泥鳅血清凝集素的研究

采用DEAE-Sepharose FF离子交换层析和Superdex 200 10/300 GL凝胶过滤层析技术,从泥鳅血清中分离出两种天然凝集素MSL-I和MSL-Ⅱ.SDS-PAGE检测表明,MSL-I及MSL-Ⅱ均显示两条蛋白质着色带.采用凝胶过滤法测得MSL-I相对分子质量为25 000,MSL-Ⅱ相对分子质量为82 000.根据凝胶过滤测定及SDS-PAGE检测结果可知,MSL-Ⅱ由相对分子质量分别为29000和13 600两种亚基组成,MSL-I的亚基组成有待进一步研究.MSL-Ⅱ能结合多种糖,其中对乳糖的亲和力最强.MSL-Ⅱ具有较强的热稳定性,在pH 4～11范围内具有凝集活性,最适pH为6～8.     

中华绒螯蟹血清凝集素的分离纯化与性质研究

运用硫酸铵盐析和GlcNAc-Sepharose 6B亲合层析等方法从中华绒螯蟹的血清中分离出一种天然的凝集素,经SDS-PAGE测得其分子量约为82 kD,由单一亚基组成,由等电聚焦凝胶电泳测得其等电点(pI)为5.4.糖凝集抑制试验中检测到葡萄糖、半乳糖、甘露糖、乳糖、果糖和蔗糖对中华绒螯蟹血清凝集素均没有抑制作用,但N-乙酰氨基糖GIcNAc、GalNAc和ManNAc等能抑制其凝集活性.热变性试验结果表明,温度为10～50℃,凝集素仍保持强的凝集活性,但温度升至60℃后,凝集活性迅速下降,至80℃失去凝集活性,在pH 4.0～8.0的各缓冲液中保持较强的凝集活性,而在此pH范围外,凝集活性均有不同程度的下降.中华绒螯蟹血清凝集素可使外源红血细胞发生凝集,还具有凝集细菌或抗菌作用.     

泥鳅血清凝集素的研究

采用DEAE-Sepharose FF离子交换层析和Superdex 200 10/300 GL凝胶过滤层析技术,从泥鳅血清中分离出两种天然凝集素MSL-I和MSL-Ⅱ.SDS-PAGE检测表明,MSL-I及MSL-Ⅱ均显示两条蛋白质着色带.采用凝胶过滤法测得MSL-I相对分子质量为25 000,MSL-Ⅱ相对分子质量...     

施氏鲟卵黄蛋白的分离纯化及其性质

采用组织匀浆、饱和硫酸铵分步沉淀和Sephadex G-100葡聚糖凝胶层析的方法提取和纯化施氏鲟卵黄蛋白,并对其性质进行了研究。结果表明:采用50%和70%饱和度的硫酸铵分步沉淀与Sephadex G-100凝胶层析相结合的方法,可以获得一种卵黄蛋白纯品。Native-PAGE和SDS-PAGE分析表明,该纯化蛋白纯度为100%,由一种同源亚基组成,其亚基的相对分子质量约为30 kD。油红O、免疫印迹(Western-blot)和罗丹明B染色均为阳性,表明该蛋白为施氏鲟卵中的一种脂磷蛋白。     

中华倒刺鲃血清凝集素的研究

从中华倒刺鱼巴(Spinibarbus sinensis)血清分离到一种天然的凝集素,能够凝集多种不同的抗原物质,其组成成分为糖蛋白,具有耐热和耐酸碱性,对β-巯基乙醇敏感。凝集反应受温度、PBS离子浓度以及时间等因素的影响。经糖的专一性抑制实验显示,蔗糖和乳糖对凝集反应有抑制作用,说明这2种糖分子是凝集素在识别和凝集过程中起一定作用的糖分子。     

南方鲇血清凝集素的研究

从南方鲇(Silurusmeridionalis)血清分离到一种天然的凝集素分子,能够凝集多种不同的抗原物质,其组成成分为糖蛋白,具有耐热和耐酸碱性,对β-巯基乙醇敏感。凝集反应受温度、离子强度以及时间等因素的影响。经糖的专一性抑制实验显示,蔗糖和半乳糖对凝集反应有抑制作用,说明这2种糖分子是凝集素在识别和凝集过程中起一定作用的糖分子。     

南美白对虾血清凝集素的凝集活性及部分性质研究

利用血凝试验及糖抑制试验测定南美白对虾的血清的凝血活性及糖抑制专一性,同时进行热稳定性、pH及Ca2 影响试验。结果显示:南美白对虾血清只能凝集4种红细胞、4种单细胞藻类、4种微生物细胞或孢子。血清对家兔红细胞的凝集可被乳糖、D-甘露糖、D-果糖、D-半乳糖和D-葡萄糖5种糖所抑制,其凝集活性均依赖于Ca2 ,在pH为4 0～8 0范围内较稳定,经50℃保温10min后活性完全消失。     

草鱼肠道-菌株产纤维素酶的分离纯化及性质研究

从草鱼肠道分离出1株枯草芽孢杆菌,有较强的活性.该菌产生的纤维素酶粗酶液,经盐析、透析,并通过葡聚糖凝胶层析柱Sephadex G-75分离纯化,经SDS-PAGE后表明第2峰已纯化,得到一种相对分子质量约为62.43 kD纤维素酶.分离纯化后该酶的比活力提高了2.924倍,回收率为6.38%.酶学试验研究表明:该酶的最适反应温度为55℃,最适pH值为7.0;Lineweaver-Burk法求得动力学参数,Km和Bmax分别为1.02×10-3g/mL、2.727×10-2mg/(mL·min).     

三种梭子蟹血清凝集素的细胞凝集活性比较

利用凝集试验及糖抑制试验测定三种梭子蟹的血清对红细胞、藻类细胞、微生物细胞或孢子的凝集活性，同时进行糖抑制、热稳定性、pH及Ca^2 影响试验。结果显示：三种梭子蟹中远洋梭子蟹对红细胞最敏感，而三疣梭子蟹对单细胞藻类和微生物细胞或孢子最敏感；红星梭子蟹血清对鹌鹑红细胞的凝集可被供试的7种糖所抑制，而供试的7种糖均不能抑制远洋梭子蟹血清对鹌鹑红细胞的凝集；远洋梭子蟹血清具有广泛的pH适应性，在pH为4．0～10．0范围内对鹌鹑红细胞都具有凝集活性，且具有较强的热稳定性，经60℃保温10min后仍具凝集活性；三种梭子蟹的凝集活性均依赖于Ca^2 。     

马氏珠母贝黏液糖蛋白分离纯化及其抗氧化活性研究

该研究采用直接醇沉法、盐提醇沉法和水提醇沉法提取马氏珠母贝(Pinctada martensii)黏液中的糖蛋白,优选直接醇沉法粗品(DAS),进一步采用Sephacryl S-200纯化获得直接醇沉纯化品(DAPS),同时测定DAS及DAPS的体外抗氧化活性。结果显示,DAS的糖和蛋白质得率均最高,且3种方法提取的糖蛋白粗品蛋白质组成无明显差异;DAPS的总糖和蛋白质质量分数分别为(22.72±0.26)%和(71.18±0.62)%,分子量主要集中在245kD;DAS和DAPS对羟自由基的清除能力显著,在0.8 mg·mL-1下两者对羟自由基的清除率均高于90%,对超氧阴离子自由基及DPPH自由基也有一定清除能力。研究结果可为探索马氏珠母贝黏液功效组分提供理论基础数据。     

角叉菜凝集素的分离纯化及其性质

角叉菜（Ｃｈｏｎｄｒｕｓ ｏｃｅｌｌａｔｕｓ）经磷酸盐缓冲液抽提，２０％～７５％硫酸铵分级沉淀，牛甲状腺球蛋白－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析，可以从红藻角叉菜中纯化出角叉菜凝集素（ＣＯＬ），在ＰＡＧＥ上显示单一蛋白染色带，在等电聚焦电泳上显示单一蛋白染色带，其ＰＩ为８．３０，纯化后的ＣＯＬ的最大此外吸收峰在２８５ｎｍ，用ＳｅｐｈａｄｅｘＧ－２００分子筛层析测得其分子量为４２５２。该凝集素可以凝集     

Expression and localization of MCsialec, a sialic acid-specific lectin in the marine bivalve Manila clam, Ruditapes philppinarum

A novel sialic acid-specific lectin (MCsialec) was detected from an expressed sequenced tag (EST) sequence from Manila clam haemocytes infected with Perkinsus olseni. The cDNA of the lectin was cloned using gene-specific primers based on a previously determined EST and characterized. The full-length cDNA of MCsialec is 603 bp in length and encodes a polypeptide of 200 amino acids with a calculated molecular mass of 21.928 kDa. Sequence alignment and protein motif analyses showed that MCsialec shares identity with sialic acid-specific invertebrate lectins from Cepaea hortensis, Helix pomatia and Haliotis discus discus. The lectin was expressed in Escherichia coli M15 cells and purified using a Ni-NTA His-binding resin matrix for antibody production. The presence of the lectin in various tissues of Perkinsus-infected and uninfected Manila clams was analysed by both PCR and immunohistochemical localization assays. MCsialec was detected in each tissue of the clams; however, upon infection, the level of expression of the lectin increased in each tissue. Vibrio tapetis infection also induced high-level expression of MCsialec in the haemocytes. These data suggest that MCsialec plays a crucial role in the immune system of the Manila clam during pathogenic infection.     

Purification and characterization of a lectin from the starfish Asterias amurensis

In this study, we describe a novel lectin (designated AMUL) from the Northern Pacific starfish Asterias amurensis. AMUL was purified from the coelomic fluid by affinity chromatography followed by size-exclusion chromatography. The native molecular mass of the lectin was found to be approximately 270 kDa by size-exclusion chromatography using a Superdex 200 column. AMUL showed distinct bands at approximately 14 or 18 kDa on SDS-PAGE under nonreducing or reducing conditions, respectively. The hemagglutination activity of AMUL against rabbit erythrocytes was strongly inhibited by the two sialic acids N-acetylneuraminic acid and N-glycolylneuraminic acid. The glycoproteins tested showed no inhibition of hemagglutination. The first 28 amino acid residues of AMUL were determined by automated Edman degradation, showing that AMUL has considerable sequence homology with C-type lectins from other echinoderms. These results show that AMUL is a novel echinoderm-derived sialic-acid-specific lectin that belongs to the C-type lectin superfamily.     

黄鳝内脏铁型超氧化物歧化酶的纯化

经丙酮分级沉淀,DEAE-琼脂糖离子交换层析和Sephacry1S-200凝胶过滤,从黄鳝内脏中分离纯化获得铁超氧化物歧化酶(Fe-SOD),并对其部分性质进行分析鉴定。获得该酶的比活力为1500U mg,提纯倍数为368 5,回收率为24 7%。该酶最大紫外吸收波长为280nm。聚丙烯酰胺凝胶电泳和等电点聚焦电泳结果表明纯化酶蛋白呈一条带,测得该酶分子量约为87kD,亚基分子量约为14 5kD;等电点为pH7 1。     

Effects of lectin in the scleractinian coral Ctenactis echinata on symbiotic zooxanthellae

We report herein the presence of a lectin in the scleractinian coral Ctenactis (Fungia) echinata. The lectin bound preferentially to lactose, melibiose, and d-galactose. The purified lectin CecL was composed of several isolectins, and it was found to have a molecular mass of 67.4 kDa via gel filtration. Glycopeptidase F-treated CecL showed a single band at 32.5 kDa. The mass/charge ratios of the reduced CecL peaks were equivalent to half those of the native peaks. These results suggest that CecL is composed of two glycosylated polypeptides linked by interchain disulfide bonds. In a biological activity test using a zooxanthellal culture (Dinoflagellate Symbiodinium) clonally isolated from Fungia cf. fungites, CecL transformed the flagellated motile form of Symbiodinium into the nonmotile coccoid form, a form equivalent to the symbiotic stage. The activity of CecL on Symbiodinium cells was concentration dependent, and 100 μg/ml CecL arrested Symbiodinium cells in the coccoid form for 5 days. CecL also suppressed the growth of Symbiodinium cells, unlike the octocoral lectin derived from Sinularia lochmodes, which arrests Symbiodinium cells in the coccoid form but does not affect the growth of the coccoid. This result provides further evidence that coral lectins play a role in symbiont engagement and maintenance in zooxanthellae–coral symbiosis.     

奥尼罗非鱼血清免疫球蛋白的纯化及分子量的初步分析

通过饱和硫酸铵盐析结合 Sephadex G-200柱层析的方法,纯化制备了健康非免疫状态下奥尼罗非鱼的免疫球蛋白,进行变性还原条件下的聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹试验对血清的免疫球蛋白进行初步分析,发现SDS-PAGE电泳条件下血清重链分子量为85 kD,轻链分子量为30 kD.如果罗非鱼血清免疫球蛋白在自然状态与其他硬骨鱼类一样也为四聚体,那么其总分子量的理论值应为920 kD.     

贻贝凝集素生物活性的研究

从海洋无脊椎动物贻贝中分离出的凝集素(CGL),为N-乙酰半乳糖胺/半乳糖(GalNAc/Gal)特异性的凝集素。经SDS-聚丙烯酰胺凝胶电泳测定,其相对分子量为18 000。其血凝活性不依赖钙离子的存在。在植物凝集素(PHA)存在的条件下,CGL具有体外促进小鼠脾细胞增殖的活性。同时,它还具有抑制酶活性,其抑制50?E的浓度(IC50)为6.82 mg/m l和促进体外离体兔回肠蠕动的作用。     

Isolation of Sulfated Anticoagulant Compound from Fermented Red Seaweed Grateloupia filicina

The red alga Grateloupia filicina was fermented with 0.15 g/mL sucrose at 25 C, and anticoagulant activity was measured biweekly up to the 10th wk by activated partial thromboplastin time (APTT). A sulfated polysaccharide compound having anticoagulant activity was purified from the sixth wk of fermentation by fast protein liquid chromatography using diethylaminoethyl cellulose followed by a sepharose‐4B column. The concentration of the purified sulfated anticoagulant polysaccharide was 136.65 μg/mL, and the sulfate concentration of the purified compound was 26.33 μg/mL. The presence of just a single spot on agarose gel electrophoresis confirmed that the compound was purified, and polyacrylamide gel electrophoresis showed that the purified compound was of high molecular mass. The purified sulfated anticoagulant polysaccharide consisted of galactose, glucose, and mannose in an approximate molar ratio of 0.95 : 0.01 : 0.03. Both purified anticoagulant and commercial heparin showed >1000 sec APTT activity at 136.65 μg/mL. As the technique of fermentation is inexpensive, the purified anticoagulant compound could have significant potential in the medical and pharmaceutical industry.     

Purification and characterization of glycerolipid acyl-hydrolase from the red alga Gracilaria vermiculophylla

ABSTRACT:   A glycerolipid acyl-hydrolase was purified 19-fold with a yield of 11% from the prostaglandin-producing red alga Gracilaria vermiculophylla by ammonium sulfate precipitation, anion-exchange chromatoraphy and gel filtration chromatography. Sodium dodecylsulfate– polyacrylamide gel electrophoresis of the final preparation showed a single band corresponding to a molecular mass of 20 kDa, but Superdex 200 fast protein liquid chromatography exhibited a molecular mass of 40 kDa. Accordingly, it was suggested that the purified enzyme was a homodimer of a 20 kDa subunit. The optimal temperature and pH were 37°C and 7–8, respectively. The purified enzyme catalyzed hydrolysis of the acyl groups of both glycoglycerolipids and phospholipids, especially monogalactosyldiacylglycerol and phosphatidylcholine. These results suggest that the enzyme hydrolyze the membrane lipids of the alga to release various saturated and unsaturated fatty acids, including arachidonic acid as substrate for prostaglandin synthesis.     

Purification of vitellogenin from the air breathing catfish, Clarias gariepinus

Vitellogenin (Vtg) is a female specific glycophospholipoprotein which can be induced both in male and female with estradiol and xeno-estrogens. The basic theme behind the purification of vitellogenin from the fish is to understand the evolutionary relationship and for the purification and characterization of the Vtg receptor. The male catfish, Clarias gariepinus was administrated with estradiol over a period of time for the synthesis of Vtg and the serum was collected. The Vtg was purified from the serum using a two step chromatographic technique. The serum was passed on to DEAE-ion exchange column and the protein was eluted using a salt gradient. The fractions containing the Vtg were pooled and passed onto a gel permeation chromatography column and the pure protein was obtained. The molecular weight is around 200 kDa on the SDS-PAGE and around 520 kDa on the native gel electrophoresis.