To study the effect of the last 24 amino acids peptide(MGF-Ct24E) corredsponding to the C-terminal part of the mechano growth factor(MGF) E domain on bone formation in vitro, primary osteoblasts were obtained from neonatal rat. MTT assay and cell cycle assay were used to study the proliferation and cell cycle stage distribution of osteoblasts. The gene expression profile of osteoblasts after treat with MGF-Ct24E was measured by DNA microarray analysis. Quantitative PCR was used to verify the microarray data. The results suggested that the proliferation activity of MGF-Ct24E group was significantly higher at the first day compared with control. The significantly greater proportion of S and G2/M phase cells appeared in the MGF-Ct24E group than control. Microarray data showed that 1397 genes were identified to be differentially expressed in the MGF-Ct24E group. Among these identified genes,922 genes were up-regulated and 475 genes were down-regulated. Most of the differentially expressed genes were associated with the regulation of proliferation and differentiation, growth factor activity and binding. The effect of MGF-Ct24E on the regulation of cell proliferation and differentiation suggest a potential role in the treatment of bone repair.
Aim: To detect OAS1, its monoclonal antibody was prepared by immune mouse. Methods: BALB/c female mice were immunized with His-OAS1 expressed by E.coli BL21 cells. Monoclonal antibody (McAb) was prepared by hybridoma technology. Identify the purity and characteristics of the selected McAb purified with saturated ammonium sulfate and give preliminary evaluation of the effectiveness of its application, in which SDS-PAGE, ELISA, Western blot and other methods were used. Results: High efficient expression of OAS1 protein in vivo, a McAb can detect the OAS1 was obtained designated as C24-5. The titer of C24-5-McAb was over 9×10-10mol/L, and the affinity constant of C24-5-McAb was 7.66×107 L·mol-1. Conclusion: The preparation of C24-5-McAb is very important to further improve the detection of OAS1.
IRAK2 plays a critical role in IL1 and TLR-mediated signaling, however, the mechanisms by which IRAK2 regulates IL1/TLR signaling remain unclear. It is found that IRAK2 became modified by co-transfected IRAK1 in 293 cells. The modified IRAK2 were recognized by anti-IRAK2 Western blotting as slower mobility shift bands on SDS-PAGE. In bone-marrow derived macrophages, endogenous IRAK2 was also modified upon LPS induction. Moreover, the modified IRAK2 bands collapsed upon pretreatment by calf intestinal phosphatase, indicating that IRAK2 modification is mainly attributed to phosphorylation. By in vitro kinase assay, immunoprecipitated IRAK2 was demonstrated to be able to phosphorylate MBP in vitro, suggesting that LPS signaling induces the kinase activity of IRAK2. Furthermore, a kinase-inactive mutant of IRAK2 is made, which is unable to reconstitute the LPS-induced signaling and inflammatory gene expression in IRAK2-deficient macrophages. Taken together, it demonstrates that the kinase activity of IRAK2 is critical for LPS-induced downstream signaling and proinflammatory gene expression, in addition, phosphorylation of IRAK2, likely regulated by IRAK1 might have a key role in activating IRAK2 kinase.
Object: In order to express recombinant HA protein using the Bac-to-Bac Baculovirus Expression System. The expression of the recombinant HA protein was confirmed by Western blot and IFA. Method: A/California/04/2009(H1N1)HA gene was amplified by PCR, and the PCR product was cloned into the pFastBacHT A vector. The recombinant plasmid pFastBacHT-HA was identified by restriction enzyme digestion and gene sequencing. The recombinant plasmid pFastBacHT-HA was subsequently transformed into E. coli DH10Bac component cells. The recombinant rbacmid-HA identified by Bluo-gal blue/white selection and PCR was purified and transfected into sf 9 cells. The P1 virus obtained from sf 9 cells was amplified and stocked. The sf 9 cells infected with the baculovirus was harvested and analyzed for detection of the expression of recombinant protein by Western blot and IFA. Conclusion: The expression vector engineered for expressing A/H1N1 influence virus HA gene was constructed, which was transfected into insect cells, The recombinant baculovirus with higher viral titer was obtained. The recombinant protein was expressed by recombinant baculovirus in the infected sf 9 cell and identified by Western blot and IFA, demonstrating that the truncated HA protein can be recognized by the antibody specific to HA of 2009 H1N1 influenza virus.
Objective: To construct the pPIC-vMIP-II-TfN yeast expression vector and to express vMIP-II-TfN fusion protein. Method: PCR was employed to amplify the N-terminal half-molecular of human transferrin gene. Molecular cloning methods including digestion, ligation and transformation were used to construct the pPIC-vMIP-II-TfN yeast expression vector. The linearized pPIC-vMIP-II-TfN was transformed into Pichia pastoris X33 strain by electroporation. The transformant was induced by methanol to express target protein and ammonium sulfate precipitation, dialysis, and Ni-NTA affinity chromatography were used to purify the target protein. SDS-PAGE and Western blot was used to detect the expression and purification of vMIP-II-TfN. The activity of vMIP-II-TfN was detected by chemotaxis inhibition activity assay. Results: A 1.1kb IgG3-TfN DNA fragment containing the Xba I site was amplified and it was inserted into Xba I sites of pPIC-vMIP-II. After PCR identification and sequencing, the recombinant plasmid pPIC-vMIP-II-TfN was obtained successfully and the open reading frame was correctly. The linearized pPIC-vMIP-II-TfN by Sac I was transformed into Pichia pastoris X33 strain. After inducing with methanol for 72 h, the transformant was confirmed to express a recombinant protein with molecular mass of 48 kDa by SDS-PAGE and Western blot. After characterization, the vMIP-II-TfN fusion protein was successfully purified from the supernatant of the broth using ammonium sulfate precipitation, dialysis and affinity chromatography. Furthermore, in vitro bioacitivity assay indicated that vMIP-II-TfN has chemotaxis inhibition activity. Conclusion: The pPIC-vMIP-II-TfN yeast expression vector was constructed successfully. The transformant can express the vMIP-II-TfN fusion protein which had chemotaxis inhibition activity.
Effects of eight media on the growth, PSⅡ activity, lipids and carbohydrate accumulation of a marine microalgae Isochrysis zhanjiangensis were investigated. When the nitrogen concentration in the medium is 1.5 g/L, the highest lipid content of 39.8 % and the maximum lipid yield of 0.92 g/L were achieved, while the carbohydrate content of 11.6 % was the lowest. When the nitrogen concentration in the medium is 0.016 g/L, the lowest biomass of 0.74 g/L and the lowest lipid content of 21.1 % were obtained. However, the highest carbohydrate content of 44.4 % was obtained. Based on the result of linear fitting equation, the concentration of NO3-in the medium has a good positive correlation with total lipid content of algal cells. Therefore, the regulation in the yield of lipids and carbohydrate is realized by studying different nitrogen-level media.
Heat shock protein 60 (Hsp60), an important member of the molecular chaperonin family, plays significant roles in the transportation, assembly and folding. Here, a fast method of the purification of Hsp60 from the nematocyst of jellyfish Cyanea nozakii Kishinouye was described. The Hsp60 was achieved by a two-step separation of anion-exchange and size-exclusion chromatography. Subsequently, SDS-PAGE analysis revealed that the purified protein was an apparent and single protein band with a molecular mass of 60 kDa and the N-terminal amino acid sequences of APKEIKFGADAKSLM, which is accordance with the sequences of heat shock protein 60 and its identity was also confirmed by a highly sensitive sandwich ELISA, which is used to quantitate Hsp60 levels of the crude and isolated protein. The method of the purification of Hsp60 from the jellyfish Cyanea nozakii Kishinouye is very helpful to the study of the potential functions and further application of Hsp60.
The process of Trypsase, Alcalase protease and Papain hydrolysis of fresh antler velvet was investigated. Alcalase protease showed the highest degradation efficiency. it was founded that enzymatic concentration, hydrolysis temperature, pH and hydrolysis time were the main parameters which affect the degradation yield of fresh velvet antler. by single factor experiments. The optimal hydrolysis conditions were obtained by the orthogonal tests as follows: substrate concentration 0.08 g/ml, enzymatic hydrolysis temperature 65 ℃, pH 9.0 and hydrolysis time 6.0 h. Under the conditions,the degradation yield of fresh antler velvet is up to 92.6%, and the yield of the amino acids product reached to 12.1%.
Savinase was chemically modified using NaIO4 oxidised dextran. The molecular size and the secondary structure of modified Savinase were characterized by GPC and CD. The factors related with the activity of the modified Savinase, such as kinetic constant, temperature and pH value were studied and compared with those of native Savinase. GPC results showed that the molecular size of the protease increased after chemical modification. CD spectra revealed the difference between native and modified Savinase, which further demonstrated the conjugate of oxidized dextran and Savinase. Compared with the native Savinase, the modified one had a higher affinity to casein. The optimum reaction temperature for both of the protease was 40℃, and the modified Savinase had a better thermal stability at 30℃~50℃. The stability of modified Savinase is better than the native one at pH8.5~9.5.
In order to achieve efficient extracellular production of γ-cyclodextrin glycosyltransferase(γ-CGTase), E. coli BL21(DE3) carrying the γ-cgt gene of Alkalophilic Bacillus clarkii 7364 was investigated. By comparison of different culture media and conditions, got the optimized medium: 5g/L glycerol, 6g/L peptone, 24g/L yeast extract, 6mmol/L Ca2+, 2mmol/L Mg2+, 0.75% glycine, the optimized conditions: cultivated in 25℃, 220r/min, 30ml/250ml, pH 6.5, induced by 5g/L lactose at 10h of culture, which contained 0.02% SDS, the enzyme activity rised from 5189.2U/ml to 14683.4U/ml.
Corynebacterium crenatum AS. M7, mutated and stored is a high arginine producing strain. ArgR is one of the most important regulating proteins during arginine biosynthesis. To further confirm the effect of ArgR on arginine biosynthesis in C. crenatum, specific primers were designed and used for PCR amplification of full argR gene from wild strain C. crenatum AS 1.542 and a mutant strain C. creantum AS. M7. The amplified argR gene were aligned and compared for difference. Result showed that argR from C. crenatum AS 1.542 has ORF of 516 bp, encoding a protein consists of 172 amino acids. In comparison, argR from C. crenatum AS. M7 has a C instead of T on position 109, resulting termination of ArgR expression in C. crenatum AS. M7. Meanwhile, argR gene from C. crenatum AS 1.542 was cloned into pXMJ19 vector and transformed into C. crenatum AS. M7 to form a recombinant strain. Arginine production with recombinant strain was evaluated by fermentation in triangle flask. SDS-PAGE and Western blot were used for argR gene expression analysis, result showed a decrease of arginine production from 7.8 mg/ml to 2.5 mg/ml (appr. 67.9%).
The open reading frame of sesquiterpene cyclase cDNA (AF304444) from Artemisia annua was subcloned into a bacterial expression vector pET30a(+) in frame with N-terminal and C-terminal HIS6-tag, then recombinant vector pET30SESQ was introduced into Escherichia coli strain BL21(DE3). Recombinant sesquiterpene cyclase was induced at 28℃ by adding 0.5 mmol/L IPTG (Isopropyl-beta-D-thiogalactoside) and purified using immobilized metal affinity chromatography on Ni2+ columns. GC-MS analysis showed that the recombinant sesquiterpene cyclase can catalyze the formation of farnesol from FPP.
Newcastle disease viruses (NDV) are detected by three detecting tests and make a comparison of advantages and disadvantages about these tests. The allantoic fluid are obtained through inoculating the NDV virulent F48E9 and avirulent Lasota to SPF chicken embryo and detecting by double antibody sandwich ELISA, suspension array system and RT-PCR. After measure the concentration about antibody 6C4 and 4D9 against NDV, 6C4 was selected as biotin labeled antibody, alternative 4D9 as solid phase capture antibody, the double antibody sandwich detection is based on biotin-streptavidin amplification. Design a group of universal primes against NDV after analyzing the F gene in NDV virulent and avirulent viruses which have been published on genebank and measure the detection limit by RT-PCR. The results show that the detection sensitivity of NDV virulent and avirulent viruses is 1:160 by ELISA, but operation is cumbersome and time-consuming; the detection limit is 1:160 and 1:320 through suspension array system, respectively, however, its operation is more convenient and the equipments are expensive compared with ELISA. The detection limits of RNA in virulent and avirulent viruses are 259pg and 14pg, compared with previous two methods, RT-PCR is a measurement on nucleic acid level, it should have high sensitivity in theory, but the necessary reagents are considerable expensive and the laboratory personnel need training in certain skills.
The trypsin-resistance of β-mannanase MAN47 from Armillariella tabescens was improved by site-directed mutagenesis method. In order to determine the amino acid for mutation, homology modeling were used. Firstly, the structure of β-mannanase MAN47 was constructed by homology modeling and the contact of intramolecular trypsin cleavage sites (K and R) with the surrounding solvent (exposure extent) was analyzed on the basic principles of the catalytic characteristics of trypsin. Among those trypsin cleavage sites, K280 with most exposure extent to the surrounding solvent was chosen as the candidate mutation site. Then K280 was subject to simulating mutation. And K280N was determined as the suitable mutant after calculating the changes in H-bond length and the structure of enzymes before and after simulating mutation. Then the gene of the mutant K280N by site-directed mutagenesis were obtained, and it was cloned into secreted expression vector pYCα. The amplified recombinant plasmids in DH5α cells were transformed into Saccharomyces cerevisiae BJ5465 for the mutant enzyme expression. Subsequently, transformants were screened out by incubation with simulated intestinal fluid (pH 6.8 10mg/ml trypsin solution). And both the wild-type and mutated enzymes were characterized and evaluated on trypsin-resistance. Results showed that the mutant enzyme K280N had a half-life of 173 min after incubation with trypsin at 40°C, which was 74 min longer than that of the wild-type enzyme (99 min). But the optima pH and temperature of both the two enyzmes were similar. In conclusion, the trypsin-resistance of the β-mannanase MAN47 was successfully improved after site-directed mutagenesis.
Objective: To construct a waaL knockout mutant of Escherichia coli O157:H7. Methods: Using plasmid pKD4 as a template,the kanamycin-resistant gene flanked by homologues of waaL gene was amplified by PCR. The PCR products were electro-transferred into the O157:H7,with the help of the Red recombinant system,WaaL gene were replaced by the kanamycin-resistant gene. Then the kanamycin-resistant gene was eliminated by FLP-promoted recombination system. Result: waaL gene of the E. coli O157:H7 was completly eliminated and the kanamycin-resistant gene was also eliminated.
Gene targeting is a well established technique which allows researchers to create virtually any desired modification in the genome of a living mouse. The first step to generate a gene knockout mouse is to construct a targeting vector for homologous recombination in mouse embryonic stem cells. Since traditional construction strategy which mainly recruits PCR, restriction digestion and ligation is difficult to get longer DNA fragments for homologous recombination, A strategy using modified recombineering to generate gene targeting vectors was reported. Unlike other methods which use much longer PCR regions for construction, a method used short 50bp homologous regions which were synthesized in PCR primers as the homologous arms to clone more than 10 kb genomic DNA fragment from BAC which contains Gpr56 genomic DNA into a low copy vector through cap repair. Then the 2~5 exon region of Gpr56 was replaced by an ASCI-flanked cassette through a second recmobineering. After that, a DNA fragment which contains a Flp-FRT-based self-excision NEO cassette was then cloned into the vector with ligation through ASCI sites. After DNA sequencing of selected regions, it was confirmed that the Gpr56 targeting vector was what desired.
To establish a protein fingerprint database of common clinical Gram positive cocci, which can be applied for rapid identification of bacteria, 185 clinical bacterial isolates including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Enterococcus faecium, Enterococcus faecalis were collected and studied. These isolates were assigned into training set and testing set. Bacterial proteins were detected by surface enhanced laser desorption & ionization time-of-flight mass spectrometry (SELDI-TOF MS). Protein fingerprint was analyzed by ProteinChip and Biomarker Wizard software and a protein fingerprint database of some Gram positive cocci was established by using self-constructed Fingerwave software. Similarity analysis between the protein fingerprint of testing set and the constructed database was conducted to evaluate its diagnostic value. A small protein fingerprint database was developed for five common Gram positive cocci. All the tested strains using the database were successfully identified with a result perfectly consistent with those obtained by using automated microbiology analysis system and molecular biological method. Thus, construction of a large-scale protein fingerprint database is desired, which will provide a practicable tool for rapid identification of bacteria.
By Chromosome walking PCR technique, the 3'flanking sequence of the exogenous gene in the pAHC25 plasmid of wheat ‘B73-6-1’ was successfully cloned. An event specific transgenic detection method for ‘B73-6-1’ was established by PCR with the specific primers based on the 3′border. The amplification fragment include part sequence of both the exogenous DNA and wheat genome. This assay was applied to detect typical genetically modified crops, with high specificity for B73-6-1, and the limit of detection was 0.1%. The results showed that the qualitative PCR assay was accurate, rapid and efficient for detection of B73-6-1.
Fusarium is an important genus of fungal pathogen, its intrusion into plant causes fungal diseases, which results in a huge damage to the production of crop and other plants. Plant is one major source of resistance genes. With the rapid development of molecular biotechnology, a large number of resistance genes and resistance candidate genes for Fusarium have been isolated and identified from different plants, and applied to genetic engineering breeding for plant resistance to Fusarium. The resistance candidate genes as well as the classes and mechanism of resistance genes for Fusarium have been summarized, and the interaction mechanism and gene regulation between Arabidopsis and Fusarium were reviewed.
PAs are serine proteases of tryptic specificity which can convert the proenzyme, plasminogen, into an active enzyme plasmin. For the assay of PAs activity, four techniques have been established. Fibrin plate method and colorimetric assay using chromogenic substrates are two early methods which can be used for semi-quantitative analysis. Reverse fibrin autography and fibrin zymography are two new qualitative techniques which can be used to detect the molecular weight or isoelectric point of unknown PAs. The developing process and principle of these four techniques were reviewed, and the potential applications, advantages and disadvantages were discussed.
