ABSTRACT
AFLP analysis was carried out with 14 Pleurotus ostreatus strains from different areas. Optimal conditions of the AFLP fingerprinting analysis for P. ostreatus were first tested and the results showed that the primer pairs E-3/M-3 especially E-AGC/M-CAT and E-AGC/M-ACC could give more amplified DNA fragments than others like E-2/M-1 or E-2/M-3. From the fingerprinting map of the primer pair E-AGC/M-CAT, 184 clear and stable DNA bands were observed, including 101 polymorphous bands that are accounted for 54.89%. The genetic similarity coefficient and genetic distance were calculated from AFLP data among 14 P. ostreatus strains. The genetic distance between these strains ranged from 0.192 to 0.754, indicating that P. ostreatus was rich in genetic diversity. UPGMA cluster analysis was also performed. It's shown that 14 P. ostreatus strains are divided into six groups, and strains from same areas or with similar best-growth temperature usually have higher comparability in the UPGMA tree. P. ostreatus P17 and P. ostreatus Za3, P. ostreatus Min31 and P. ostreatus Yiping have intimate genetic relationships with each other respectively which were consistent with its geographical distribution and best growth temperature. P. ostreatus Ce5 showed remarkable genetic differentiation, which has farther genetic relationships compared with other P. ostreatus strains. In addition, the reasons for cluster results of strains from AFLP data consistent with morphology, geographical distribution and optimal condition of AFLP fingerprinting analysis for P. ostreatus were discussed.
Subject(s)
DNA Fingerprinting/methods , Pleurotus/genetics , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment LengthABSTRACT
An inhibitor of the apoptosis protein (IAP) family gene from Trichoplusia ni, Tn-IAP1v, a variant of lepidopteran Tn-IAP1, was cloned by RT-PCR. There are six single nucleotide polymorphisms between the two Tn-IAP1 variants, resulting in three predicted single amino acid polymorphisms. With the GST fusion expression system, soluble recombinant Tn-IAP1v was highly expressed in Escherichia coli and then purified by affinity chromatography. Caspase inhibition assays indicated that recombinant Tn-IAP1v could specifically inhibit human caspase-9 in vitro instead of caspase-3, -7, and -8, which was further confirmed by the observation that recombinant Tn-IAP1v can directly bind caspase-9 in the protein pull-down assay. These results suggested that Tn-IAP1v might serve as an initiator caspase inhibitor in vivo in the conserved mitochondria apoptotic pathway.
Subject(s)
Cysteine Proteinase Inhibitors/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Insect Proteins/metabolism , Insect Proteins/pharmacology , Lepidoptera , Proteins , Animals , Caspase 9 , Caspases/metabolism , Cell Line , Cloning, Molecular , Cysteine Proteinase Inhibitors/genetics , Cytochrome c Group/antagonists & inhibitors , Deoxyadenine Nucleotides/antagonists & inhibitors , Humans , Inhibitor of Apoptosis Proteins , Insect Proteins/genetics , Polymorphism, Single Nucleotide , Ubiquitin-Protein LigasesABSTRACT
In order to reduce the human anti-murine antibody (HAMA) in radioimmunotherapy, the gene encoding the heavy chain variable region (V(H)) of the murine monoclonal antibody with high specificity and affinity to carcinoembryonic antigen was fused to the human C(gamma3) gene to construct the murine-human chimeric heavy chain antibody gene, then was linked to core-streptavidin which can specifically bind to biotin, facilitating its purification and radioisotope labeling. The fusion gene was expressed in E.coli at high level, accounting for 24% of the total bacteria protein, and was characterized by SDS-PAGE and Western-blots. When using RIA the content of inclusion bodies was denatured and followed by renaturation, it was shown by using RIA to possess ability to bind to its specific antigen of CEA. Using horseradish peroxidase (HRP) labeled biotin as antibody in Western-blots, one band of 70 kD only was detected, demonstrating the fusion protein not only had the ability to bind CEA, but also could bind biotin specifically.
