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At least 37 records · Page 2Linked to original sources
PubMed1975

[Ultrastructure of Bac. anthracis and Bac. cereus].

A study was made of the ultrastructure of Bac. anthracis and Bac. cereus (strains 1835 and 205). There were established definite morphological differences between the bacilli under study. The cell wall was much thicker in the causative agent of anthrax, it was permeated by tubules; the surface of the wall was covered by a thick layer of osmiophilic material. The spore membrane of Bac. anthracis was multistratal in character; osmiophilic filaments were seen on the surface of exosporium. Fungoid processes from the cell wall were present in Bac. cereus (strain No. 205); intraplasmic membrane structures were considerably developed; a crystal located on the contralateral end of the cell was formed with the spore, both processes coinciding in time.

Bacillus anthracis↗
PubMed2003

[Establishment of Spodoptera exigua multicapsid nucleopolyhedrovirus BAC-TO-BAC expression system].

Present studies describe the successful establishment of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) BAC-TO-BAC expression system. The mini-F-lacZ-attTn7-kan fragment (Luckow et al, 1993) was inserted into SeMNPV US1 isolate (SeUS1) at polyhedrin gene locus by directly cloning. The recombinant virus containing low-copy-number mini-F replication, which named bacmid, can propagate in Escherichia coli. Because SeUS1 isolate is make up of several genotypes and one bacmid carries one SeMNPV genotype, the SeUS1 BAC library is established by all SeMNPV bacmids (SeBAC). REN analysis for 111 SeBAC shows that SeUS1 consists of the genotype with whole SeMNPV genetic information and several genotypes with various different deletions. Progeny virus can be produced in insect cell line after transfection with SeBAC10, which carries the whole SeMNPV genome. So SeBAC10 is a shuttle vector that can replicate in eukaryocyte as well as prokaryocyte. Considering the insert mutation of SeMNPV polyhedrin gene (Seph) in SeBAC10, Seph was reintroduced into the bacmid by site-specific transposon-mediated insertion at attTn7, the target site for the bacterial transposon Tn7. The derived recombinant SeBAC10 was named SeBAC10ph. After SeBAC10ph was transfected into Se301 cells (a susceptible insect cell line to SeMNPV), cytopathogenic effect was shown and polyhedra appeared, which indicate that the foreign gene (Seph) is expressed.

Animals↗
PubMed2006

Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system.

Superoxide dismutase (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and, thus, form a crucial part of the cellular antioxidant defense mechanism. In this paper, we used the total fat body RNA of silkworm, Bombyx mori L. to clone and sequence a 648-bp Mn-SOD cDNA fragment through RT-PCR. Furthermore, a newly established Bac-to-Bac/BmNPV Baculovirus expression system was used to overexpress the recombinant Mn-SOD enzyme in silkworm larvae. The hemolymph was collected from the infected larvae 96 h post-infection and subjected to a 12 % SDS-PAGE and Western blotting. A 18.0-kDa protein was visualized after rBacmid/BmNPV/SOD infection. The SOD enzyme activity was determined with a tetrazolium salt for detection of superoxide radicals generated by xanthine and xanthine oxidase and its peak appeared in 96 h post-infection with 2.7 times of the control larvae. The availability of large quantities of SOD that the silkworm provides should greatly facilitate the future research and testing of this protein for potential application in medicine.

Animals↗
PubMed2004

Cloning and expression of human UDP-glucuronosyltransferase 1A4 in Bac-to-Bac system.

UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from uridine diphosphate-glucuronic acid (UDP-GA) to compounds with amine, hydroxyl, and carboxylic acid moieties. N-glucuronidation is an important pathway for elimination of many tertiary amine therapeutic agents used in humans. UGT1A4 has been reported to be specific for glucuronidating primary, secondary, and tertiary amines, forming N-glucuronides. To further investigate the drugs metabolized by UGT1A4, the Bac-to-Bac expression system was used to express the recombinant UGT1A4 with His-tag on the C-terminal. The His-tagged recombinant UGT1A4 expressed in Spodoptera frugiperda (Sf9) cells were detected using anti-His antibody and the molecular weight of the recombinant protein was approximately 55kDa. The enzyme activity towards imipramine in cell homogenate protein was found to be 83.14+/-15pmol/min/mg protein (n=3) with 0.5mM imipramine by HPLC, but was not detectable in blank Sf9 cells. It paved the way for the further studies for drug glucuronidation by UGT1A4. The purification of the UGT1A4 can be done by Ni-resin. This is helpful to do research on the structure of the UFT1A4.

Animals↗
PubMed2004

Characterization of an Aedes aegypti bacterial artificial chromosome (BAC) library and chromosomal assignment of BAC clones for physical mapping quantitative trait loci that influence Plasmodium susceptibility.

Previous studies have confirmed a genetic basis for susceptibility of mosquitoes to Plasmodium parasites. Here we describe our efforts to characterize a bacterial artificial chromosome genomic library for the yellow fever mosquito, Aedes aegypti, and to identify BAC clones containing genetic markers that define quantitative trait loci (QTL) for Plasmodium gallinaceum susceptibility. This library (NDL) was prepared from the Ae. aegypti Liverpool strain and consists of 50 304 clones arrayed in 384-well microplates. We used PCR analysis with oligonucleotide primer pairs specific to 106 genetic markers (as sequence-tagged sites or STS) to screen the NDL library. Each STS identified between one and thirteen independent clones with an average of 3.3 clones. The average insert size was 122 kb and therefore the NDL library provides approximately 7.87-fold genome coverage. The availability of the NDL library should greatly facilitate physical mapping efforts, including positional cloning of QTL for traits of interest such as Plasmodium susceptibility and for whole genome sequence determination and assembly.

Aedes↗
PubMed2005

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system.

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.

Amino Acid Sequence↗
PubMed2004

To BAC or not to BAC: marine ecogenomics.

Most microbes in the ocean are still resistant to our collective cultivation efforts. Environmental microbial genomics provides science with the means for accessing and assessing the genomes, diversity, evolution and population dynamics of uncultured microorganisms--the ocean's hidden majority.

Chromosomes, Artificial, Bacterial↗
PubMedDate not supplied

Comparison of the biological activities of human immunodeficiency virus 1 P24 and GP41 expressed in Spodoptera frugiperda cells by use of bac-to-bac system.

Recombinant transposing plasmids pFH24 and pFH41 were constructed by cloning the human immunodeficiency virus 1 (HIV-1) p24 and gp41 genes, respectively, into the transposing vector pFastBacHTa. Recombinant bacmids rBH24 and rBH41 were obtained by transposing pPolh/p24 and pPolh/gp41 expression cassettes from recombinant plasmids pFH24 and pFH41, respectively. Recombinant viruses rAcH24 and rAcH41 were generated by transfection of the Spodoptera frugiperda (Sf9) cells with the DNAs of plasmids rBH24 and rBH41, respectively. Analysis of the expressed p24 or gp41 proteins with an antiserum to HIV-1 (HIV-1 antiserum) by an enzyme-linked immunosorbent assay (ELISA) and dot blot assay showed high biological activity of these proteins; p24 was more active than gp41. Also a Western blot analysis showed stronger bands for p24 than for gp41. The high reactivities of p24 and gp41 with the HIV-1 antiserum suggest that these proteins could also be used as specific standard antigens in HIV-1 diagnostics.

Animals↗
PubMed2001

Production of human papillomavirus type 16 early proteins in Bac-To-Bac Expression System (GibcoBRL).

Human papillomavirus type 16 (HPV16) is a major agent in cervical cancer etiology. Its early proteins are responsible for virus persistence, replication and initiation of neoplastic disease. In the present study we describe a use of baculovirus-insect cell expression system for production and study of HPV16 E2 and E4 proteins. The E2 protein binds specifically to viral DNA and E4 protein shows characteristic cytopathic effects on cells.

Animals↗
PubMed2000

[Construction of a novel Bm NPV Bac to Bac system].

A Bi-Shuttle vector Bm-Bacmid was constructed by co-infecting Bm N cells with wild type genomic DNA from BmNPV and Ac-Bacmid DNA. It could not only replicate in E. coli cells as a large plasmid and but also remain infectious when induced into Bm N or Sf9 cells. Recombinant virus rBmHBe was obtained after transposition of a donor plasmid carrying Hepatitis Be antigen gene (HBeAg) into att Tn7, and was demonstrated by Southern blotting. SDS-PAGE analysis showed that HBeAg gene were highly expressed in Bm N cells. By ELISA testing, the highest antigenecity titer of HBeAg protein in cell cultural medium was up to a dilution of 1:32,000. Although HBeAg protein also presented in the Bm N cells the titer was only 1:2000. The HBcAg protein was much fewer than HBeAg (< 1:160) whatever in culture medium and in cells. The results showed that Bm N cells was able to recognize the signal peptide sequence and cut it correctly for HBeAg protein's excreting production.

Animals↗