赤潮异弯藻抗体的制备及酶联免疫检测方法的建立

赤潮异弯藻(Heterosigma akashiwo)是一种广泛分布于世界近岸海域的有害针胞藻类(Raphidophyte),也是我国近海常见的赤潮生物,曾经有多次引发赤潮的报道[1,2].在赤潮发生的早期,自然水体中赤潮生物的细胞密度相对较低,且赤潮藻个体微小,传统的鉴定方法,即海水样品采集后利用光学显微镜直接观察,主要用于藻的定性和分离,当样品量较多时,定量计数过程则烦琐、费时、费力,工作量大.     

大弹涂鱼(Boleophthalmus pectinirostris) Gal-8L基因序列及其细菌凝集活性的鉴定

半乳糖凝集素-8基因(galectin-8,Gal-8)属于凝集素家族一员。已知Gal-8在哺乳动物的先天免疫中发挥重要作用,然而在鱼类中却鲜有报道。本研究通过转录组测序获得大弹涂鱼(Boleophthalmus pectinirostris)半乳糖凝集素-8样基因(galectin-8 like,Bp Gal-8L)c DNA序列。首先确定其序列特征,其次采用实时定量PCR(real-time quantitative PCR,q PCR)的方法鉴定Bp Gal-8L组织表达分布及其与迟缓爱德华氏菌(Edwardsiella tarda)感染相关性;最后分别通过原核表达获得N端和C端CRD区重组蛋白Bp Gal-8L-N和Bp Gal-8L-C,并采用玻片法观察这两个CRD区的细菌凝集活性和糖类结合特性。多重序列比对显示,Bp Gal-8L蛋白含有两个糖识别结构域(carbohydrate recognition domain,CRD),但只有C端的CRD区存在Gal家族典型的β-半乳糖苷糖类结合基序。系统进化树表明Bp Gal-8L属于鱼类Gal-8的一种亚型。q PCR结果显示,Bp Gal-8L基因m RNA在各组织中普遍表达,并且在迟缓爱德华氏菌感染后大弹涂鱼肝和脾中表达量呈上调趋势。细菌凝集实验结果显示,只有Bp Gal-8L-C重组蛋白对检测的5种革兰氏阴性菌及2种革兰氏阳性菌有凝集作用,并与乳糖和脂多糖特异性结合。综上所述,Bp Gal-8L与大弹涂鱼抗菌免疫密切相关,且其N端和C端CRD区发挥不同作用。     

SCREENING OF AGGLUTININS IN MARINE ALGAE FROM FUJIAN COAST OF CHINA

Thirty-three species of marine algae belonging to Rhodophyta, Phaeophyta and Chlorophyta from the Fujian coast were examined for agglutinins with different animal and human erythrocytes. Protein extracts from 26 species were active against at least one type of the erythrocytes tested. There were 3 species (Grateloupia imbricata, lshigefoliacea and Entermorpha prolifera) whose extracts could agglutinate all the erythrocytes used. The lowest protein concentration required to produce erythrocyte agglutination varied remarkably, from 3.1μg/ml to 500μg/ml . The strongest activity was found in the agglutina-tion of rabbit erythrocytes by Gloiopeltis furcata extract. Inhibition assays performed with nine mono- and bisaccharides indicated that agglutinations of rabbit erythrocytes by extracts of 7 species were inhibited by one or more types of the sugars assayed. The agglutinating activity shown by extracts of most species wasnot affected when the test solution was heated to 90℃, but was lost at 95℃ - 100℃. A few extracts losttheir activity at 60 RS, 65 RS and 75 RS, respectively.     

Screening of Lectins in Crab and Shrimp from Fujian Coast of China

Twelve species of crustacean from Fujian coast were examined for lectins with different animal erythrocytes. Serum extracts fromall of 12 species showed agglutinating activity against at least two types of the erythrocytes used, which revealed the existence of lectins in these species. There were five species ( Penaeus japonicus, Lophosquilla costata, Charybdis feriatus, Portunus pelagicus, Scylla serrata ) whose serums could agglutinate all the erythrocytes tested. The lowest serum protein concentration required to produce erythrocytes agglutination varied remarkably, ranging from 0.7 μg/mL to 8 080 μg/mL. The strongest activity was shown in the agglutination of rabbit erythrocyte by serum of Penaeus vanaminas. Inhibition assays performed with seven mono- and bisaccharides showed that agglutination of quail erythrocytes by serums of three species (Portunus pelagicus, Scylla serrata and Sesarma sp. ) were not inhibited by any sugars, while others were inhibited by at least three types of sugars. The assay of temperature influence on agglutinating activity showed that only Penaeusjaponicus retained activity when the serum was heated to 90℃, and other serums lost their activity at 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ for 10 minutes, respectively.     

两种微藻凝集素对几种不同类型细胞的凝集作用

经离子交换和分子筛层析从紫球藻（Porphyridium cruentum）和海水小球藻（Chlorella sp．）中分离纯化得到2种凝集素,研究它们对几种不同类型细胞即动物和人血红细胞、海洋和淡水微藻、细菌和真菌的凝集活性。研究结果表明,2种微藻凝集素对它们当中一些类型的细胞或种类具有不同程度的凝集活性,其中对水生聚胞藻（Synechocystis aquetilis）的凝集活性最强,最低质量浓度只需0．0313g／L。     

环境胁迫对栉孔扇贝(Chlamys farreri)血浆蛋白及其抗菌活性的影响

采集采自山东某养殖场的栉孔扇贝(Chlamys farreri)健康贝和病贝的血淋巴,测定其血浆蛋白浓度、血浆蛋白图谱和相对分子质量,以及血浆抗菌活性、凝集素活性等几种重要的体液免疫因素,研究环境胁迫对血浆蛋白及其抗菌活性的影响。结果表明:栉孔扇贝(C.farreri)血浆蛋白质量浓度约1 260~1 650μg/mL;血浆蛋白SDS-PAGE图谱显示3条蛋白带,相对分子质量分别为34 000、25 000和14 000;健康贝血浆抗菌活性指数BI为0.34,病贝BI为0.21;凝集素活力水平为1.602~2.204。氨氮胁迫下血浆蛋白浓度下降约780μg/mL,凝集素活力下降为1.301;高温胁迫下血浆蛋白质量浓度下降约为900μg/mL,凝集素活力为1.903;高盐胁迫下血浆蛋白浓度下降约为990μg/mL,凝集素活力为2.204。     

稀杯盔形珊瑚钙网蛋白基因的克隆及功能分析

钙网蛋白（calreticulin,CRT）是高度保守的内质网钙结合蛋白和分子伴侣,主要参与调节钙离子水平和指导蛋白质正确折叠。在这里,我们克隆稀杯盔形珊瑚（Galaxea astreata）的CRT基因,命名为Gacrt,并分析其促进细菌凝集的能力。Gacrt全长1792bp,其中5’端非翻译区(5''UTR)为77 bp,3’端非翻译区(3''UTR)为380bp,开放阅读框(ORF)1335bp,编码444个氨基酸残基,包含信号肽结构域、内质网检索信号序列（KDEL）、两条保守的钙网蛋白家族结构和一组三重复序列。通过构建原核表达系统得到CRT重组蛋白,纯化后的重组蛋白可以促进革兰氏阳性菌藤黄微球菌和革兰氏阴性菌大肠杆菌凝集,具有细菌凝集活性,支持GaCRT作为免疫相关分子参与机体对细菌防御的观点。     

丽文蛤血清中外源凝集素的凝集性能

丽丽文文蛤蛤（（ＭＭｅｅｒｒｅｅｔｔｒｒｉｉｘｘｌｌｕｕｓｓｏｏｒｒｉｉａａ））属属瓣瓣鳃鳃纲纲 ,,帘帘蛤蛤科科 ,,文文蛤蛤属属。。是是沿沿海海滩滩涂涂养养殖殖的的主主要要品品种种之之一一 ,,内内含含丰丰富富的的蛋蛋白白质质。。具具有有高高营营养养、、易易养养殖殖的的优优势势。。但但随随着着丽丽文文蛤蛤养养殖殖的的连连年年持持续续 ,,其其病病害害的的发发生生率率正正逐逐年年提提高高。。而而凝凝集集素素作作为为软软体体动动物物体体内内参参与与体体液液免免疫疫的的重重要要免免疫疫因因子子 ,,其其在在免免…     

松江鲈肌酸激酶的基因克隆、表达与功能分析

前期蛋白质质谱分析发现,松江鲈（Trachidermus fasciatus Heckel）经鳗弧菌（Vibrio anguillarum）刺激后,其皮肤黏液中的肌酸激酶（creatine kinase,CK）的表达明显上调。目前,肌酸激酶在鱼类中的功能研究尚未深入开展。本文利用RACE技术克隆获得了松江鲈CK基因的全长cDNA序列（命名为TfM-CK）。TfM-CK基因的cDNA全长为1 474 bp,开放阅读框为1 146 bp,编码381个氨基酸。序列比对分析显示,TfM-CK序列高度保守。实时荧光定量PCR显示,TfM-CK mRNA广泛表达于松江鲈各组织,在肌肉中的相对表达量最高,其次为血液。鳗弧菌刺激后,TfM-CK mRNA在肌肉、皮肤、脾脏和头肾中均上调表达。其中,脾脏中的表达量高达对照组的900多倍。原核重组表达纯化的TfM-CK （rTfM-CK）蛋白酶活测定实验结果显示,重组蛋白的酶活为22.0 U/mg。rTfM-CK对鳗弧菌、大肠杆菌（Escherichia coli）、巨大芽孢杆菌（Bacillus megaterium）和金黄色葡萄球菌（Staphylococcus aureus）有较强的凝集作用。由以上实验结果推测,TfM-CK可能通过细菌凝集作用参与到了松江鲈抵抗病原菌的先天免疫防御过程中。本研究为认识鱼类肌酸激酶的功能及其在病原菌防御过程中的分子免疫机制机制提供了参考。     

农用矿产资源的开发利用

农用矿产资源，依其理化性质可分为土壤改良矿物、肥料矿物、饲料矿物和农药矿物四大类。农用矿产资源对提高土壤肥力、植物的生长和动物的繁衍，具有不可替代的作用。