Objective:To study the role of recombinant human midkine (rhMK) in the repair of acute partial-thickness defects of knee articular cartilage. Methods:The animal model of acute partial-thickness defects in rats was constructed, then normal saline or rhMK (20μg/kg、60μg/kg、180μg/kg)was injected into knee cavity 24 hours after surgery.All rats were sacrificed on the 8th week after surgery, knee joints were taken to make paraffin sections, then sections were stained with Toluidine blue and observed with microscope to determine optimum dose of injection.Results:Different dose of rhMK could repair partial-thickness defects in different degree, the optimum dose of injection was 180μg/kg. For pharmacokinetic analysis, cartilage was harvested at 1 hour and 1, 3, 6, 9, 12 and 15 days after knee injection of 180μg/kg rhMK (n=4), the concentration of rhMK in cartilage tissue firstly increased and then decreased, the terminal T1/2 in cartilage tissue was 8.69 days.Conclusion: rhMK could repair partial-thickness defects of knee articular cartilage in rats obviously, the optimum dose was 180μg/kg and the optimum interval of time was 8 days.
Nucleophosmin (NPM1) mutations, described recently, play an important role in acute myeloid leukemia. To investigate the role of NPM1 mutations in K562 leukemic cell proliferation and invasion in vitro, the pEGFPC1-NPM1-mA plasmid vector with NPM1 mutation A (NPM1-mA) was transfected into K562 cells, and the K562-mA cells with stably expressed NPM1-mA protein were established. Cell growth curve was used to determine the proliferation potential in vitro. FCM was used to detect the cell cycle. Cell adhesion assay and Transwell were used to detect the adhesion, migration and invasion capability. The results showed that the proliferation potential of K562-mA cells was decreased. Compared with control groups, the percentage of cells in the G1 phase was increased remarkably and that in the S phase was decreased significantly. In addition, the migration ability of K562-mA cells was enhanced, but the adhesion and invasion capability of cells were attenuated.NPM1 mutations may inhibit K562 cells proliferation and invasion in vitro, which provide a better foundation for studying the molecule mechanisms of NPM1 mutations in leukemiagenesis.
Objective: Investigate the correlation between ANXA2 and Hepatocellular Carcinoma (HCC) proliferation, apoptosis and motility. Method: Four siRNAs recombinants targeting to ANXA2 were introduced into SMMC-7721 cells using an optimized calcium phosphate transfection method. ANXA2 mRNA and protein levels were evaluated by using semi-quantitative RT-PCR and Western blotting. The cell proliferation, motility, and apoptosis were determined with MTT, wound healing assay, and Hoechast33258 staining methods. Results: Four siRNAs suppressed ANXA2 expression effectively with time dependence (p<0.05). Cell proliferation and motility were inhibited after ANXA2 knockdown (p<0.05), and the inhibitory efficiency was positively correlated with ANXA2 knockdown level. Conclusion: It is concluded that the over-expression of ANXA2 in HCC is closely associated with the cell malignant behaviors, and siRNA can inhibit the ANXA2 expression efficiently in SMMC-7721 cells.
Ribonuclease inhibitor (RI) is a wide spread cytoplasmic protein regulating RNase activity in vivo, and extensively used in a variety of molecular biology applications where RNase contamination is a potential problem. Commercial mouse RI (mRI) was suggested to be more resistant to oxidative denaturation. In order to obtain the soluble active mRI product, recombinant expression vectors for mRI were constructed. Recombinant plasmid of pQE80L was more efficiently transformed into BL21 host cells than vector of pQE30, suggesting a toxic effect on the strains caused by basal expression of mRI. Upon induction with IPTG, mRI protein was expressed in both supernatants and inclusion bodies in different strains. BL21 strain yielded a similar amount of soluble products with higher RNase inhibiting activity than Top10 and TG1 strains. The yield of mRI fused to His-tag was about 4~8 mg/L. Expression of mRI fused to TrxA was also induced, which showed a partial inhibitory activity to RNase and restored to full activity after cleavage of TrxA from the protein. When mRI was treated with H2O2, the same oxidative stability was observed as to human RI (hRI), contradicting to the prediction that mRI should be more resistant to oxidative denaturation based on amino acid sequence analysis.
The plant 2-Cys peroxiredoxin BAS1 is a thiol-dependent peroxidase capable of reducing hydrogen peroxide through the reactive catalytic cysteine, which is regenerated by NADPH dependent thioredoxin reductase from chloroplast (NTRC). Maize contains two BAS1, 2-Cys peroxiredoxin A (2-Cys PrxA) and 2-Cys peroxiredoxin B (2-Cys PrxB).The BAS1 gene encoding the mature 2-Cys PrxA from maize leaves was amplified by RT-PCR, and Cys34 was mutated into Ser34 in the BAS1 by site-directed mutagenesis. The SDS-PAGE analysis displayed that the subunit of the purified BAS1 wild type and mutant C34S has the apparent molecular mass about 23kDa. The protein binding assay showed the purified BAS1 mutant C34S is associated with the purified NTRC through the intermolecular disulfide bridge. Non-reducing SDS-PAGE analysis proved the purified BAS1 wild type formed the dimer through the intermolecular disulfide bridge, whereas the purified BAS1 mutant C34S formed the monomer. The results of modification BAS1 with thiol-reactive labeling reagent AMS and the activity analysis identified that the reducing BAS1 is essential for the catalysis of the reduction of hydrogen peroxide, which is kept by NTRC and its cofactor NADPH.
A full length cDNA named MaBTB (BR-C, ttk and bab or pox virus and zinc finger), which contained 1 080bp with an open reading frame, was isolated by SSH combined with RACE technology. Sequence analysis revealed that this gene shared high identities of 86%、85%、84%、84%、86% and 87% to those of BTB/POZ from Oryza sativa(OsBTB), Medicago truncatula(MtBTB/POZ), Arabidopsis thaliana(AtBPM4), Vitis vinifera(VvBTB), Zea mays (ZmBTB)and Picea sitchensis(PsBTB)), respectively. Phylogenetic analysis showed that MaBTB was closely related to Medicago truncatula (MtBTB) and Arabidopsis thaliana (AtBPM4).The expression feature of MaBTB was investigated by RT-PCR. The result showed that under salt, ethylene and ultraviolet radiation treatment, the expression of MaBTB gradually increased. The accumulation of MaBTB decreased and then gradually increased with cold and FOC race 4 treatment. However, the accumulation of MaBTB was not distinct variety by wounding treatment.
Aspergillus niger produces three classes of cellulases for cellulose degradation and among these enzymes the expression level of cellobiohydrolases is very low, affecting the cellulose digestion efficiency by the cellulase system of Aspergillus niger.In order to construct an engineered strain with high cellulase enzyme activity, a hybrid gene containing strong promoter fragment of glucoamylase gene glaA and coding region of cellobiohydrolase gene cbhB was constructed via in vitro synthesis. The hybrid gene was subsequently cloned into the binary vector pCAMBIA1301 and the recombinant plasmid was introduced into conidiospores of Aspergillus niger by Agrobacterium tumefaciens-mediated genetic transformation. The transformants were hygromycin resistant with the hybrid gene inserted into chromosome DNA of Aspergillus niger via T-DNA illegitimate recombination. In total, 48 transformants were seleted and their cellulase activities were tested. The results showed that transformant A3-9 had the highest CMC activity which was 1.31-fold as high as the wild type strain of Aspergillus niger, while both transformants B1-7 and A3-6 had the highest FPA activity which was 2.51-fold as high as wild type strain of Aspergillus niger. Furthermore, induction conditions for the hybrid gene expression were also analyzed.
Based on combination of bioinformatics, The similarity between the cloning gene and corresponding gene of Acetobacter suboxydans reach to 80%. Then, recombinant plasmid was constructed by inserting arDH genes into vector pET22b and functionally expressed into E. coli JM109(DE3). The molecular mass of recombinant D-arabitol dehydrogenase was about 30 kDa, so it belonged to the short-chain dehydrogenase. The recombinant enzyme was purified by His Trap and subjected to enzymological characterization. The ArDH could not only oxidize D-arabitol to D-xylulose, but also reduce D-xylulose to D-arabitol. When catalyzing oxidative reaction, the Km value for D-arabitol was 60.67 mmol/L, Vmax was 0.803 U/mg; it could use NAD+ and NADP+ as cozyme, but it preferred to depending on NAD+; the optimum pH and temperature of oxidative reactions was 12.0 and 30℃. However, when catalyzing reductive reactions, the Km value for D-xylulose was 36.93 mmol/L, Vmax was 1.71 U/mg; the optimum pH and temperature of reductive reactions was 7.0 and 30℃.
In order to determine the optimal culture conditions for hydrogen production of Chlamydomonas reinhardtii strain 849 and its transgenic alga lba, the orthogonal experiments were designed to test the effects of such three factors as light intensity, cell density and the sulfur content in the culture medium at three levels, meanwhile O2- evolution activity and pH value were detected. The results showed that at 25 ℃, the optimal culture conditions for hydrogen production for both strain 849 and the transgenic alga lba were under 60μmol/(m2·μs) of light intensity, 12.5μg/ml of the chlorophyll content as cell density and 0μmol/L of sulfate content in the medium. Under such condition, the maximal hydrogen yield of strain 849 and the transgenic algae lba reached 349μl/mg chl and 634μl/mg chl respectively. The net O2- evolution activity of transgenic alga lba was lower than 849.The results provided basic data for the utilization of leghemoglobin in the improvement of hydrogen production of C. reinhardtii with bioengineering methods in the future.
Eight biogas slurry samples were collected from some rural household biogas digesters in Sichuan, Zhejiang and Shanxi Provinces or from biogas project in Beijing. The result of biogas fermentation experiment in 500ml reaction system under different temperatures (21℃ 、18℃ and 15℃ respectively) shown that five of the samples Z-2、 Z-3、Z-6、Z-8 and Z-10 could produce biogas under different temperatures. Three of the biogas slurry samples Z-3、Z-6 and Z-10 which produce higher yield of biogas were chose for further biogas fermentation experiment in 2 500 ml reaction system under 15℃ lower temperature, a higher yield biogas fermentation system Z6 with the biogas yield of 0.15m3/m3day was selected from the three samples according to the results of two times continuous biogas fermentation experiments. The metagenomic DNA of microbial community from the Z-6 biogas fermentation system was extracted by direct extraction method, purified through Wizard DNA Clean-Up System, partial digested by Sau3AⅠand then ligated with plasmid vector of pBluescript SK (+). A metagenomic library containing 10 000 transformations was constructed, stochastic analysis of the library indicated that the average size of the foreign DNA fragments was 5 kb and the capacity of foreign DNA in the library was 48 Mb. The diversity of the restriction types of the foreign DNA fragments was higher. The results laid a foundation for utilization of gene resources of the key hydrolytic enzymes in biogas fermentation.
The experiment took chicken manure and pig manure to conduct composting experiment and researched on changes and interrelationships of enzyme activity and microbe quantity. The results showed that: the catalase activity and cellulose activity kept at a higher level in the early stage of composting, and then decreased quickly. Finally, the catalase activity between 9ml/g and 12ml/g, and the cellulose activity between 12.37mg/(kg·h) and 15.07mg/(kg·h). The trend of urease activity was "highed-lowed-highed". The trend of bacteria quantity was "low-high-low", actinomycetes quantity was "high-low" and fungi was "high-low-high". Through correlation analysis, the actinomycetes may was important factor that affected the change of catalase activity and cellulose activity. The actinomycetes had a significantly positive correlation with catalase in chicken manure, and had a positive correlation with catalase and cellulose in pig manure, and had a significantly positive correlation with catalase and cellulose in mixed chicken and pig manure.
Objectives: To express the outermembrane segment of mouse CTLA-4 in eukaryotic cells. Study the peptide's binding to B7 of antigen present cells could rescue the T cells activation after stimulated, and therefor promote the T cells proliferation. Methods: The total RNA were purified from mouse spleen cells. By RT-PCR the whole gene of CTLA-4 was amplified. Primers designed according to the pcDNA3.1 and CTLA-4 outer membrane sequences, and employed for PCR. Amplified sequence together with the plasmid were cut and reconstructed. The right plasmid was further transferred into Hepa1-6 cells, and the expression was examined by western-blot. Results: CTLA-4 outer membrane segment expressing Hepa 1-6 cells were achieved. The expressed peptide were positive to anti-His antibody and CTLA-4 antibodies on western blot. Conclusion: The recombinant peptide were obtained and will be applied for cell and animal expreiments.
Objective:To clone, express and characterize the C-terminal gene fragment of P1 protein. Methods:On the basis of successful clone and sequence analysis of the C-terminal gene fragment of P1,the gene fragment were ligated into pET32a(+).An expression vector pET32a(+)/ P1(3 520~4 563bp)were constructed and expressed in E.coli rosetta induced by IPTG. recombinant protein was purified through GSTrap 4B affinity chromatography column. ELISA and Western blotting were used to determine the antigenic of the recombinant protein. Immune mouse with recombination protein, monoclonal antibody was prepared. Results:The recombinant gene can be overexpressed in E.coli. SDS analysis revealed that the expressed protein was identical to C-terminal gene fragment of P1 protein in molecular weight with a purity of 95%. ELISA and Western blotting analysis showed that the recombinant protein can displayed high antigenic activity toward Mycoplasma pneumoniae convalescent antiserum. Two monoclonal antibody cell line were obtained. Conclusion:The C-terminal gene fragment of P1 protein of MP has been successful cloned and expressed, monoclonal antibody was prepared which could be useful for developing diagnose reagents or vaccine of MP.
Corynebacterium acetoacidophilum was used as the original strain for stepwise mutation by whole genome fragmentation mutation (UV and NaNO2) and subsequent sulfaguanidine resistance screening. A L-proline over-producing mutant was obtained. The optimum contents of glucose, biotin and thiamin in shake-flask fermentation were 16%, 300μg/L and 400μg/L respectively, and pH is 6.8~7.0, where 25ml medium was cultured in 500ml shake-flask. After 72h by flask-shaking batch fermentation, the production of L-proline reached 75.6g/L, 5% higher than with the control. To investigate the effect of cell growth on the L-proline production, 50L fed-batch culture was performed at the specific growth rate (μ) of 0.06/h,0.08/h and 0.1/h.The results showed that the specific L-proline production rate reached 0.091g/(g.h) and the highest production was 82.1g/L, increased by 14% compared with the control when the specific growth rate (μ) was 0.08/h.
p14ARF is a newly discovered tumor suppressor gene which functions in cell cycle regulation. P14ARF mainly locates in nucleoli with some others positioned on the nuclearplasma. p14ARF also inactivated in some human tumor high frequencily. Now more and more attention focus on the mechanism of p14ARF abnormalities leading to tumor as well as applying P14ARF as a target for cancer gene therapy.The signaling pathway for P14ARF was reviewed.
As a key pathogenic factor in tumorigenesis, p53 had been researched widely. MicroRNA involved in the cancer disease also become a undisputed factor, but the relationship between p53 and microRNA regulation was unclear.Recent studies that shed light on p53 and miRNA possible regulation model and the action of p53 in miRNA function and synthesis were discussed. Finally, the schematic illustration of putative p53-microRNA regulatory pathway also was presented and discussed.
With the end of human genome sequencing project, the research of gene product becomes a hotspot, thus lead the rapid development of proteomic. Serum secretome plays a significant role in cancer. So far, many secretary proteins are considered as tumor biomarks and used for clinical detection.The basic research methods and latest advance in serum secretome of cancer were reviewed.
MicroRNA (miRNA) is a class of -21nt non-coding RNAs that regulate mRNAs at the post-transcriptional level, and widely play roles in eukaryotic growth and development, metabolism and stress responses. However, the biological functions of most miRNAs remain unclear. Investigating miRNAs’ spatial and temporal expression pattern through sensitively quantitative detection methods in different tissues, which is an important job to explore their functions. Therefore, focused on the basic principles and laboratory procedures of two classes and seven quantitative detection methods which based on PCR technology, and the differences and applications among them were analyzed.
The main active ingredient of Sinopodophyllum emodi (Wall.) Ying is podophyllotoxin, which has an important application potential in medical field. Currently, the main source of podophyllotoxin for commercial production come from wild Sinopodophyllum emodi (Wall.) Ying, which has become endangered in the world for overcollecting.Both botanical characteristics of Sinopodophyllum emodi (Wall.) Ying and the research progress of podophyllotoxin production by cell engineering (such as tissue culture, rapid propagation, largescale cell culture, hairy root culture)and endophytic fungi fermentation were introduced with a view to providing background information for the related researches.
There is a huge market potential of shikonin and its derivatives in medicine, food, cosmetics and dyeing fields. How to improve their production has become the research hotspot of the field. The biosynthesis and accumulation methods of shikonin and its derivatives, including the screening of high yield strains, the improvement of productionmedium, the effects of additional materials and conditions, the regulation of genetic engineering, the effects of bioreactors the analytical methods, the extraction and purification techniques, and so on are summarized. The expectation about the direction of shikonin and its derivatives in the future is also discussed.
D-amino acid oxidase (EC 1.4.3.3, DAAO) is a FADdependent enzyme that plays an important role in metabolism of endogenous D-amino acids in living cells. The D-amino acid oxidase (DAAO) catalyzes oxidative deamination of D-amino acids yielding hydrogen peroxide, ammonium and the corresponding αketo acids. The recent data about this enzyme was summarized, including its physiological role and practical applications, optimization of expression, and the characteristics of some singlepoint mutant enzyme which designed by a rational approach.
As the key achievement of transgenic breeding R&D, transgenic event and its intellectual property (IP) protection lay solid foundation for the commercialization of transgenic crops. It is found that currently majority of the transgenic events of major crops are held by foreign agro-biological companies, such as Monsanto, Syngenta and Bayer. About 40 percent of the events are filed for patent protection, 22 applications are also claimed in China with 6 applications already granted. The number of claims is around 20 per patent on the average, which emphatically cover the key technical features like flanking sequence and insertion sequence. The boundaries almost prevent any unauthorized commercial use. In contrast, China has not filed any actual patents on events, although the patent of "Huahui No.1" looks very similar, the scope of protection is rather limited. Thus, in order to dominate the industrialization of biotechnology crops in China, Self-owned IP on events should be fostered, the dynamic trends of targeted foreign patents should be analyzed, and higher degree of patent protection on events must be achieved. Eventually, it is needed to set up a fully- integrated IP protection system for the agro-biotechnology industry.
