scholarly journals Efficient and flexible strategies for gene cloning and vector construction using overlap PCR

2017 ◽  
Author(s):  
Zhongtian Liu ◽  
Tingting Zhang ◽  
Kun Xu
Keyword(s):  
Gene Cloning ◽  
Rna Extraction ◽  
Plasmid Vector ◽  
Open Reading Frames ◽  
Expression Vectors ◽  
Functional Study ◽  
Vector Construction ◽  
Dna Elements ◽  
Pcr Method ◽  
Overlap Pcr

AbstractGene cloning and vector construction are basic technologies in modern molecular biology for gene functional study. Here, we present flexible and efficient strategies for gene cloning and vector construction using overlap PCR. We firstly cloned the open reading frames (ORFs) of the porcine MSTN, chicken OVA and human α-glucosidase genes by overlap PCR-based assembling of their exons, which could be amplified with genomic DNAs as the templates without RNA extraction and RT-PCR reaction. Secondly, we generated additionally three designed functional cassettes by overlap PCR-based assembling of different DNA elements, which facilitated the construction their expression vectors greatly. Moreover, we further developed an interesting overlap-circled PCR method for fast plasmid vector construction without any cutting and ligating procedure. These advanced applications of overlap PCR provide useful alternative tools for gene cloning and vector construction.

A Porcine Reproductive and Respiratory Syndrome Virus Vaccine Candidate Based on PRRSV Glycoprotein 5 and the Toll-Like Receptor 5 Agonist Salmonella typhimurium Flagellin

2015 ◽  
Vol 25 (1) ◽  
pp. 56-59 ◽  
Author(s):  
Hongqin Song ◽  
Dan Xiong ◽  
Jing Wang ◽  
Xianyue Zhai ◽  
Guangcheng Liang
Keyword(s):  
Salmonella Typhimurium ◽  
Neutralizing Antibodies ◽  
Expression Vectors ◽  
Respiratory Syndrome Virus ◽  
Toll Like Receptor ◽  
Pcr Method ◽  
Overlap Pcr ◽  
Toll Like Receptor 5 ◽  
Cellular Immune ◽  
Glycoprotein 5

Glycoprotein 5 (GP5) from porcine reproductive and respiratory syndrome virus (PRRSV) is a key inducer of neutralizing antibodies. A truncated GP5 gene lacking the signal peptide and transmembrane sequences was amplified via an overlap PCR method and inserted into prokaryotic expression vectors, pET32a or pGEX-6p-1, to add an His or GST tag, respectively. His-tagged GP5 was induced with IPTG, verified by SDS-PAGE and Western blotting, and purified to serve as an immunogen accompanied with the <i>Salmonella typhimurium</i> flagellin (FliC), a Toll-like receptor 5 (TLR5) agonist. Levels of TLR5 and cytokine mRNAs in spleens of mice following injection with FliC were detected by qRT-PCR to verify the activation of innate immunity. FliC was used as an adjuvant and administered with the GP5 to C57BL/6 mice via intraperitoneal injection. Coadministration of GP5 with FliC induced a significantly enhanced GP5-specific IgG and IFN-&#947; response compared with administration of GP5 alone, and the GP5-specific titer in the GP5 + FliC coadministration group was elevated almost twofold after the third immunization. These results indicate that FliC is an effective adjuvant, increasing the induction of antibodies against GP5 with the induction of both humoral and cellular immune responses.

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

scholarly journals Characterization of the IS200/IS605 Insertion Sequence Family in Halanaerobium Hydrogeniformans

Genes ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 484
Author(s):  
Michael Sadler ◽  
Melanie R. Mormile ◽  
Ronald L. Frank
Keyword(s):  
Insertion Sequence ◽  
Open Reading Frames ◽  
Insertion Sequences ◽  
Genome Plasticity ◽  
Mobile Dna ◽  
Dna Elements ◽  
Left And Right ◽  
Evolutionary Role ◽  
Reading Frames

Mobile DNA elements play a significant evolutionary role by promoting genome plasticity. Insertion sequences are the smallest prokaryotic transposable elements. They are highly diverse elements, and the ability to accurately identify, annotate, and infer the full genomic impact of insertion sequences is lacking. Halanaerobium hydrogeniformans is a haloalkaliphilic bacterium with an abnormally high number of insertion sequences. One family, IS200/IS605, showed several interesting features distinct from other elements in this genome. Twenty-three loci harbor elements of this family in varying stages of decay, from nearly intact to an ends-only sequence. The loci were characterized with respect to two divergent open reading frames (ORF), tnpA and tnpB, and left and right ends of the elements. The tnpB ORF contains two nearly identical insert sequences that suggest recombination between tnpB ORF is occurring. From these results, insertion sequence activity can be inferred, including transposition capability and element interaction.

scholarly journals High-throughput subcellular protein localization using cell arrays

2005 ◽  
Vol 33 (6) ◽  
pp. 1407-1408 ◽  
Author(s):  
Y.-H. Hu ◽  
D. Vanhecke ◽  
H. Lehrach ◽  
M. Janitz
Keyword(s):  
High Throughput ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Open Reading Frames ◽  
Expression Vectors ◽  
Sequence Information ◽  
Cell Array ◽  
Single Experiment ◽  
Cell Arrays

Accomplishment of the human and mouse genome projects resulted in accumulation of extensive gene sequence information. However, the information about the biological functions of the identified genes remains a bottleneck of the post-genomic era. Hence, assays providing simple functional information, such as localization of the protein within the cell, can be very helpful in the elucidation of its function. Transfected cell arrays offer a robust platform for protein localization studies. Open reading frames of unknown genes can be linked to a His6-tag or GFP (green fluorescent protein) reporter in expression vectors and subsequently transfected using the cell array. Cellular localization of the transfected proteins is detected either by specific anti-His-tag antibodies or directly by fluorescence of the GFP fusion protein and by counterstaining with organelle-specific dyes. The high throughput of the method in terms of information provided for every single experiment makes this approach superior to classical immunohistological methods for protein localization.

scholarly journals Molecular Characterization of a Phage-Encoded Resistance System in Lactococcus lactis

1999 ◽  
Vol 65 (5) ◽  
pp. 1891-1899 ◽  
Author(s):  
Stephen McGrath ◽  
Jos F. M. L. Seegers ◽  
Gerald F. Fitzgerald ◽  
Douwe van Sinderen
Keyword(s):  
Dna Replication ◽  
Topoisomerase I ◽  
Plasmid Vector ◽  
Dna Interaction ◽  
Open Reading Frames ◽  
Phage Resistance ◽  
High Copy Number Plasmid ◽  
Specific Fragment ◽  
Reading Frames

ABSTRACT A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.

scholarly journals Simultaneous cloning of open reading frames into several different expression vectors

BioTechniques ◽  
2003 ◽  
Vol 35 (3) ◽  
pp. 520-526 ◽  
Author(s):  
Clement A. Stanyon ◽  
Thawornchai Limjindaporn ◽  
Russell L. Finley
Keyword(s):  
Open Reading Frames ◽  
Expression Vectors ◽  
Reading Frames

scholarly journals Conjugative Plasmid from Lactobacillus gasseri LA39 That Carries Genes for Production of and Immunity to the Circular Bacteriocin Gassericin A

2009 ◽  
Vol 75 (19) ◽  
pp. 6340-6351 ◽  
Author(s):  
Yoshiyuki Ito ◽  
Yasushi Kawai ◽  
Kensuke Arakawa ◽  
Yoshiko Honme ◽  
Takashi Sasaki ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Lactobacillus Reuteri ◽  
Plasmid Vector ◽  
Open Reading Frames ◽  
Conjugative Plasmid ◽  
Lactobacillus Gasseri ◽  
Plasmid Maintenance ◽  
Circular Bacteriocin ◽  
Derivative Plasmid ◽  
Host Killing

ABSTRACT Gassericin A is a circular bacteriocin produced by Lactobacillus gasseri strain LA39. We found a 33,333-bp plasmid, designated pLgLA39, in this strain. pLgLA39 contained 44 open reading frames, including seven genes related to gassericin A production/immunity (gaa), as well as genes for replication, plasmid maintenance, and conjugative transfer. pLgLA39 was transferred from LA39 to the type strain of L. gasseri (JCM 1131) by filter mating. The transconjugant exhibited >30-fold-higher more resistance to gassericin A and produced antibacterial activity. Lactobacillus reuteri LA6, the producer of reutericin 6, was proved to harbor a plasmid indistinguishable from pLgLA39 and carrying seven genes 100% identical to gaa. This suggests that pLgLA39 might have been transferred naturally between L. gasseri LA39 and L. reuteri LA6. The seven gaa genes of pLgLA39 were cloned into a plasmid vector to construct pGAA. JCM 1131T transformed with pGAA expressed antibacterial activity and resistance to gassericin A. pGAA was segregationally more stable than a pGAA derivative plasmid from which gaaA was deleted and even was more stable than the vector. This suggests the occurrence of postsegregational host killing by the gaa genes. pLgLA39 carried a pemIK homolog, and segregational stabilization of a plasmid by the pLgLA39-type pemIK genes was also confirmed. Thus, pLgLA39 was proved to carry the genes for at least two plasmid maintenance mechanisms, i.e., gaa and pemIK. Plasmids containing a repA gene similar to pLgLA39 repA were distributed in several L. gasseri strains.

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