Molecular Gene Cloning Research Articles

Application of Biotechnology towards Diagnosis and Treatment in Veterinary Medicine in Africa: Potentials and Future Developments

Biotechnology is an already established technique in several areas of medicine, but its application in the field of veterinary medicine has only started to emerge with the potential to revolutionize veterinary practice. This paper therefore reviews the potential applications of biotechnology in veterinary medicine towards diagnosis and treatment in Africa which includes; molecular gene cloning, production of recombinant biotechnology derived vaccines, application of polymerase chain reaction (PCR), Real time polymerase chain reaction (RT-PCR), Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and Bioinformatics to diagnosis of infectious and parasitic diseases , gene therapy, disease diagnosis, treatment delivery systems and many more. These components occur in other parts of the world and are therefore presumed to be consolidated into the African delivery framework as a private enterprise in a foreseeable future. Whilst it is sensible to postulate that biotechnology application and its peculiar evolution will imminently transform veterinary medicine, there is immense treat, amidst stakeholders in the industry, about food health and safety and other civil and ethical concerns which can hinder this novel scientific breakthrough. The ethical concerns which include; theory of the Three Rs (Reduction of animal population, Refinement of enactments and farm managements to curtail affliction and despair, Replacement of animals with non-animal surrogate wherever necessary. Limitations regarding the application to veterinary practices are extensively discussed. This review has implication on the future of revolutionalisation of veterinary practice and increase in animal protein source for human consumption

Molecular gene cloning and sequencing of glutamate decarboxylase gene from Lactobacillus delbrueckii and Lactobacillus reuteri

Glutamate decarboxylase enzyme produces γ-aminobutyric acid (GABA) in a non-reversible decarboxylation reaction of glutamate. GABA is a major inhibitory neurotransmitter of the brain and it is also present at high concentration in other organs such as pancreatic islets. GABA has effects on blood pressure, diabetes, inflammation, sleeplessness and depression. Some bacteria such as Lactobacillus strains are capable of GABA production. Identification of these bacteria is important both for researchers and industry. The aim of this study was molecular gene cloning and sequencing of glutamate decarboxylase ( gad ) from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and Lactobacillus reuteri ATCC 23272 . These strains were cultured in MRS medium at 37 ○ C for 24 hours. For cloning gad gene from these strains, polymerase chain reaction (PCR) was performed using specific primers designed by Oligo7 software. PCR production was extracted from agarose gel and was inserted into PGEM-T vector using T4 DNA ligase enzyme and then it was transformed to E. coli XL1Blue . In the final step, white colonies were selected and after plasmid extraction, the existence of gad gene in recombinants was confirmed by PCR. Gad gene was cloned from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and Lactobacillus reuteri ATCC 23272 . It is for the first time that the gad gene sequences from these bacteria were registered on NCBI with accession numbers KF751355 and KF751352 respectively. The result of this research indicates that the two aforementioned bacteria contain glutamate decarboxylase gene and therefore they possibly can be used for industrial γ-aminobutyric acid production.

Heterologous expression of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Triticum aestivum and Arabidopsis thaliana

Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) plays a key metabolic role in higher plants. Purification to homogeneity of enzymes found in relatively low abundance in plants represents a major technical challenge that can be solved by molecular gene cloning and heterologous expression. To apply this strategy to np-Ga3PDHase we performed the cloning of the gapN gene from Arabidopsis thaliana and Triticum aestivum, followed by the heterologous expression in Escherichia coli by two different strategies. Soluble expression of the Arabidopsis enzyme in the pET32c+ vector required a chaperone co-expression system (pGro7). The system using E. coli BL21-CodonPlus ® cells and the pRSETB vector was successful for expression of a soluble His 6-taged recombinant wheat enzyme producing 2.5 mg of electrophoretically pure protein per liter of cell culture after a single chromatographic purification step. Both systems were effective for the expression of functional plant np-Ga3PDHases, however the expression of the Arabidopsis enzyme in pRSETB was affordable but not as optimal as for the wheat protein. This would be associated with a different codon usage preference between this specific plant and E. coli. Considering the relevant role played by np-Ga3PDHase in plant metabolism, it is experimentally valuable the development of a procedure to obtain adequate amounts of highly purified enzyme, which envisages the viability to perform studies of structure-to-function relationships to better understand the enzyme kinetics and regulation, as well as carbon and energy metabolism in higher plants.

Molecular gene cloning and overexpression of the phytase from Debaryomyces castellii CBS 2923

The ORF encoding the Debaryomyces castellii CBS 2923 phytase was isolated. The deduced 461-amino-acid sequence corresponded to a 51.2kDa protein and contained the consensus motif (RHGXRXP) which is conserved among phytases. No signal sequence cleavage site was detected. Nine potential N-glycosylation sites have been predicted. The protein shared 21–69% sequence identities with various phytases of yeast or fungal origin. Heterologous expression of the D. castellii CBS 2923 phytase in the methylotrophic yeast Pichia pastoris was tested under both the P. pastoris inducible alcohol oxidase (AOX1) promoter and the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Maximum production levels obtained were 476Uml−1, with the AOX1 expression system and 16.5Uml−1 with the GAP one. These productions corresponded to a 320-fold and a 10-fold overexpression of the protein, respectively as compared to the homologous production. The biochemical characteristics of the recombinant phytase were identical to those of the native enzyme.

Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds

A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans) but did not exhibit antibacterial action against Escherichia coli. Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH(2)-terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins.

Molecular gene cloning, expression, and characterization of bovine brain glutamate dehydrogenase.

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The Km and Vmax values for NAD+ were 0.1 mM and 1.08 micromol/min/mg, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - 100 microM, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.

Knockout mice highlight the promise of purines

ATP is now recognized as an important signalling molecule that mediates its biological effects via cell surface receptors known as P2 receptors that are both ionotropic (P2X) and metabotropic (P2Y). Seven P2X receptor subunits have been characterized by molecular gene cloning, most of which are located in nervous tissues throughout the body, suggesting a fundamental role for ATP and its receptors in neuronal signalling. However, experimental progress has been hampered by the lack of specific or selective drugs to distinguish between the P2X subtypes. In addition, the presence of different subtypes of receptors in cells can result in the formation of heteromers that display unique properties compared with the individual subunits; the rules governing the composition of these heteromers are still being determined....one P2X receptor subtype, the P2X3, has conclusively been shown to be essential for detecting some kinds of sensory stimuli.

Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis.

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55 degrees C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.

Nitrocellulose immunoblotting for identification and molecular gene cloning of Eimeria maxima antigens that stimulate lymphocyte proliferation.

An immunoblotting technique was used to identify lymphostimulatory antigens within sized polypeptide fractions of Eimeria maxima sporozoites. Six fractions contained polypeptides that specifically stimulated the proliferation of immune lymphocytes in an in vitro assay, and polyclonal antisera were made in rabbits against these fractions. cDNA clones, isolated with antisera against a lymphostimulatory fraction of around 70 kDa, were found to encode four different antigens including a classical hsp70, a molecule homologous to an endoplasmic reticulum chaperonin (BiP/GRP), and a calcium-dependent serine/threonine protein kinase that appears homologous to a recently described molecule from Plasmodium falciparum. The protein kinase cDNA clone was overexpressed in Escherichia coli, and the recombinant antigen was found to induce both antibody and lymphoproliferative responses in chickens when administered subcutaneously. Thus, immunoblotting, in combination with in vitro lymphoproliferation assays, can be used as an initial screen for the identification of lymphostimulatory antigens from a complex pool of polypeptides, and a combination of cDNA cloning, expression, and immunization allows assessment of the lymphostimulatory activity of individual polypeptides. These studies should facilitate further evaluation of antigens that are potential candidates for inclusion in a recombinant vaccine against poultry coccidiosis.

Molecular gene cloning and nucleotide sequencing and construction of an aroA mutant of Pasteurella haemolytica serotype A1

The aroA gene of Pasteurella haemolytica serotype A1 was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. The nucleotide sequence of a 2.2-kb fragment encoding aroA predicted an open reading frame product 434 amino acids long that shows homology to other bacterial AroA proteins. Several strategies to inactivate aroA were unsuccessful. Gene replacement was finally achieved by constructing a replacement plasmid with aroA inactivated by insertion of a P. haemolytica ampicillin resistance fragment into a unique NdeI site in aroA. A hybrid plasmid was constructed by joining the aroA replacement plasmid with a 4.2-kb P. haemolytica plasmid which encodes streptomycin resistance. Following PhaI methylation, the replacement plasmid was introduced by electroporation into P. haemolytica NADC-D60, a plasmidless strain of serotype 1A. Allelic exchange between the replacement plasmid and the chromosome of P. haemolytica gave rise to an ampicillin-resistant mutant which grew on chemically defined P. haemolytica medium supplemented with aromatic amino acids but failed to grow on the same medium lacking tryptophan. Southern blot analysis confirmed that aroA of the mutant was inactivated and that the mutant was without a plasmid.