Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

1991 ◽  
Vol 229 (2) ◽  
pp. 301-306 ◽  
Author(s):  
Setsuzo Tada ◽  
Katsuya Gomi ◽  
Katsuhiko Kitamoto ◽  
Kojiro Takahashi ◽  
Gakuzo Tamura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Aspergillus Oryzae ◽  
Fusion Gene

Immunogenicity Evaluation of Chimeric Subunit Vaccine Comprising Adhesion Coli Surface Antigens from Enterotoxigenic Escherichia coli

2019 ◽  
Vol 29 (1-6) ◽  
pp. 91-100
Author(s):  
Dorna Khoobbakht ◽  
Shohreh Zare Karizi ◽  
Mohammad Javad  Motamedi ◽  
Rouhollah Kazemi ◽  
Pooneh Roghanian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Subunit Vaccine ◽  
Fusion Gene ◽  
Intestinal Epithelial Cells ◽  
High Titer ◽  
Chimeric Protein ◽  
Amino Acid Sequences ◽  
Intestinal Epithelial ◽  
E Coli

Enterotoxigenic <i>Escherichia coli</i> (ETEC) is the most common agent of diarrhea morbidity in developing countries. ETEC adheres to host intestinal epithelial cells via various colonization factors. The CooD and CotD proteins play a significant role in bacteria binding to the intestinal epithelial cells as adhesin tip subunits of CS1 and CS2 pili. The purpose here was to design a new construction containing <i>cooD</i> and <i>cotD</i> genes and use several types of bioinformatics software to predict the structural and immunological properties of the designed antigen. The fusion gene was synthesized with codon bias of <i>E. coli</i> in order to increase the expression level of the protein. The amino acid sequences, protein structure, and immunogenicity properties of potential antigens were analyzed in silico. The chimeric protein was expressed in <i>E. coli</i>BL21 (DE3). The antigenicity of the recombinant proteins was verified by Western blotting and ELISA. In order to assess the induced immunity, the immunized mice were challenged with wild-type ETEC by an intraperitoneal route. Immunological analyses showed the production of a high titer of IgG serum with no sign of serum-mucosal IgA antibody response. The result of the challenge assay showed that 30% of immunized mice survived. The results of this study showed that CooD-CotD recombinant protein can stimulate immunity against ETEC. The designed chimera could be a prototype for the subunit vaccine, which is worthy of further consideration.

scholarly journals Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans

2019 ◽  
Vol 21 (1) ◽  
pp. 297 ◽  
Author(s):  
Triinu Visnapuu ◽  
Aivar Meldre ◽  
Kristina Põšnograjeva ◽  
Katrin Viigand ◽  
Karin Ernits ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Aspergillus Oryzae ◽  
Hydrolysis Product ◽  
Hydrolytic Activity ◽  
Glycoside Hydrolases ◽  
Arxula Adeninivorans ◽  
Sequence Identity ◽  
Diabetes Drug

Genome of an early-diverged yeast Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two α-glucosidases of GH13 family. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. We showed that maltose, other maltose-like substrates (maltulose, turanose, maltotriose, melezitose, malto-oligosaccharides of DP 4‒7) and sucrose were hydrolyzed by BaAG2, whereas isomaltose and isomaltose-like substrates (palatinose, α-methylglucoside) were not, confirming that BaAG2 is a maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 µM) and Tris (Ki = 70.5 µM). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—glucose. At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Saccharomyces cerevisiae maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to BaAG2. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae.

Fusion Expression on the Esat-6 and cfp-10 Genes of Mycobacterium bovis in Escherichia coli

2013 ◽  
Vol 421 ◽  
pp. 354-358
Author(s):  
Jia Ming Lin ◽  
Chun Fang Wang ◽  
Jia Ning Guan ◽  
Hong Xia Ma ◽  
Shuang Hou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Bovis ◽  
Expression Vector ◽  
Fusion Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigenic Activity ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Single Antigen

Based on the (Gly4Ser)3 linker, theesat-6andcfp-10gene were fused for raising the antigenicity of single antigen. The DNA fragments ofesat-6andcfp-10were fused by splicing by overlapping extension (SOE) polymerase chain reaction (PCR),and the fusion gene esat-6-cfp-10 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-esat-6-cfp-10. pMD-esat-6-cfp-10 and pET28a (+) were digested byBamHI andEcoRI double enzymes. The purified mpb esat-6-cfp-10 fusion gene was subcloned into the expression vector pET28a (+),and the prokaryotic expression vector pET-esat-6-cfp-10 was constructed. Plasmid containing pET-esat-6-cfp-10 was transformed into competenceEscherichia coliBL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),approximately 25 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic activity ofMycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of DNA vaccine; living carrier vaccine; subunit vaccine and diagnostic reagents against bovine tuberculosis.

Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene

Gene ◽  
1988 ◽  
Vol 62 (1) ◽  
pp. 127-134 ◽  
Author(s):  
Margareta Kolar ◽  
Peter J. Punt ◽  
Cees A.M.J.J. van den Hondel ◽  
Helmut Schwab
Keyword(s):  
Escherichia Coli ◽  
Penicillium Chrysogenum ◽  
Fusion Gene ◽  
Lacz Fusion ◽  
Selection Markers

Expression of an Escherichia coli β-galactosidase fusion gene in Aspergillus nidulans

Gene ◽  
1985 ◽  
Vol 40 (1) ◽  
pp. 99-106 ◽  
Author(s):  
Robert F.M. van Gorcom ◽  
Peter H. Pouwels ◽  
Theo Goosen ◽  
Jaap Visser ◽  
Henk W.J. van den Broek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Aspergillus Nidulans ◽  
Fusion Gene
Sign in / Sign up

Export Citation Format

Share Document