Objective To construct and apply gold-labeled immunology assay for the detection of okadaic acid.Methods Gold-labeled immunology testing technology for fast detecting okadaic acid was established and optimized by a series of procedures,including purification of monoclonal antibody of okadaic acid,preparation of colloidal gold,label of monoclonal antibody,preparation of strip,simulated sample extraction and spiked recovery detection,followed by extraction and testing of exposed shellfish.Results 30 nm colloidal gold particles were used to mark the test strip,the concentration and coated amount of gold-labeled monoclonal antibody were 200 μg/mL and 2.5 μL/cm,respectively,and the concentration of second antibody was 1.0 mg/mL.The colloidal gold test strip detection in spiked shellfish and exposed shellfish were applied,with detection time of 3-5 minutes and detection limit of less than 25 ng/mL.Conclusion Gold-labeled immunology assay for fast detecting okadaic acid was established,meeting the safe threshold of okadaic acid in shellfish food ruled by many countries and proving technical foundation for detection of okadaic acid in shellfish.

A method for the detection of okadaic acid(OA) by liquid array was established.Applying the principle of indirect competitive immunoassay,coupled with fluorescent beads coated with OA-OVA as a carrier and using luminex Xmap 200TM liquid array system to detect the content of OA and the sensitivity of the method.The specificity of the method was conformed by the detection of OA,DTX-1,GYM,PTX2,SPX1,YTX,STX and MC-LR.Meanwhile,the average recovery of samples added OA was conformed.This method can be used for detecting the limit of OA in shellfish.The results showed that the concentration detection limit to OA was 0.129 μg/L,and the dynamic ranges were 0.2～63 μg/L,which were higher than the sensitivity of corresponding conventional Idc-ELISA.There was not the cross reaction with other toxins except for DTX-1.The average recovery of samples added OA was between 84.96%～90.35%.This method can be used for detecting the limit of OA in shellfish.

DNA extraction of red tide-forming causative species Amphidinium sp.was performed.PCR technology was used to amplify 18S rDNA.The result of amplification indicated that the length of PCR productions of Amphidinium sp.was 1 736 bp,G+C was 47%.Then,homology analysis was performed using BLAST in NCBI,and the result indicated that the fragment of 18S rDNA of Amphidinium sp.we cloned had a high homology(99%)with that of Amphidinium carterae,95% homology with any other Amphidinium sp.in GenBank,85% homology with Gymnodinium and Prorocentrum.There was no distinct homology with human genomic and mouse genomic.The results show that,18S rDNA was an effective method for rapid identification of genus,while not quite sure for the species identification.

The way of " extracting-sahing-chromatography" was used to purify the phycoerythrin and phyeoeyanin from Porphyra yezoensis in process scale-up. First, by comprehensive comparison of efficiency, the Sephadex G-25 was selected from four resins (Sephadex G-25 、G-100、S-300 and CL-6B) as the best choice used in crude extract desalting of phycobiliprotein. Then the preparation process of phycobiliprotein was scaled-up with raw material(Porphyra yezoensis) increased from 1 g to 20g, and finally to 400g. The results indicated that the yields of purified phyeoerythrin and phycocyanin (absorption spectra purity above 3. 2) increased during according to process scale-up,with 0.323% phycoerythrin and 0. 148% phycocyanin obtained from 400g frozen Porphyra yezoensis blades respectively. It is no doubt that the process involved in the experiment is a potential way for large scale preparation of phyeobiliproteins of high purity.

The crude extract of phycobiliproteins was prepared from thallus of Porphyra yezoensis with combination of dissolving-bulging and tissue-homogenator methods.Effects of solvent volume,treating time,ammonium sulphate precipitation time,and hydroxylapatite(HA)chromatography on purity and yield of phycobiliprotein were studied.The results showed that the optimum conditions for purification,including the ratio of thallus to buffer(1:5),treatment with dissolving-bulging,precipitation with ammonium sulphate for four times,all of which increased the purity of phycoerythrin and phycocyanin up to 1.71 and 0.98,respectively.Due to HAP chromatography according to Siegelman,the phycoerythrin and phycocyanin purity increased to 4.73 and 4.42,with the yield of 0.144% and 0.042%.

To optimize the chemical conjugation between R-phycoerythrin (R-PE) and antibody, different molar ratios of the heterobifunctional reagent N-succinimidyl-3-2-pyridyldithio propionate (SPDP) to R-PE were tested for R-PE derivation into R-PE-PDP, and different molar ratios of dithiothreitol (DTT) to IgG were tested for IgG thiolation. The results showed that in terms of best product yields determined by ultraviolet (UV) spectrophotometry, the optimal molar ratio of SPDP to R-PE was 40:1 for PE derivation, and that of DTT to IgG was 500:1 for thiolation. R-PE-labeled secondary antibody was produced by cross-linking PE-PDP and thiolated IgG. After further purification, UV spectra and native polyacrylamide gel electrophoresis determined its high purity and molecular weight. Finally, in conjunction with antigen-specific first antibodies, the R-PE-labeled IgG was applied in fluorescence immunoassays as secondary antibody and successfully detected antigens spotted on nitrocellulose membrane as well as intracellular antigen in SMCC-7721 cells. This study provides a feasible method of fluorescence antibody preparation from R-PE of Porphyra yezoensis and demonstrates high fluorescent labeling efficiency and good immunologic reactivity of the product.

以生长快速、细胞具多个蛋白核的大型海藻条浒苔作为材料研究CO 2 浓度对条浒苔Rubisco酶在蛋白核和叶绿体基质之间迁移的影响。应用金标免疫电镜分子定位技术对Rubisco酶集中蛋白核程度进行数值化分析。电镜下可观察到标记Rubisco的金颗粒大部分集中分布在蛋白核中。根据Morita（1997）提出的方法,设定PR-ratio值（蛋白核内分布的Rubisco酶总量与蛋白核外类囊体基质中的Rubisco酶总量之比）作为衡量Rubisco集中蛋白核程度的分析指标。不同CO 2 浓度对于Rubisco酶分布的长期影响和短期影响研究均显示CO 2 浓度升高时,Rubisco倾向于向叶绿体基质中扩散;CO 2 浓度较低或无CO 2 培养时,Rubisco酶不断向蛋白核中集中。研究结果显示,蛋白核可能在光合作用和CCM机制中具有重要作用。