Abstract Objective To evaluate the immunogenicity of HIV-1 clade C/B’ vaccine based on modified vaccinia virus Ankara (MVA) vector in mice. Methods Mice were inoculated with 3-dose HIV vaccine by intramuscular injection. Blood sample were collected every second week, and then the antibodies against HIV were detected. At week 6, mice were killed and cellular immune responses were examined by ELISPOT. Result The number of spot forming cells in the 107 pfu/mL -dose group was more than those of 105 pfu/mL -dose and 106 pfu/mL -dose groups significantly. HIV specific antibodies emerged at week 2 and elevated rapidly at week 4 and week 6. The level of specific IgG in the 107 pfu/mL -dose group was more than those of 105 pfu/mL -dose and 106 pfu/mL -dose groups significantly. Conclusion The ADMVA induces both humoral immunoresponse and cellular immune responses.
The intracellular hepatitis B surface antigen (HBsAg) content per cell was increased by 7.2-fold in the culture with 1.5% dimethyl sulfoxide (DMSO) compared with that in the control without DMSO, while the extracellular HBsAg production and specific productivity were only improved by 70% and 3.2-fold, respectively. Electron microscope has been employed to reveal large dilated structures within recombinant CHO cells in the presence DMSO. The dilated structures have a distribution within whole cytoplasm, and some dilated areas were engulfed in the nucleus. These large, dilated structures were not observed in the control. Immunogold labeling was used to discover the accumulated HBsAg was localized within these dilated areas, and some HBsAg-specific labels were detected in the nucleus membrane, owing to the encroachment of the dilated areas upon nucleus. This work could help to reveal the mechanism of intracellular HBsAg accumulation in the presence of DMSO.
Abstract: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, Apo-2L) is a mediator of cell death that preferentially targets cancer cells. The C-terminal extracellular region of Apo-2L (amino acids 114-281) exhibits a homotrimeric subunit structure. Soluble Apo-2L(sApo-2L) induces extensive apoptosis in lymphoid as well as non-lymphoid tumor cell lines. But it is limited in its efficacy by the short circulating half-life and low stability. sApo2L-Fc is a fusion protein consisting of Apo-2L and a fragment(hinge, CH2, CH3 domains) of the Fc of the IgG1. The DNA was constructed in the pcDNA3.1（+） eukaryotic expression vector. Using DOTAP-mediated gene transfer technique, pcDNA3.1- sApo2L-Fc was transformed into Chinese hamster ovary(CHO) cells. The selective medium containing G418 was then employed to select the positive colony. After purified with protein A affinity chromatography, the product was detected with SDS-PAGE and Western blot. A protein band with molecular weight of 44KDa was found in the SDS-PAGE. Western blot displayed the recombinant product had strong immunological activity with mouse anti-human Fc monoclonal antibody. After purification, the biological activity of sApo2L-Fc was more than 1.0×105IU/mg.
By means of genetic cloning and recombinant techniques, full genome cDNA sequences of rotavirus strain TB-Chen were isolated from an infantile hospitalized with acute gastroenteritis. Nucleotide sequences analyses showed that the full genome of strain TB-Chen contains 18613 nucleotides, encoding 5791 amino acids. Genotyping results showed that the strain TB-Chen belongs to genotype G2P[4]/NSp4[A]. This is the first report on a full genome of Group A rotavirus in China, and has important significance for deep understanding structure and functions of rotaviruses and developing rotavirus vaccines. Key words: Group A human rotavirus Full genome Genotyping analysis
A strain NK13 was screened for a certain extent asymmetric hydrolyze the rac-ketoprofen Chloroethyl ester and identified as Bacillus megaterium. For the preparation of gene libraries, a positive clones was obtained from the tributyrin flat. The sequence of this esterase gene had been analysised, and contained the whole ORF of an esterase gene with the length of 933bp. The esterase gene of NK13 was compared with the esterase genes of GenBank and the result showed that the esterase gene of NK13 was a novel gene（GenBank accession nember DQ196347）.The new esterase gene was inserted into the plasmid pET21b+, then the recombinant plasmid transformed E.coli BL21. After being induced by IPTG, it was expressed in the host strain. SDS-PAGE analysis showed that the relative molecular mass of the esterase was about 34KDa. The result of TLC and HPLC showed that the recombinant strain had higher conversion ration than templet strain. 47.4%of the rac-ketoprofen Chloroethl ester was hydrolyzed to ketoprofen by the recombinant strain in 45min. The (S)- Ketoprofen enantiomeric excess, in the later ,was 55.46%, which indicated that the esterase could hydrolyze (S)-Ketoprofen Chloroethyl este firstly.
Abstracts Objective: To prepare chitosan nanoparticles carrying gene，study its transfection efficiency and immunological enhancement in mice. Methods: The nanoparticles was based on the foot and mouth disease DNA vaccine which designed by our laboratory,it was prepared by complex coacervation , and its morphology was observed by transmission electronic microscopy (TEM) . Particle size , polydispersity and zeta potential were determined by nanoparticle size analyser. The molecular localization of pDNA in the chitosan nanoparticles was determined by gel retardation assay. Transfection efficiency of pDNA-chitosan nanoparticles was determined by gene transfection experiment in vitro. Balb/c mice were immunized by pDNA- chitosan nanoparticles to test both humoral and cellular immune responses. Results: The morphology of the nanoparticles were mostly spherical. The average particle size was 150nm, and the polydispersity of particle size＜0.26,the zeta potential of the nanoprticles was 21 mV. The results of gel retardation showed chitosan could completely combine the pDNA by electronic combination and pDNA was mostly encapsulated within the nanoparticles.The gene transfection experiment in vitro showed that the pDNA-chitosan nanoparticles can transfect BHK celles , and the gene can express in these celles. Immunity test proved that the nanoparticles not only induced high cellular immunogenecity, but also induced a good humoral immunogenecity in mice. Conclusion: chitosan nanoparticles can deliver gene into cell and express in these celles. Immunity test proved that it has favourable immunological enhancement.
The NSP2 gene of porcine reproductive and respiratory syndrome virus （PRRSV）S1 strain was partly amplified and cloned into a prokaryotic expression vector pGEX-6P-1，and a fusion protein GST-tNSP2 with molecular weight of 50 kD was expressed in E. coli. The purified GST-tNSP2 protein showed a strong reaction with the PRRSV-positive sera in Western-blotting assay. BALB/c mice were immunized with the purified protein, and the splenocytes of the immunized mice were fused with murine myeloma cells SP2/0. After subcloning by 3 times, two hybridoma clones which produced McAbs steadily were screened by ELISA, named 3H3 and 2B5. They all reacted strongly with the PRRSV S1 infected Marc-145 cells in IFA, but not with the PRRSV SY0608 strain. Both of the McAbs were belong to IgG1 isotype, and their light chain were Κ. It indicated that the expressed GST-tNSP2 protein and McAbs could be used for identification of PRRSV isolates and functional analysis of NSP2.
Peroxiredoxin (Prx), one of the ubiquitous enzymes in organisms, plays an important role in eliminating oxidative stress. The cDNA encoding peroxiredoxin(Prx) of Fenneropenaeus chinensis protein was cloned and expressed in E. coli. The recombinant protein was expressed as inclusion body after IPTG induced. LC–ESI–MS analysis showed that four peptide fragments of the recombinant protein were identical to the corresponding sequence of F. chinensis Prx. The fusion protein was purified using the immobilized-metal affinity chromatography. After refolded, the purified recombinant protein was shown to reduce H2O2 in the presence of dithiothreitol. The fusion protein is useful for studies on the function of Prx in immune defense and against oxidative stress of F. chinensis.
A full-length sequence coding for Taxane 13α-hydroxylase(13OH) was cloned from Taxus cuspidata cDNA library.The DNA was verified by sequencing after it was inserted into pGM-T easy vector. Compared with the nucleotide and amino acid sequence in the Genbank, nucleotide of the cloned DNA in our paper showed 99.38% identity with that of the reported taxane 13α-hydroxylase of Taxus cuspidate and its deduced amino acid sequence showed 99.18% identity with that of the reported taxane 13α-hydroxylase of Taxus cuspidata. To construct the plant expression vector pC13OH, the full-length DNA was inserted into pCambia1305.1. PCR and restriction enzyme analysies confirmed the correctness of the construct, then the recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 by electroporation. The transformation of this engineered Agrobacterium strain with tobacco(Nicotiana Tabacu L.) was studied. Plants were regenerated from the infected leaf dics under the selection of hygromycin. PCR ananlysis with 13OH-specific primers shows 4 plants were positive. Among them, 3 palnts were GUS positive after doing the assay of the fusion GUS reporter gene.
Preliminary study on antagonistic activity of antifungal substance from biocontrol bacterium Bacillus subtilis B29 was made by analyzing the inhibitory effect of cell-free supematant to mycelium growth and conidial spore germination of Fusarium oxysporum f．cucumerinum．The results showed that the antifungal substance secreted by the strain B29 inhibited the growth of Fusarium oxysporium f．cucumerinum and conidial spore germination．The optimum fermentation conditions of antifungal substance from strain B29 were original pH7.5, 75ml/250ml at 30℃ for 120h. The antifungal substance was precipitated by (NH4)2SO4 from 30% to 70%. The crude antifungal substance of strain B29 was stable to heat at some extent, partially tolerant to proteinase K, and more sensitive to trypsinase and pepsin.
According to PDA plate culture, shake-flask culture and solid-state fermentation with straw, we chose the best strain which has growth advantage and yields high activity lignin-degrading enzymes from 6 common white-rot fungus. In order to degrade and utilize the straw obviously, we used the two-step mixed fermentation by Pteurorus ostreatus and Trichoderma Koningii. Through the different combination ways, we found that H6-T10 can get the best straw degradation results. The lignin degradation rate reached 44.77%, and the cellulose degradation rate was 41.48%. All of this could make preparation for biogas fermentation by stalks.
In this paper, artificial hapten Hg-ITCBE was synthesized using ITCBE (Isothiocyanobenzylethyl enediamine tetraacetic acid) as the bifunctional chelating agent. Antigens were synthesized through the coupling the hapten with carrier protein keyhole limpet hemocyanin(KLH) and bovine serum albumin (BSA). After the assay for concentration of the antigen with bicinchoninic acid (BCA) method, the hapten antigen and carrier protein were scanned with ultraviolet spectrophotometer, and verified with SDS-PAGE. The degree of conjugate substitution was quantified using the trintrobenzenesulfonic acid(TNBS) method. Concentration of mercury ion in antigen determined with graphite-furnace atomic absorption spectrometry(AAS). The results indicated antigens were synthesized successfully. The extent of substitution of free lysine residues was 34.75 ± 4.60％ for Hg-ITCBE-KLH, 40.61 ±0.99％for the Hg-ITCBE-BSA and 61.27 ±0.69％ for the ITCBE-BSA. The concentration of mercury ion were 38.4±0.5μg/ml, 125.5±0.9μg/ml and 0μg/ml , respectively.
ABSTRACT：Objective：To search for the biomarkers for differential diagnosis between human esophageal squamous cell carcinoma cells and normal esophageal epithelial cells. Methods：Laser capture microdissection was used to separate human esophageal squamous cell carcinoma cells and normal esophageal epithelial cells, Proteome alterations were observed using two dimensional polyacrylamide gel electrophoresis(2-DE) technique and electrospray ionisation tandem mass spectrometry．Westernbloting was used to detect the different protein expressions in ESCC and esophageal epithelial cells, Results : The 2-DE patterns of human esophageal squamous cell carcinoma cells and normal esophageal epithelial cells were established. 14-3-3 protein ε、S100A9 were identified differentially expressed between ESCC and esophageal epithelial cells. Conclusion ：Laser capture microdissection is a key technique in proteomic study which can effectively resolve the problem of irrelevant tissues．14-3-3 protein ε、S100A9 may serve as abiomarker for differential diagnosis of human esophageal squamous cell carcinoma cells and normal esophageal epithelial cells.
Object: To develop a new quantitative determination method for the biological activity of recombinant human ciliary neurotrophic factor. Methods: Dorsal root ganglions were derived from the chick embryo and dispersed into single neuron cell，The rhCNTF was added to neuron cells and incubated for 64 hours，The activity of acid phosphatase in neuron cells was determined and the biological activity of rhCNTF was analyzed quantificationally. Result: rhCNTF could promote original era dorsal root neuron cells of chick embryo surviving，the livability of neuron cells was positively related to the amount of rhCNTF added to the culture. Conclusion: A quantitative determination method for the biological activity of rhCNTF was developed by testing the activity of acid phosphatase in neuron cells. Compared with the typical ways，this method was quantificational easily，repeatable better and with much fewer disturbance factors.
Phytoene synthase is a rate-limiting enzyme in the pathway of carotenoid biosynthesis. This paper is aimed at establishing a transformation of Ginseng callus cells, elevating the nutritive value through encouraging the composition of corresponding carotenoid. Taking Ginseng callus cells as acceptor, PSY gene were transformed into cells via Agrobacterium-mediated. PCR and RT-PCR analysis from the transferred gene plants proved that PSY gene has been transferred into Ginseng callus cells and can be expressed. The content of β-carotene was increased by an average of 26 times. This research established a base of improving and raising the contents of carotenoid in the Ginseng callus cell.
Cucumber mosaic virus was detected from infected Basella rubra L. with the indirect enzyme-linked immunosorbent assay. Total RNA was extracted from infected leaves and the cDNAs of coat protein gene of CMV-Ba were obtained by RT-PCR. The amplified cDNA fragments were then cloned into pMD 18-T vector and sequenced，the result showed that the CP gene was 657 nucleotides in length. This sequence was aligned with the obtained CP gene and some CMV strains or isolates of subgroup Ⅰ and subgroup Ⅱ in GenBank using DNA MAN software. The results showed that CMV-Ba shared 90.9% -93.8% and 76.1%-76.9% identity with the known CP genes of subgroup Ⅰ and Ⅱ respectively in nucleotide level，on the other hand，amino acids deduced from CMV-Ba CP gene shared 92.7% -97.7% and 72.4%-78.1% identity with the known CP protein of subgroup Ⅰ and Ⅱ，respectively. This suggested that CMV-Ba CP gene belongs to CMV subgroupⅠ.
Gambogic acid-loaded Polylacticacid nanoparticles (GA-PLA-NPs) were prepared by modified emulsification solvent diffusion. The shape of nanoparticles was observed by transmission electron microscope (TEM).The size distribution and mean diameter were measured by laser particle size analyzer. The entrapment efficiency and content of drug loading were determined by Ultraviolet Spectrophotometer after ultracentrifugation. GA-PLA-NPs release behavior in vitro was carried out. The acute toxicity were carried out to study the Security of GA-PLA-NPs. The preparation process adapted to the formulation was as follows: the volume ratio of the aqueous and organic was 2∶1(v/v), the surfactant concentration in aqueous was 0.5%,the drug concentration in organic was 0.1%(w/v), GA∶PLA was 1∶4（w/w）. The mean diameter was 51.36nm for the nanoparticles prepared by above conditions.The entrapment efficiency and content of drug loading were 98.87 % and 13.3 %. The release behavior of drug in vitro showed an initial burst effect with subsequently a slower rate stage. The LD50 value of GA-PLA-NPs on Mouse was 26.3 mg/kg. The results showed that the GA-PLA-NPs were well prepared with stable quality and high dispersion. PLA-NPs might be used as a new carrier for Gambogic acid.
MicroRNAs (miRNAs) are a newly identified class of non-protein-coding small RNAs that play important roles in multiple biological processes. Recent evidence indicates that the expression of many miRNAs is both temporally and spatially regulated by RNA editing, differential processing and tissue-specific enhancers, and the potential for ultimately designing molecular medicines based on the modulation of miRNAs seems good. A better understanding of the mechanism which regulates miRNAs is very helpful to reveal the pathogenesis of some diseases, discover novel molecular targets for treatment by interference, and even develope an effective gene therapy. Therefore, the latest progress in the mechanism regulating miRNAs is summarized in the paper.
Amino acids are crucial to plants and play an important role in plant's biological process. Plants must synthesize all needed amino acids. Once the amino acid biosynthesis being inhibited, the plants will be difficult to survive, hence the key enzymes concerned with the amino acid biosynthesis remain to be the important targets during the development of new herbicides. Herbicides designated as amino acid biosynthesis inhibitors take up a large proportion among the commercialized herbicides，at the same time, along with the development of plant transgenic techniques, a great deal of transgenic plants tolerant to herbicidal inhibitors of amino acid biosynthesis come out and become the main body of transgenic herbicide-tolerant plants gradually. This article reviewed the currently commercialized herbicidal inhibitors of amino acid biosynthesis，functional mechanism and the advance in transgenic herbicide-tolerant plants.
Biodiesel, a nontoxic,cleaning, renewable and biodegradable fuel, is expected as a substitute for conventional fossil diesel. There are three main approaches to produce biodiesel, alkali-catalysis processing, enzymatic-catalysis processing and supercritical processing. With the unique property of energy-saving and environment-friendly , enzymatic-catalysis appears a great potential for industrial application. The main bottleneck of this technology is high cost and low stability of the lipase, as well as the inactivation of lipase by methanol and so on.. To settle the problem, several methods have been used including the fixed-bed bioreactor, enzyme immobilized processing, whole-cell biocatalyst, changing addition method of methanol, developing of novel acyl acceptor, enhancing methanol resistance of lipase. The main problems and the relative strategy research of the enzymatic-catalysis technology were sum up in this paper.
