Chimeric Plasmid Research Articles

Long terminal repeats are not the sole determinants of virulence for equine infectious anemia virus

The long terminal repeats (LTRs) of equine infectious anemia virus donkey leukocyte-attenuated virus (EIAV-DLA) were substituted with those of the wild-type EIAV-L (wt EIAV-L, the parent virus of EIAV-DLA). The resulting chimeric plasmid was designated pOK-LTR DLA/L. Purified pOK-LTR DLA/L was transfected into monocyte-derived macrophage (MDM) cultures prepared from EIAV-negative, heparinized whole blood from a donkey. Eighth-passage cell cultures developed the typical cytopathogenic effects (CPE) of EIAV infection, and virions with typical EIAV profiles were observed with an electron microscope. Horses were inoculated with the chimeric virus or EIAV-DLA and challenged with the wt EIAV-L strain six months later. All of the horses inoculated with either the chimeric virus or EIAV-DLA were protected from disease, whereas the control horses died with typical EIA symptoms.

HSP70 gene fused with Hantavirus S segment DNA significantly enhances the DNA vaccine potency against hantaviral nucleocapsid protein in vivo

Heat shock proteins (HSPs) have been shown to act as adjuvants when coadministered with peptide antigens or given as fusion proteins and enhance the vaccination efficiency. To evaluate the enhancement of the potency of Hantaan virus (HTNV) nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), we developed a novel chimeric HTNV S-HSP70 DNA vaccine plasmid by genetically linking HSP70 gene to the full-length HTNV S segment DNA (HTNV S DNA). C57BL/6 mice were immunized with this plasmid followed by a subsequent boost with homologous recombinant protein. The levels of HTNV NP-specific antibody and cellular immune response were measured by use of ELISA, fluorescence activated cell sorter (FACS) analysis, cytotoxicity assay, and IFN-γ ELISPOT assay. We found that HTNV S-HSP70 DNA vaccination significantly increased the levels of HTNV NP-specific antibody, IgG2a/IgG1 ratio, IFN-γ producing CD8 + T-cell precursor frequencies, and cytotoxic T lymphocyte (CTL) response when compared with immunization with HTNV S DNA alone or HTNV S DNA physically mixed with HSP70 DNA. By contrast, HSP70 DNA or vector DNA immunization could not induce appreciable levels of specific antibodies and CTL response. Thus, we demonstrate for the first time that HSP70-based HTNV S DNA can induce both humoral and cellular immune response specific for HTNV NP and is a promising candidate DNA vaccine for HTNV infection.

Green fluorescent protein is a reliable reporter for screening signal peptides functional in Lactobacillus reuteri

A signal peptide (SP)-probe vector pNICE-gfpSP, which employed a green fluorescent protein (Gfp) as the SP-selection marker, was constructed for use in Lactobacillus reuteri. This chimerical plasmid allowed cloning and screening DNA fragments with the SP function by direct visualization of the expressed fluorescence activity around cells. Assay of fluorescent intensity in their culture supernatant with spectrofluorometry further enabled quantifying the secretion efficiency of the identified SP fragment.

Green tea catechin increases cholesterol 7a‐hydroxylase mRNA and promoter activity in HepG2 cells

In the present study, we investigated the effect of green tea catechins on the mRNA level and promoter activity of CYP7A1 gene in human hepatocellular blastoma cells (HepG2). The CYP7A1 mRNA level was measured using real time PCR. The −1312/+358 bp of CYP7A1 promoter was subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. The CYP7A1 promoter/luciferase chimeric plasmid was transfected into HepG2 cells using superfect. Different kinds of catechin, (−)-epicatechin gallate (ECG), (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), and (−)-epicatechin (EC) up-regulated the CYP7A1 mRNA level by 5.5-, 4.2-, 2.9- and 1.9-fold, respectively, compared to the control. Also, ECG or EGCG significantly increased CYP7A1 promoter (−1312/+358 bp) activity by 6.0- and 4.0-fold, respectively compared to the control (P<0.05). The effect of EGCG, a major compound from green tea catechins, was investigated at different concentrations. The EGCG stimulated CYP7A1 gene expression showing increase the CYP7A1 mRNA level and promoter activity (P < 0.05) at dose-dependent manner. These results suggest that the expression of CYP7A1 gene, the rate-limiting enzyme in regulating cholesterol metabolism, may be up-regulated by green tea catechin, at the transcriptional level through the promoter activation. *This work was supported by research grants from the Korea Science and Engineering Foundation (M10510130005-05N1013-00500)

Plasmids in the aphid endosymbiont Buchnera aphidicola with the smallest genomes. A puzzling evolutionary story

Buchnera aphidicola, the primary endosymbiont of aphids, has undergone important genomic and biochemical changes as an adaptation to intracellular life. The most important structural changes include a drastic genome reduction and the amplification of genes encoding key enzymes for the biosynthesis of amino acids by their translocation to plasmids. Molecular characterization through different aphid subfamilies has revealed that the genes involved in leucine and tryptophan biosynthesis show a variable fate, since they can be located on plasmids or on the chromosome in different lineages. This versatility contrasts with the genomic stasis found in three distantly related B. aphidicola strains already sequenced. We present the analysis of three B. aphidicola strains (BTg, BCt and BCc) belonging to aphids from different tribes of the subfamily Lachninae, that was estimated to harbour the bacteria with the smallest genomes. The presence of both leucine and tryptophan plasmids in BTg, a chimerical leucine-tryptophan plasmid in BCt, and only a leucine plasmid in BCc, indicates the existence of many recombination events in a recA minus bacterium. In addition, these B. aphidicola plasmids are the simplest described in this species, indicating that plasmids are also involved in the genome shrinkage process.

Construction and Characterization of Nisin-Controlled Expression Vectors for Use inLactobacillus reuteri

The Nisin-controlled gene expression (NICE) system, which was discovered in Lactococcus lactis, was adapted to Lactobacillus reuteri by ligating nisA promoter (PnisA) and nisRK DNA fragments into the Escherichia coli-Lb. reuteri shuttle vector pSTE32. This chimerical plasmid (pNICE) was capable of expressing the heterologous amylase gene (amyL) under nisin induction. Optimization of induction factors for this Lb. reuteri/pNICE system, including nisin concentration (viz. 50 ng/ml), growth phase of culture at which nisin be added (viz. at the early exponential phase), and the best time for analyzing the gene product after inoculation (viz. at the 3rd h), allowed the amylase product to be expressed in high amounts, constituting up to about 18% of the total intracellular protein. Furthermore, the signal peptide (SP) of amyL gene (SPamyL) from Bacillus licheniformis was ligated to the downstream of PnisA in pNICE, upgrading this vector to a NICE-secretion (NIES) level, which was then designated pNIES (Sec+, secretion positive). Characterization of pNIES using an amyL-SPDelta gene (amyL gene lacking its SP) as a reporter revealed the 3rd h after induction as the secretion peak of this system, at which the secretion efficiency and the amount of alpha-amylase being secreted into the culture supernatant were estimated to reach 77.6% and 27.75 mg/l. Expression and secretion of AmyL products by pNIES in Lb. reuteri was also confirmed by SDS-PAGE and immunoblotting analysis.

P.034 Construction of hepatitis B surface antigen vectors using natural restriction sites located within or around the hydrophilic a determinant region: development of chimeric plasmids containing the HVR1 of hepatitis C virus E2 protein

P.034 Construction of hepatitis B surface antigen vectors using natural restriction sites located within or around the hydrophilic a determinant region: development of chimeric plasmids containing the HVR1 of hepatitis C virus E2 protein

Synthesis and assembly of an adjuvanted Porphyromonas gingivalis fimbrial antigen fusion protein in plants

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease by binding to saliva-coated oral surfaces. To assess whether edible plants can synthesize biologically active P. gingivalis fimbrial antigen, for application as an oral vaccine, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein (FimA), was cloned into a plant expression vector immediately downstream of a cDNA fragment encoding the cholera toxin B subunit (CTB). The chimeric plasmid was transferred into potato ( Solanum tuberosum) cells and the ctb– fimA cDNA fragment detected in transformed leaf genomic DNA by PCR amplification methods. A novel protein band of 21 kDa was detected in transformed potato tuber extracts by immunoblot analysis. Oligomeric CTB–FimA (266–337) fusion protein was identified in the extracts through the binding of anti-CTX and anti-native fimbriae antibodies. The pentameric structure of CTB–FimA fusion protein was confirmed by ELISA measurements of G M1 ganglioside receptor binding. Quantification of the CTB–FimA fusion protein by ELISA indicated that the chimeric protein made up about 0.33% of total soluble tuber protein. The biosynthesis of immunologically detectable CTB–FimA fusion proteins and the assembly of fusion protein monomers into biologically active pentamers in transformed potato tuber tissues demonstrate the feasibility of synthesizing adjuvanted fimbrial protein in edible plants for development of adjuvanted mucosal vaccines against P. gingivalis generated periodontal disease.

Transgenic patchouli plants produced by Agrobacterium-mediated transformation

A successful transformation procedure using Agrobacterium was established for the most important aromatic crop, patchouli (Pogostemon cablin Benth.). To avoid inhibition of Agrobacteriuminfection by patchouli oil accumulated in leaf tissues, complete plants regenerated in vitrowhich possessed no or trace amounts of patchouli oil in leaf tissues were used as an explant source. Conditions for transformation were examined using two A. tumefaciensstrains containing a different chimeric plasmid. Leaf explants were infected with A. tumefaciensstrain EHA101/pIG121-Hm carrying β-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes. Following co-cultivation for 3 days and selection by 50 mg l−1 hygromycin B, greenish calli with adventitious shoots were selected, from which putative transformants with roots were regenerated. Histochemical assay showed that GUS expression is detected in every organs of transformants, which was confirmed by the detection of high activity of GUS. Using another strain LBA4404/pBI 121-PaCP1 encoding the coat protein precursor gene of patchouli mild mosaic virus (CP-P) and neomycin phosphotransferase (NPTII) gene, putative transformants were also obtained after co-cultivation for 7 days and selection by 100 mg l−1 kanamycin. Using total DNAs from the transformants, the full length of CP-P was detected by PCR reaction. Comparing between two strains examined,it was noted that prolonged co-cultivation period and higher dose of a selection drug were indispensable for successful infection with LBA4404/pBI 121–PaCP1.

Functional Characterization of the Iron-regulatory Transcription Factor Fep1 from Schizosaccharomyces pombe

In response to excess iron, Schizosaccharomyces pombe cells repress transcription of genes encoding components involved in iron uptake through the Fep1 transcription factor. Fep1 mediates this control by interacting with the consensus sequence 5'-(A/T)GATAA-3', found in iron-dependent promoters. In this report, we show that Fep1 localizes to the nucleus under both iron-replete and iron-starved conditions. The Fep1 DNA binding domain (amino acids 1-241) contains two GATA-type zinc finger motifs. Although we determine that the Fep1 C-terminal zinc finger (ZF2) is essential for DNA binding, we show that the N-terminal zinc finger (ZF1) enhances DNA binding affinity approximately 5-fold. Between the two zinc finger motifs of Fep1 resides an invariant amino acid sequence, denoted the Cys-rich region (amino acids 68-94), in which four highly conserved Cys residues are found. Cells harboring mutant alleles in which two or more of the conserved Cys residues were substituted by alanine exhibited elevated fio1(+) mRNA levels. We determine that the dissociation constant for the resulting complex between each of the Cys mutants and the sequence 5'-(A/T)GATAA-3' reflects a much lower affinity that correlates with failure to repress fio1(+) gene expression. Deletion analysis identified two heptad repeats (amino acids 522-536) within the C-terminal region of Fep1 that are necessary and sufficient to mediate Fep1 dimerization. Moreover, mutations that impair dimerization also negatively affect transcriptional repression. Together these findings reveal several novel features of Fep1, a non-canonical GATA factor required for iron homeostasis.

404. Roles of Inverted Terminal Repeats (ITRs) and Capsid Proteins from Novel NHP AAVs in rAAV Mediated Gene Transfer

Recombinant AAV2 genomes transcapsidated with the capsid from a number of novel primate AAVs including AAV8 and rh.39 have been proven to be more robust gene transfer vehicles than the original AAV2 vector. We have recently isolated and characterized infectious molecular clones of AAV8 and AAVrh.39 and created recombinant AAV vector genomes carrying ITRs from AAV8 and AAVrh.39. Our study on the cleavability of those ITRs by AAV2 Rep in an in vitro nicking assay suggested the possibility of packaging those novel vector genomes with chimeric packaging plasmids consisting of the rep gene from AAV2 and cap genes from other AAVs.

Development of a genetic transformation system for benzene-tolerant Rhodococcus opacus strains

Rhodococcus opacus B-4 and B-9 are tolerant to various organic solvents including benzene, toluene, ethylbenzene, xylenes and styrene, and are suitable bacterial hosts for the production of chemical products from hydrophobic substrates. A 4.4-kb endogenous plasmid (pKNR 01) was isolated from R. opacus B-4 and sequenced completely. Plasmid pKNR 01 encodes proteins that share similarity to replication proteins from the enteric bacterial and actinomycete theta-replication plasmids. A 7.4-kb chimeric plasmid, designated pKNR 01.1, was constructed by fusing XhoI-digested pKNR 01 and Escherichia coli vector pSTV 28. Plasmid pKNR 01.1 had the ability to replicate in B-4 and B-9. A protocol for transformation of B-9 by electroporation was optimized employing pKNR 01.1. Frequencies of 4.1 x 10(5) transformants per mug of plasmid DNA were obtained for B-9 cells, whereas B-4 harboring naturally occurring pKNR 01 was transformed at lower frequencies (approximately 1 x 10(4) transformants per mug of plasmid DNA). Deletion analysis of pKNR 01.1 showed that the 1.9-kb SphI-XhoI region containing the repA and rep B genes and the 0.6-kb region upstream of repA was essential for plasmid maintenance in R. opacus strains.

Molecular Cloning of Mouse Type 2 and Type 3 Inositol 1,4,5-Trisphosphate Receptors and Identification of a Novel Type 2 Receptor Splice Variant

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R2 and IP(3)R3, respectively) from mouse lung and found a novel alternative splicing segment, SI(m2), at 176-208 of IP(3)R2. The long form (IP(3)R2 SI(m2)(+)) was dominant, but the short form (IP(3)R2 SI(m2)(-)) was detected in all tissues examined. IP(3)R2 SI(m2)(-) has neither IP(3) binding activity nor Ca(2+) releasing activity. In addition to its reticular distribution, IP(3)R2 SI(m2)(+) is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca(2+) ionophore stimulation, most of the IP(3)R2 SI(m2)(+) is in clusters. IP(3)R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca(2+) ionophore stimulation. IP(3)R2 SI(m2)(-) does not form clusters in either resting or stimulated cells. IP(3) binding-deficient site-directed mutants of IP(3)R2 SI(m2)(+) and IP(3)R3 fail to form clusters, indicating that IP(3) binding is involved in the cluster formation by these isoforms. Coexpression of IP(3)R2 SI(m2)(-) prevents stimulus-induced IP(3)R clustering, suggesting that IP(3)R2 SI(m2)(-) functions as a negative coordinator of stimulus-induced IP(3)R clustering. Expression of IP(3)R2 SI(m2)(-) in CHO-K1 cells significantly reduced ATP-induced Ca(2+) entry, but not Ca(2+) release, suggesting that the novel splice variant of IP(3)R2 specifically influences the dynamics of the sustained phase of Ca(2+) signals.

Secretion of noninfectious dengue virus-like particles and identification of amino acids in the stem region involved in intracellular retention of envelope protein

DNA plasmids that express flavivirus premembrane/membrane (prM/M) and envelope (E) proteins in the form of virus-like particles (VLPs) have an excellent potential as DNA vaccine candidates against virus infection. The plasmid-expressed VLPs are also useful as safe, noninfectious antigens in serodiagnostic assays. We have constructed plasmids containing the prM/M and E gene regions for DENV-1, -3, and -4 that express and secrete VLPs when electroporated into Chinese hamster ovary cells. Constructs containing the full-length DENV-1 E protein gene did not secrete VLPs into tissue culture fluid effectively. However, a 16-fold increase in ELISA titers of DENV-1 VLPs was achieved after replacing the carboxy-terminal 20% region of DENV-1 E protein gene with the corresponding sequence of Japanese encephalitis virus (JEV). DENV-3 plasmids containing either the full-length DENV-3 E protein gene or the 20% JEV sequence replacement secreted VLPs to similarly high levels. Whereas DENV-4 VLPs were secreted to high levels by plasmids containing the full-length DENV-4 E protein gene but not by the chimeric plasmid containing 20% JEV E replacement. Domain substitutions by replacing prM/M protein stem-anchor region with the corresponding prM/M stem-anchor region of JEV or DENV-2 in the chimeric DENV-4 construct failed to promote the secretion of DENV-4 VLPs. Using the DENV-2 chimeric plasmid with carboxy-terminal 10% of JEV E gene, the sequence responsible for intracellular localization of E protein was mapped onto the E-H1 α-helix domain of DENV-2 E protein. Substitution of three amino acids from the DENV-2 sequence to the corresponding amino acids in the JEV sequence (I398L, M401A, and M412L) in the E-H1 was sufficient to promote extracellular secretion and resulted in detectable titers of DENV-2 VLP secretion.

Hormones and Nutrients Regulate Acetyl-CoA Carboxylase Promoter I in Rat Primary Hepatocytes

This study investigated the regulation of acetyl-CoA carboxylase (ACC) promoter activity by hormones and nutrients. Genomic clones including promoter I (PI) of the ACC gene were isolated and sequenced. ACC PI fragments (-1,049/+100 or -220/+21 bp) were subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. The ACC PI/luciferase chimeric plasmids were transfected into primary rat hepatocytes using lipofectin. Insulin treatment increased the activity of -1,049/+ 100 and -220/+21 ACC PI by 3.0- and 3.5-fold, respectively, compared to the control. The activity of both constructs was also increased by dexamethasone (Dex) and triiodothyronine (T3), with the greatest effects seen with all three hormones present. With -1,049/+100 or -220/+21 ACC PI, the addition of glucose increased luciferase activity compared to glucose-free control (p<0.05). On the other hand, polyunsaturated fatty acids (PUFA) reduced the activity of the -1,049/+100 ACC PI construct, with eicosapentaenoic acid and docosahexaenoic acid showing the greatest effect (about 70% of the control). However, the addition of PUFA to the culture media did not affect the activity of -220/+21 ACC PI. Therefore, insulin, Dex, T3, glucose, and PUFA regulate ACC gene expression, at least in part, through the PI promoter.

Sulfur‐substituted and α‐methylated fatty acids as peroxisome proliferator‐activated receptor activators

FA with varying chain lengths and an alpha-methyl group and/or a sulfur in the beta-position were tested as peroxisome proliferator-activated receptor (PPAR)alpha, -delta(beta), and -gamma ligands by transient transfection in COS-1 cells using chimeric receptor expression plasmids, containing cDNAs encoding the ligand-binding domain of PPARalpha, -delta, and -gamma. For PPARalpha, an increasing activation was found with increasing chain length of the sulfur-substituted FA up to C14-S acetic acid (tetradecylthioacetic acid = TTA). The derivatives were poor, and nonsignificant, activators of PPARdelta. For PPARgamma, activation increased with increasing chain length up to C16-S acetic acid. A methyl group was introduced in the alpha-position of palmitic acid, TTA, EPA, DHA, cis9,trans11 CLA, and trans10,cis12 CLA. An increased activation of PPARalpha was obtained for the alpha-methyl derivatives compared with the unmethylated FA. This increase also resulted in increased expression of the two PPARalpha target genes acyl-CoA oxidase and liver FA-binding protein for alpha-methyl TTA, alpha-methyl EPA, and alpha-methyl DHA. Decreased or altered metabolism of these derivatives in the cells cannot be excluded. In conclusion, saturated FA with sulfur in the beta-position and increasing carbon chain length from C9-S acetic acid to C14-S acetic acid have increasing effects as activators of PPARalpha and -gamma in transfection assays. Furthermore, alpha-methyl FA derivatives of a saturated natural FA (palmitic acid), a sulfur-substituted FA (TTA), and PUFA (EPA, DHA, c9,t11 CLA, and t10,c12 CLA) are stronger PPARalpha activators than the unmethylated compounds.

Construction and expression of the eukaryotic expression vector containing human soluble transforming growth factor beta receptor II

To construct a eukaryotic expression vector encoding the gene of extracellular region of type II transforming growth factor beta receptor (sTGFbetaR II), to express the protein in CHO cell line and to determine its biological activity. The extracellular region (amino acids 1-159) of the human TGFbetaR II cDNA was amplified by PCR from a TGFbetaR II chimeric plasmid,and the eukaryotic expression plasmid pCDNA3.1/myc-his(-)B-sTGFbetaR II(pCDNA-sTGFbetaR II) was constructed by inserting the sTGFbetaR II cDNA into the EcoR I/Hind III-digested pCDNA. The DNA sequence was confirmed by double digestion and the pCDNA-sTGFbetaR II plasmid was transfected into the CHO cell line. The sTGFbetaR II protein was confirmed by Western blotting analysis and its biological function was determined. The specific protein was observed in western blotting, and the protein abrogated the growth-inhibitory effects of TGF-beta1 on mink lung epithelial cells (Mv1Lu). The eukaryotic expression plasmid pCDNA-sTGFbetaR II has been successfully constructed and the sTGFsTGFbetaR II protein with biological activity obtained.

Human adipocyte fatty acid-binding protein (aP2) gene promoter-driven reporter assay discriminates nonlipogenic peroxisome proliferator-activated receptor gamma ligands.

Peroxisome proliferator-activated receptors (PPARs) regulate storage and catabolism of fats and carbohydrates. PPARgamma activity increases insulin sensitivity and adipocyte differentiation at the expense of adipogenesis and weight gain. The goal of this study was to 1) clone the promoter of the human adipocyte fatty acid binding protein (aP2) gene, namely fatty acid-binding protein-4, 2) characterize its pharmacological regulation, and 3) determine its putative predictability for adipogenesis. Among the selected PPAR agonists, rosiglitazone and pioglitazone displayed the highest maximal efficacy (E(max)) on reporter-gene assays in COS-7 cells cotransfected by either a galactosidase 4-response element-based or a human aP2 promoter-based Luc reporter vector, along with either chimeric or full-length human PPAR expression plasmids. The non-subtype-selective 2-(4-[2-(3-[2,4-difluorophenyl]-1-heptylureido)ethyl]phenoxy)-2-methyl-butyric acid (GW-2331) and the compounds [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propoxyl]phenoxy]-acetic acid (L-165041), (4-((2S,5S)-5-(2-(bis(phenylmethyl)amino)-2-oxoethyl)-2-heptyl-4-oxo-3-thiazolidinyl)butyl)-benzoic acid (GW-0072), and indomethacin behaved as partial agonists relative to pioglitazone in full-length human aP2-PPARgamma2. Beyond their partial PPARgamma agonist properties, these compounds elicited a lower maximal up-regulation of mouse aP2 mRNA in 3T3-L1 adipocytes as compared with pioglitazone; these properties paralleled a time-dependent increase in neutral lipids. By contrast, the selective PPARalpha agonist 2,2-dichloro-12-(4-chlorophenyl)dodecanoic acid (BM-17.0744) neither stimulated the human aP2-PPARalpha promoter reporter-gene assay, thus demonstrating a specific interaction between PPARgamma and the aP2 promoter, nor affected lipogenesis in 3T3-L1 cells. Altogether, these data characterized a functional promoter of the human aP2 gene; its in vitro pharmacological regulation in PPARgamma-mediated reporter-gene assay may represent an interesting complement or an alternative to time-consuming procedures aiming at discriminating PPAR ligands with low lipogenic properties.

Construction of exogenous multiple epitopes of helper T lymphocytes and DNA immunization of its chimeric plasmid with HBV pre-S2/S gene.

To design and construct an exogenous multiple epitope of helper T lymphocytes (HTL), and to evaluate its effect on anti-HBs response through DNA immunization. Artificial HTL epitope, PADRE and four other HTL epitopes from different proteins were linked together using splicing by overlap extension to generate exogenous multiple epitopes of HTL, MTE5. pcMTE5 and pcHB were generated by cloning MTE5 and fragments of HBV pre-S2/S gene into mammalian expression plasmid pcDNA3. Four chimeric plasmids were constructed by cloning MTE5 into the region of pre-S2 gene (Bam HI), 5' terminal of S gene (HincII, Xba I) and 3' terminal of S gene (Acc I) of pcHB respectively. BALB/c mice were used in DNA immunization of the recombinant plasmids. Anti-HBs was detected using Abbott IMx AUSAB test kits. The sequences of MTE5 and the 6 constructs of recombinant plasmids were confirmed to be correct by DNA sequencing. The anti-HBs response of the co-inoculation of pcHB and pcMTE5 was much higher than that of the inoculation of pcHB only (136.7+/-69.1 mIU/mL vs 27.6+/-17.3 mIU/mL, P<0.01, t = -6.56). Among the 4 chimeric plasmids, only the plasmid in which MTE5 was inserted into the pre-S2 region had good anti-HBs response (57.54+/-7.68 mIU/mL), and had no significant difference compared with those of pcHB and the co-inoculation of pcHB and pcMTE5. Exogenous multiple epitopes of HTL had immune enhancement when they were co-inoculated with pre-S2/S gene or inoculated in the chimeric form at a proper site of pre-S2/S gene of HBV. It might suggest that it was possible to improve hepatitis B vaccine using exogenous multiple epitopes of HTL. The antibody responses were very low using DNA immunization in the study. Thus, the immune enhancement effect of exogenous multiple epitopes of HTL has to be confirmed and the effect on overcoming the drawback of the polymorphism of HLA II antigens should also be evaluated after these chimeric plasmids are expressed in mammalian cell lines.

Molecular cloning and expression of uricase gene from Arthrobacter globiformis in Escherichia coli and characterization of the gene product

Arthrobacter globiformis FERM BP-360 produces uricase (urate oxidase; EC 1.7.3.3) intracellularly. A genomic library of the bacterium, prepared in the plasmid vector pUC118, was screened with probes based on the amino acid sequence of the purified uricase. We found that a chimeric plasmid in the library, designated pUOD1, carries a 2.0-kb DNA insert from the Arthrobacter DNA that hybridizes with the probe. The DNA insert contains an ORF consisting of 302 amino acids with a calculated molecular mass of 33,858. The protein translated from the ORF displays the highest identity (67%) to uricase from a bacterium, Cellulomonas flavigena. X-ray fluorescence analysis showed that the Arthrobacter uricase contains copper ion. However, we found that the catalytic activity of uricase is inhibited by the excessive addition of copper ion. Although the production of A. globiformis uricase is induced by the addition of uric acid to the culture medium, Escherichia coli harboring pUOD1 produced 20-fold higher uricase than the original Arthrobacter strain, even without an inducer.