expression level of F1 was much higher than its parent lines.The expression level of three genes was positively correlated with the salt resistance of F2.The content and composition of carotenoids, biomass, photosynthetic indicators, content of chlorophyll and free proline were detected. The results showed that the salt tolerance of F1 was significantly higher than the parent lines. In conclusion, the high expression of TPS2, TPS3 and GGPS4 has a certain correlation with the resistance of salt and other abiotic stress of F1.
The aims are to find the effect of GSH on the two bacteria Ketogulonicigenium vulgare and Gluconobacter oxydans in vitamin C one-step fermentation process.It was found that addition of 1mg/ml glutathione to the 5L fermentation by K. vulgare-G. oxydans consortium significantly enhanced the production of 2-KGA by 22.8%. According to the 16S rDNA realtime fluorescence quantitative PCR analysis, the final biomass of K. vulgare increased to 148% and G. oxydans decreased to 61% relative to the control strain. Using the metabolomics methods, it is found that glutathione could promote pentose phosphate pathway, citric acid cycle, the sulfate and other metabolic pathways of K. vulgare, and glutathione can at the same time slow down the consumption of L-sorbose by G. oxydans to improve fermentation efficiency of the consortium.
The rational design method was applied to increase the thermostability of bovine enterokinase. Molecular dynamics software Gromacs v4.5.5, FlexService and B-FITTER were used to predict the flexibility profile of the bovine enterokinase structures. Fragment 64~69 and Fragment 85~90 were confirmed to represent the flexible region. Subsequently, β-turn sequence statistics and stereoscopic criteria of introducing proline were combined to pinpoint appropriate substitution sites by prolines. Finally, site-directed mutations (S67P, R87P and Y136P) were introduced within the fexible region using Quik ChangeTM method and the thermostability of wild-type and the enterokinase mutants were investigated. The result demonstrated that the half-life (t1/2) and half inactivation (T5010) temperature of the R87P mutant increased by 3.1 min and 11.8℃ when compared to that of the wild-type enzyme. Meanwhile, the catalytic efficiency (Km/kcat) of the R87P mutant enzyme remained nearly unchanged. This strategy has potential to be applied to engineer thermostability of other enzyme, which is beneficial for the wider application of biocatalysts in industry.
DNA sequence modification is a basic technology in genetic engineering. One aspect of DNA modification is insertion or substitution with large heterogeneous fragments. Sometimes fragments need to be inserted or substituted to a sequence with a scanning model, to obtain an optimal sequence meeting specific purpose. Traditional cloning method using restriction digestion and ligation reactions might become laborious and time-consuming, or even unpractical in these cases. A strategy combining Golden gate method, mixing cloning, and rapid identification of clone (named Gmix) was reported. By using Gmix, the Gaussia gene was inserted or substituted to specific location in a HBV replication plasmid with 4 different scanning models. With high successful rate, easy handling and low cost, Gmix is suitable for many other DNA modification works resembling that described here.
For heterologous synthesize natural carotenoid zeaxanthin in microorganism,a β-carotene producing Saccharomyces cerevisiae was chozen as the host cell to construct engineered yeast with synthetic biology method. The key enzymes β-carotene hydroxylase encoding gene CrtZ from nine sources were integrated in the chromosomal, separately. As the result, the strain carrying CrtZ from Erwinia uredovora achieved the highest titer of zeaxanthin. Moreover, the conversion from farnesyl pyrophosphate (an important precursor for terpenoid natural products) to farnesol was reduced by knocking out gene Lpp1 and Dpp1, providing more precursors for zeaxanthin synthesis. The zeaxanthin yield increased 1.27 fold (from 29 mg/L to 36.8 mg/L) accordingly. Furthermore, a titer of zeaxanthin was achieved as 96.2 mg/L in shake-flask through increasing the CrtZ gene copy number and regulating its promoter's activity,which is the highest reported microbial titer known.
Lunasin is a 43 amino acid polypeptide originally isolated from soybean with bioactive properties such as antihypertensive, antioxidant activity, cancer prevention and therapy, anti-inflammation, hypocholesterolemic activity, anti obesity and immunomodulation. In order to achieve efficient production of Lunasin, the methanol fed methods (DO feedback fedding and index-constant speed feeding) and induction temperature of recombinant Pichia pastoris GS115 LN were investigated. Furthermore, the feeding strategies of mixed carbon sources during induction phase were investigated. The results show that the best feeding strategies and induced temperature were index-constant speed feeding, 24℃. Consequently,the highest expression of Lunasin was 3.27g/L by feeding 1% soy peptone and 0.02% aspartic acid,which was 1.27 times higher than the single methanol induction process.
In the feeding process, the decomposition of the digestive tract proteases is one of the important reasons affecting the efficiency of feed enzymes. Therefore, the feed enzymes having protease resistance is a very important property. The rational design of protein modification with resistance to protease based on the method of the biocatalysis and computational chemistry was used, which has been established to improve Bacillus subtilis 168 1,4-β-endoxylanase with resistance to protease and to get the excellent properties. Single site mutation to R121 and K39 and double sites mutations to R121/K98 were utilized to get the fine properties of mutant enzymes XynAR121C, XynAR121C/K98Q and XynAK39I. Fortunately, the excellent properties were gotten finally. The optimal temperature of XynA, XynAR121C, XynAR121C/K98Q was 60℃,while XynAK39I was 40℃,and its temperature tolerance was lower than the wild type. The optimal pH of the wild and mutant enzymes was 6.0. During the treatment with simulated intestinal fluid (pH 6.8, 10mg/ml trypsin solution), the remained enzyme activity of mutants was much higher than the wild type. As for XynAR121C, its half-life period was 193 min, 1.52 times higher than XynA. As for XynAR121C/K98Q, its half-life period was 257 min, 2.02 times higher than XynA. As for XynAK39I, after incubation with simulated intestinal fluid at 40℃ for different time, its half-life period was 90 min, only 37min shorter than XynA.
Natural antimicrobial peptides (AMPs) are small cationic peptides with potent and broad-spectrum antimicrobial activities which have received great attention as a promising antibiotic candidates to overcome the global epidemic of antibiotics-resistant infections. Natural AMPs has provided a wealth of information on the structure-activity relationship accounting for antimicrobial activity to design novel synthetic AMPs with improved protease-resistance, reduced cost of production and less hemolysis and toxicity, which greatly promotes the potential of synthetic peptides as anti-infectious agent. Firstly the general strategies and technologies employed in the design and optimization of synthetic peptides, i.e., chemical modification, protein engineering, in silico design and screening, and minimalist de novo design were summarized. Finally, the synthetic AMPs in clinical trail with outstanding therapeutic potentials and future perspectives of improved AMPs for therapeutic applications were highlighted.
Bone diseases is one of the common clinical chronic disease in the elderly, and serious damage to health. Previous studies show that FGF family members can treat bone-related diseases, as osteoporosis, osteoarthritis and other syndrome caused by these two disease. However, it's still not completely clear for the mechanism. In addition, different FGF species, ages have different therapeutic effects on bone. Therefore, the different FGF for different bone-related diseases was summarized.
Flavonoid glycosides are a large group of plant-based phenolic nature products. The glycosylation of flavonoids increases their solubility and stability relative to flavonoid aglycones, mainly catalyzed by glycosyltransferases (Gts).Uridine diphosphate glycosyltransferases play a key role in decorating flavonoids with different sugars, resulting in numerous structurally diverse flavonoid glycosides. With the rapid development of synthetic biology and metabolic engineering, engineered yeast, Escherichia coli and other microorganisms have been utilized to construct the biosynthesis pathways of sugar donors and flavonoid aglycones. The recent progress on glycosylated flavonoids in engineered microorganisms, as well as the clustering of glycosyltransferase, biosynthetic pathway of sugar donors were summarized, and its future trend was explored.
Mixed culture of photosynthetic bacteria and other microorganisms under illumination has been extensively studied in recent years. The characteristic and applications (wastewater treatment, new energy production and materials production with high quality) of photosynthetic microbial mixed culture were analyzed, as well as factors on growth, metabolism and reproduction of mixed culture. The results showed that cooperation among microorganisms could promote the growth and reproduction of microorganisms, enhance the substrate utilization, and improve the production rate. Photosynthetic microbial mixed culture had the advantages of simple process, low cost, and high efficiency in wastewater treatment, new energy production and materials production with high quality. Among the factors that influence photosynthetic microbial mix culture, the most important ones were microbial dosage, proportion of mixed microorganisms, and pH of the culture. Based on the shortcomings of photosynthetic microbial mixed culture, some suggestions on the development were proposed.
Taking the database of national intellectual property as the statistic source, the statistics of the patent document about biomedical technological study were analysed, that offering the general development trend of biomedical patent in Jiangsu, its regional distribution, IPC technical composition and prolific institutions of patent. Meanwhile, based on the patent portfolio in the technical field, comparative analysis has been made with representative regions and provinces in China like Beijing, Shandong and Shanghai as well as the USA. Finally, the focus is on the application and regional distribution of biomedical enterprises in Jiangsu, that provides Jiangsu and other provinces with patent references for working out more appropriate strategies in terms of technological development.
