In order to decrease the potential side-effects of human basic fibroblast growth factor (hbFGF) caused by its broadspectrum mitogenic activity, a single residue of hbFGF, the residue serine 108, was replaced with neutral alanine residue to construct a mutant of hbFGF (mhbFGF) with reduced mitogenic activity. The mutant was overexpressed in Escherichia coli BL21(DE3) by IPTG induction. The expression level of mhbFGF was about 30% of the total cellular protein. The expressed mhbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. Measured by MTT method, the effect of mhbFGF on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF. The purified recombinant mhbFGF was prepared and sufficient for the following pharmacological and safety studies.

The expression cDNA library of A. tabescens was constructed by SMART technique, which useλTriplEx2 as a vector. The titer and the percentage of the constructed library were about 1.0 × 106pfu/mland 98.3% respectively, and the titer and the capacity of the amplified library were about 3.1 × 108pfu/mland 4.2 × 1010. The library was used to provide expressed sequence tags (ESTs). 147 Expressed SequenceTaqs (ESTs) were gained from 176 clones, which were selected randomly and sequenced at the 5'end. Thesequences were submitted to the EMBL database. Blasting the sequences in the GenBank, 43 of them werefound that they have significant similarity with data in GenBank. EST AJ620046 was has significantsimilarity with the arabinosidase of Bacteroides thetaiotaomicron. Using SMART-RACE a full-length cDNA ofAJ620046 was successfully obtained. In order to initially characterize the biochemical properties ofAJ620046, the ORF of AJ620046 named AF was cloned and expressed in Pichia Pastoris yeast.Recombinant pHIL-S1-AF constructed by inserting AF into pHIL-S1 was transformed into Pichia PastorisGS115. Preliminary experiments indicated that AJ620046 was expressed as a 32 kDa protein in recombinantyeast.

An effective method has been developed for laboratory scale production of IgG. Hybridomas were cultured in serum-free media with 2% IgG-free ascites. Cell density of up to 3.55 × 10 6cells/ml and antibody concentration of 135μ g/ml after purification were abtained, which is four time more than total production of that of IgG concentration in serum-free media. This in vitro method allows great improvement in antibodies production in batch tissue culture. The method reported here is easy to handle and is economical and universally adaptable.

Objective: To research the activity of antibacterial proteins isolated from Musca domestica during larvae-pupae metamorphosis. Methods: The 5-day-old larvae of Musca domestica were injured with a needle. 24h later, the larvae which had changed into pupae were selected. The antibacterial protein was isolated by a four-step protocol including grinding, heating, CM-sepharose F. F. cationexchange chromatography and another CM-sepharose F. F. chromatography. Antibacterial activities of samples were tested by an ultrasensitive radial diffusion assay using Staphylococcus aureus as the indicator organism. The molecular weight of the isolated protein was determined by SDS-PAGE.Results: The molecular weight of this isolated protein was 43kDa, and its antibacterial activity was strong. Conclusions: A kind of protein with strong antibacterial activity can be produced in the Musca domestica during larvae-pupae metamorphosis.

To improve 3-ketosteroid-△1-dehydrogenase (KSDH) activity and the transformation level for androst-4-ene-3,17-dione,3-ketosteroid-△1 -dehydrogenase gene (ksdD) from Arthrobacter simplex was cloned into plasmid pWB980 and expressed in B. subtilis WB600 under the control of promoter P43. The molecular weight of expressed enzyme was about 55kDa by SDS-PAGE analysis. The activitities assayed by spectrophotometrical method of intracellular and extracellular soluble enzyme were 110 ± 0.5mU and 15 ± 0.6mU per milligram of protein respectively. The transformation rate of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was 45.3%. Compared with Arthrobacter simplex, the enzyme activity of KSDH expressed in B. subtilis was improved about 30 fold, and the transformation level of androst-4-ene-3,17-dione by the B.subtilis recombinant cells was improved about 10 fold. The recombinant B. subtilis cells used in biotransformation of steroids provided a new way for steroid medicines production.

Enhanced green fluorescent protein( EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin.Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo - peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his -EGFP B in frame with EGFP, myc and 6 × His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6 × his did not interfere each other and octo- peptide linker owned certain flexibility. The results suggest that pcDNA6/myc-his-EGFP B may be useful as a genetic tool for mammalian cells and a vector for gene therapy.

Antheraea pernyi nucleopolyhedrovirus (ApNPV) PstI-B and C fragments were cloned and sequenced. ApNPV PstI-B was 7406 bp long, contained seven open reading frames (orfs)/genes, including p87, he65, pnk/pnl, odv-ec43 and Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) orf107,orf108 homologue on either strands of genomic DNA. ApNPV PstI-C was 6663 bp long, contained eleven orfs/genes, including pk-1, orf1629, polh, lef-2, ptp-2, ctl-1, ptp-1 and OpMNPV orf5, orf7, orf8, orf1 1 homologue on either strands of genomic DNA. Among the eighteen baculovirus genes identification, he65 and orf1629 were two diverse genes, while polh and lef-2 were two conserved genes. ApNPV was the third baculovirus found to contain pnk/pnl gene, the fourth baculovirus found to contain both ptp-1 and ptp-2 gene.

The biological activities i. e. antineoplastic activities and antiviral activity of the two novel kinds of interferons: hIFN-λ1 and hIFN-ε were studied and compared. First the fusion expression vector: pcDNA3.1A-hIFN-λ1-His and pcDNA3.1A-hIFN-ε-His by PCR was constructed, then the two kinds of plasmids were transfected into WI-38 (human embryonic lung cells ) with liposome. And cytopathic effect (CPE) suppression test was used to study and compare the antiviral activities of rhIFNλ1-His and rhIFN-ε-His, meanwhile MTT assay was used to detect their antineoplastic activities. It was found that, antiproliferative activity and MxA protein induction shown by rhIFN-λ1-His is more powerful than of rhIFN-ε -His. The antiviral molecular mechanisms of both hIFN-λ1 and hIFN-ε are related to MxA. The foundation for further study on the bioactivities and mechanism of action of hIFN-λ1 and hIFN-ε was established.

E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria.We described a high expression and efficient purification scheme and kinetic assay of interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 E.coli strain induced by IPTG. SDS-PAGE analysis revealed that the expected protein with a molecular weight 20.6kDa was soluble and amounted to about 30% of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90%. The resulting SSB protein was a correctly folded tetramer analyzed by gel filtration. It could bind ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 mol/L as determined by surface plasmon resonance.

Potato cultivar Atlantic is widely grown for potato chips in the world. However, this economically important potato cultivar exhibits very poor yields and traits under severe environmental stress. To develop an efficient plant transformation system that could be used to produce large scale transgenic potato plants with enhanced tolerance to environmental stress and therefore would be beneficial for potato processing industry, Agrobacterium-mediated transformation of internodal stem explants using both superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible SWPA2 promoter was performed. Comparing to leaf explants, stem internodal explants were less liable to damage during manipulation, more amenable to in vitro conditions. The addition of silver thiosulfate to the selection medium considerably promoted the shoot induction from explant-derived callus. Seven to nine shoots per stem explant were obtained. By combining the best treatments, this system yielded shoot induction frequency of 94.2% and transformation frequency of 80% of internodal stem explants. Stable integration of the transgenes was confirmed by PCR and Southern blot analyses. In conclusion, short duration (7～8 weeks), high efficiency and easy process make this system well suited for wider commercial applications of transgenic Atlantic potato plants.