Abstract: Production of specific antibody responses against hTSH is a cumbersome process due to the high identity between the hTSH and the other members of the glycoprotein hormone family (FSH, LH and HCG) and the high identity between the human hTSH and host animals for antibody production. Therefore, two polyclonal antibodies were purified against two recombinant proteins. Four possible ELISA tests were designed based on these antibodies. These ELISA tests were checked against hTSH and other glycoprotein hormones, and their sensitivity and specificity were assessed. Bioinformatics tools were used to analyze the immunological properties. After the immunogen region selection from hTSH protein, c terminal of B hTSH was selected and applied. Two recombinant genes, with these cut pieces (first: two repeats of C terminal of B hTSH, second: tetanous toxin+B hTSH C terminal), were designed and sub-cloned into the pET32a expression vector. Standard methods were used for protein expression, purification, and verification. Thereafter, immunizations of the white New Zealand rabbits were performed and the serums of them were used for antibody titration, purification and characterization. Then, four ELISA tests based on two antibodies were employed to assess the hTSH and other glycoprotein hormones. The results of these assessments were compared with standard amounts. The obtained results indicated that the desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. The raised antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum. Among the four designed tests, the test in which the antibody against first protein was used as capture antibody, and the antibody against second protein was used as detector antibody did not show any hook effect up to 50 miu/l. Both proteins have the ability to induce highly sensitive and specific antibody responses against the hTSH. One of the antibody combinations of these antibodies has the highest sensitivity and specificity in hTSH detection.
Abstract: Aim of this work was to study the genetic basis for oil
accumulation in olive fruit via tracking DGAT2 (Diacylglycerol
acyltransferase type-2) gene in three Egyptian Origen Olive cultivars
namely Toffahi, Hamed and Maraki using molecular marker
techniques and bioinformatics tools. Results illustrate that, firstly:
specific genomic band of Maraki cultivars was identified as DGAT2
(Diacylglycerol acyltransferase type-2) and identical for this gene in
Olea europaea with 100% of similarity. Secondly, differential
genomic band of Maraki cultivars which produced from RAPD
fingerprinting technique reflected predicted distinguished sequence
which identified as DGAT2 (Diacylglycerol acyltransferase type-2)
in Fragaria vesca subsp. Vesca with 76% of sequential similarity.
Third and finally, specific genomic specific band of Hamed cultivars
was identified as two fragments, 1- Olea europaea cultivar Koroneiki
diacylglycerol acyltransferase type 2 mRNA, complete cds with two
matches regions with 99% or 2- Predicted: Fragaria vesca subsp.
vesca diacylglycerol O-acyltransferase 2-like (LOC101313050),
mRNA with 86 % of similarity.
Abstract: Annotation of a protein sequence is pivotal for the understanding of its function. Accuracy of manual annotation provided by curators is still questionable by having lesser evidence strength and yet a hard task and time consuming. A number of computational methods including tools have been developed to tackle this challenging task. However, they require high-cost hardware, are difficult to be setup by the bioscientists, or depend on time intensive and blind sequence similarity search like Basic Local Alignment Search Tool. This paper introduces a new method of assigning highly correlated Gene Ontology terms of annotated protein sequences to partially annotated or newly discovered protein sequences. This method is fully based on Gene Ontology data and annotations. Two problems had been identified to achieve this method. The first problem relates to splitting the single monolithic Gene Ontology RDF/XML file into a set of smaller files that can be easy to assess and process. Thus, these files can be enriched with protein sequences and Inferred from Electronic Annotation evidence associations. The second problem involves searching for a set of semantically similar Gene Ontology terms to a given query. The details of macro and micro problems involved and their solutions including objective of this study are described. This paper also describes the protein sequence annotation and the Gene Ontology. The methodology of this study and Gene Ontology based protein sequence annotation tool namely extended UTMGO is presented. Furthermore, its basic version which is a Gene Ontology browser that is based on semantic similarity search is also introduced.