Expression Of Recombinant Phycoerythrin In E.coli And Its Bioactivity Studies; Cloning Of Cam From Alexandrium Catenalla And Its Expression Pattern

Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained widely concernings. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect bioactivity of gene-engineering expressed PE. Recombined plasmid pGEX-PE was expressed in E.coli BL21, and the expression products were validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E.coli cells were collected from large-scale cultures, lysed by sonication. The supernatants were reserved for the tumor cytotoxic and scavenging hydroxyl radical experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested of the sonicating and heating obtained expression products. For the sonicated products, the scavenging rates of the expression products supernatants of two plasmids containing PE gene, pBGL and pGEX-PE, were significantly higher than the negative control pGEX-4T (P<0.02), and as the protein content increasing, the scavenging rates took on a slow upward trend. For the heating sample, except for the 0.2mg/ml pBGL products, the scavenging effect of expression products of pBGL and pGEX-PE were stronger than that of pGEX-4T. However, the effect intensity was not positive correlated with the elevation of the protein concentration. Though a decreased partial hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This research showed that phycoerythrin protein expressed by E.coli has the potential medical and sanitarian value.Harmful algal blooms (HABs) or'red tides'have become one of main oceanic disasters. Alexandrium catenella is an important causative dinoflagellate of HABs.The study on its cell cycle and signal transduction can help elucidate its burstgrowing mechanism. Disclosing the molecular and genetic mechanism of bloom burst will give facilities for the forecast of red tide.Calcium signal transduction pathway plays important roles in many Eukaryotic cell processes, such as biotic or abiotic stress-response and intrinisic cell proliferation. Calmodulin (CaM) and CaM-related proteins are main celluar Ca2+ sensors, which can act as intermedium in the signal transduction pathway.In this paper, to elucidate the Ca2+ signal transduction pathway in Alexandrium catenella, calmodulin gene (cam) cDNA was isolated and analysed. The partial cam cDNA of 464bp was obtained, including 3'untranslated 106bp region with no detection of obvious poly(A) signal. 102 amino acids were translated from ORF of this cDNA sequence according to other species. Homology analysis of this partial protein sequences shows high conservativity, 98% similarity of other four dinoflagellate species, 98% of higher plants Arabidopsis thaliana,Oryza sativa,Barley, 86% of animals Drosophila melanogaster,Mus musculus,Homo sapiens, and 84% of Chlamydomonas incerta. Conserve domain and second structure analysis showed there are conservative EFh motif,three Ca2+ binding sites and six helixes contained in this protein sequence.Real-Time PCR was performed to explore the expression pattern of cam gene during the whole growth phases. The results showed that the transcription amount of cam gene coincided with the group growth rate, as the cells enter the obvious exponential stage, the expression of cam gene took on significant augment; and as the cells enter the lagging stage with cam gene down-transcripted. During the exponential period the transcription amount of cam gene exhibited variation. And it was conferred that Ca2+/CaM pathway may take effect in response to nutrient salt alteration and cysts formation of A.catenella. The study on CaM gene of A.catenella can make basis on elucidating the role of calcium signal transduction pathway in dinoflagellate.