罗非鱼肠蛋白酶的分离纯化及其性质

以罗非鱼肠为原料,采用超声波辅助提取技术、电泳技术和层析技术等对罗非鱼肠道蛋白酶进行研究,结果表明:将鱼肠匀浆经超声波提取的粗酶液,用30%～70%的硫酸铵盐进行盐析、HitrapTM Q FF阴离子交换柱纯化及Sephadex G-100凝胶柱分离纯化,得到罗非鱼肠蛋白酶纯品,其比活为335U/mg,得率为32.8%;SDS-PAGE电泳为单一蛋白酶带,分子量为28ku。该酶最适pH为8.0～8.5,在pH7.0～9.0的条件下稳定;最适温度为37～42℃,热稳定性好;该酶的Km值和Vmax值分别为0.605g/L和9.407μg/min。金属离子Ag+、Pb2+对蛋白酶有完全抑制作用,Na+、K+对该酶无抑制作用。丝氨酸蛋白酶抑制剂能完全抑制该酶活性,胃蛋白酶抑制剂和脲素对该酶有一定抑制作用,EDTA没有明显抑制作用,DTT能激活该酶活性,该酶为丝氨酸蛋白酶。     

欧洲鳗血清免疫球蛋白纯化及部分特性分析

采用饱和硫酸铵分步盐析法结合柱层析提取，纯化欧洲鳗血清免疫球蛋白（Ｉg) ,实验证实欧洲鳗Ｉg主要分布在硫酸铵饱和度３０％－５０％的区间内，Ｓephacry1-S200进一步提纯的Ｉg主要存在于第一个蛋白峰，而Ｓepharose-4B柱层析提纯的Ｉg则存在于第二个蛋白峰，ＤＥＡE-52脑子交换柱层析可进一步纯化Ｓepharose-4B柱层析的产物，ＥＬＩＳＡ和Western-blot分别证实上述提取物具有抗体的免疫学活性，ＳＤＳ－ＰＡＥＧ分析纯化的Ｉg，显示了洲鳗Ｉg重链约为６８kD，轻链约为２６kD.     

花鲈免疫球蛋白的分离纯化及部分特性分析

分离纯化了花鲈(Lateolabrax japonicus)血清免疫球蛋白(Ig),并对其特性进行初步研究与分析.采用山羊-IgG(Goat-IgG)免疫花鲈并制备血清,分别采用硫酸铵分级沉淀法、A蛋白亲和层析法及Goat-IgG偶联亲和层析法提取花鲈Ig,对提取组份进行了蛋白浓度测定、间接ELISA效价测定、SDS-PAGE和Western-blotting分析.结果表明,采用硫酸铵分级沉淀法提取的花鲈Ig主要集中在硫酸铵饱和度为30%～50%的区间,其中45%饱和度硫酸铵富集的的抗体比活最高,ELISA效价为2×104/mg蛋白,比血清ELISA效价提高2.2倍;A蛋白柱亲和层析可得单一蛋白峰,洗脱峰集中在洗脱体积0.8～1.6 mL,处,占洗脱蛋白总量的76.2%,峰值的ELISA效价为1.37×105/mg蛋白,抗体得率为2.57%;Goat-IgG偶联Sephrose 4B亲和柱层析也可得单一蛋白峰,洗脱峰集中在洗脱体积1.4～2.8 mL处,占洗脱蛋白总量的72.4%,峰值的ELISA效价为1.39×105/mg蛋白,抗体得率为2.63%.以上3种提取方法均可得到79 kD的重链和29 kD轻链,表明可得到纯化的免疫球蛋白;采用鼠抗花鲈Ig血清和单抗AA5经Western-blotting证实提取成份为花鲈Ig.本研究表明,A蛋白柱和Goat-IgG偶联柱亲和层析均可用于花鲈Ig的提取,其中A蛋白柱法获得的洗脱峰值更为集中,抗体效价高,且提取过程不需要Goat-IgG免疫鱼体,是一种更为有效的免疫球蛋白提取方法.     

尼罗罗非鱼卵黄脂磷蛋白的分离纯化与性质鉴定

采用Sephacryl S-300过滤层析和DEAE-Sepharose Fast Flow离子交换层析相结合的方法从尼罗罗非鱼(Oreochromis niloticus)成熟卵子匀浆液中分离纯化出了一种高分子量的蛋白。该蛋白能被Schiff试剂、甲基绿和苏丹黑B着色,Western blot显示能被金鱼卵黄脂磷蛋白(lipovitellin,Lv)多克隆抗血清特异性识别,在非变性条件下分子量约为560 k D,在SDS变性条件下分子量约为112 k D,结果表明分离纯化的蛋白是一种含有糖、磷、脂基团的蛋白,符合鱼类Lv的性质,且与金鱼Lv有免疫交叉反应,从蛋白的性质和免疫原性以及分子量大小等角度判断,本研究获得的高纯度蛋白为尼罗罗非鱼卵黄脂磷蛋白;纯化的罗非鱼Lv在反复冻融、37℃及60℃处理条件下均未出现降解,表明罗非鱼Lv比鱼类卵黄原蛋白(Vitellogenin,Vtg)更为稳定。研究结果为罗非鱼Lv抗体的制备奠定了基础。     

Purification and characterization of a nucleoplasmin-like protein from carp (Cyprinus carpio) eggs

ABSTRACT: Nucleoplasmin, first isolated from Xenopus laevis eggs, promotes nucleosome assembly. Hereby, we have purified a nucleoplasmin-like protein from carp ( Cyprinus carpio ) eggs using ion exchange and subsequent gel filtration columns. The protein was recognized by a polyclonal antiserum against Xenopus laevis nucleoplasmin and had an amino acid composition similar to other member of the nucleoplasmin family proteins. Partial amino acid sequences from the cyanogen bromide (CNBr)-cleaved fragments showed high homology with Xenopus nucleoplasmin. The protein was also found to form an oligomeric complex and to be phosphorylated. Moreover, this protein promoted sperm nuclear decondensation as well as that of nucleoplasmin from Xenopus laevis eggs. These results suggest that the fish protein isolated here is a member of nucleoplasmin family.     

牙鲆血清免疫球蛋白的分离纯化及部分特性分析

运用Sephacryl S-200凝胶层析和HiTrap rProtein ASepharose亲和层析2种方法对牙鲆(Paralichthys olivaceus)血清免疫球蛋白进行分离纯化,结果表明,牙鲆免疫球蛋白分布于33%~50%的硫酸铵饱和溶液中,其中45%的分离效果最好。凝胶层析和亲和层析样品均出现2个蛋白峰,用还原SDS-PAGE检测确定牙鲆免疫球蛋白存在于第2个蛋白峰中。牙鲆免疫球蛋白重链分子量约为75.4 kD,轻链分子量约为29.9 kD和28.2 kD,推测牙鲆血清免疫球蛋白的分子量为836 kD。制备了兔抗牙鲆免疫球蛋白多克隆抗体,免疫双扩散法检测多克隆抗体效价为1∶32,免疫斑点法检测多克隆抗体效价至少为1∶1 600。运用免疫印迹法(Western-bloting)检测了兔抗牙鲆免疫球蛋白多克隆抗体的特异性,实验证明该抗体与牙鲆全血清中免疫球蛋白重链、轻链反应均成阳性。     

Purification and characterization of a β–glucan binding protein from the haemolymph of freshwater prawn Macrobrachium rosenbergii

β‐glucan binding protein (βGBP), a pattern recognition protein was purified from the haemolymph of freshwater prawn Macrobrachium rosenbergii by heparin affinity chromatography that showed a single band in native gradient PAGE. The β‐glucan binding property of the purified protein was confirmed in a phenoloxidase (PO) assay, where addition of βGBP along with β‐glucan increased the specific PO activity compared with that of β‐glucan alone. The molecular weight of the βGBP was found to be ~316 kDa on gel filtration chromatography. In SDS‐PAGE, βGBP molecule was reduced to one polypeptide chain of molecular weight ~113 kDa. Thus the βGBP in M. rosenbergii is possibly a homotrimeric molecule. The purified sample run on unreduced condition in SDS‐PAGE also revealed a similar size band (~113 kDa) and hence, the polypeptide chains of βGBP are held by non‐covalent interactions. The purified βGBP samples run in native PAGE was stained positively with alcian blue for carbohydrates and Sudan black for lipids indicating the βGBP to be a glycolipoprotein. With rabbit polyclonal anti‐βGBP serum developed, an indirect ELISA was standardized and the normal βGBP concentration in adult M. rosenbergii serum was quantified to be ~2 mg mL^?1. Furthermore, the applicability of the developed ELISA is discussed.     

团头鲂血清抗病毒半乳糖凝集素样蛋白的分离与鉴定

将草鱼出血病病毒(GCHV)的结构蛋白与Sepherose-4B偶联制备亲和柱，经亲和层析从团头鲂血清中分离到一种蛋白，能凝集兔红细胞，其血凝性依赖于β巯基乙醇的存在。红细胞凝集抑制实验证实该蛋白与D半乳糖有最高的亲和力，其次为甘露糖。梯度PAGE测得其分子量约为240000，SDS梯度PAGE证实该蛋白由分子量为15000的单个亚基组成，亚基之间没有共价连接。以上特性与半乳糖凝集素相符，故称其为团头鲂抗病毒半乳糖凝集素样蛋白。抗体阻断试验证实，此种凝集素样蛋白是团头鲂血清抗病毒及红细胞凝集活性的主要成分。N末段的氨基酸序列为Lys-Val-Asn-Leu-Asp-Glu-Lys-Cys-Pro-Phe，检索GenBank，未发现与这一段序列同源的蛋白质。     

罗非鱼源无乳链球菌S-核糖基高半胱氨酸酶基因（1uxS）的克隆及其推导蛋白的三维结构预测

利用PCR技术对罗非鱼源无乳链球菌（Streptococcus agalactiae）S-核糖基高半胱氨酸酶（1uxS）基因全长DNA进行了扩增、克隆和序列测定,采用ExPAsy软件包预测了推导蛋白的特性,利用SwisS—Model服务器建立了luxS三维结构,利用Swiss—PDBviewer软件进行了蛋白质三维结构的分析。预测结果显示,罗非鱼源无乳链球菌luxS推导蛋白包括保守的酶活性中心和锌结合位点,具有影响生物被膜形成、毒力因子调控等特性功能;经拉氏构象图（Ramachandranplot）分析,所构建的z眦s的空间结构合理。     

罗非鱼源无乳链球菌S-核糖基高半胱氨酸酶基因（luxS）的克隆及其推导蛋白的三维结构预测

利用PCR技术对罗非鱼源无乳链球菌(Streptococcus agalactiae)S-核糖基高半胱氨酸酶（luxS）基因全长DNA进行了扩增、克隆和序列测定,采用ExPAsy软件包预测了推导蛋白的特性,利用Swiss-Model服务器建立了luxS 的三维结构,利用Swiss-PDBviewer软件进行了蛋白质三维结构的分析。预测结果显示,罗非鱼源无乳链球菌luxS推导蛋白包括保守的酶活性中心和锌结合位点,具有影响生物被膜形成、毒力因子调控等特性功能;经拉氏构象图（Ramachandran plot）分析,所构建的luxS的空间结构合理。     

Molecular characterization and induced expression analysis of the terminal complement component C9 in rohu,Labeo rohita

The complement component 9 (C9) plays a significant role in the formation of membrane attack complex (MAC) on the targeted cell surface. The current study is dealt with molecular characterization of C9 gene from rohu, Labeo rohita, an important cultured carp species in India. An open reading frame (ORF) of 1998 bp was amplified by polymerase chain reaction (PCR) that encodes a polypeptide of 666 amino acids having a signal peptide of 19 amino acids and a mature peptide of 647 amino acids. The SMART domain architecture analysis revealed two thrombospondin type‐1 domains (TSP1), a low‐density lipoprotein receptor domain class A (LDLa), a membrane attack complex and perforin (MACPF) domain, and an epidermal growth factor (EGF)‐like domain. Multiple sequence alignment and evolutionary analysis revealed a primitive C9 sequence of rohu with maximum similarity and clustering with common carp, grass carp and zebrafish. C9 was highly expressed in liver and constitutively expressed in wide array of tissues except in eye of rohu juveniles. It was expressed during early developmental days of rohu including in milt. A variable level of up‐regulation in C9 expression was noticed upon poly I:C induction, Aeromonas hydrophila and Argulus siamensis infections in liver, spleen and gill tissues of rohu at different time points. A constitutive expression of C9 in different stages of rohu during the ontogeny and in response to pathogen exposures along with high degree of sequence homology with other fish species proved it as an important primitive immune molecule of the complement system lytic pathway.     

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.     

罗非鱼肾脏细胞系的建立及其生物学特性

采用组织块移植法,对尼罗罗非鱼（Oreochromis niloticus）的肾脏组织细胞进行原代培养,建立了罗非鱼肾脏细胞系,已稳定传代培养50代以上,命名为TiK。罗非鱼肾脏细胞系为纤维样细胞,其最佳培养基为DMEM,最适培养温度为28℃,最适血清浓度为15%。在最适培养条件下,罗非鱼肾脏细胞系的群体倍增时间为45.8 h。细胞经液氮冷冻保存6个月后进行复苏,经台盼蓝染色,约（89.84±3.48）%的细胞具有活性,复苏后细胞生长旺盛。染色体分析显示,第32代罗非鱼肾脏细胞系染色体数目分布在20~66之间,众数为48。使用本实验室分离鉴定的罗非鱼病毒感染罗非鱼肾脏细胞,可产生典型的细胞病变效应,表明罗非鱼肾脏细胞对该病毒敏感。该细胞系的建立为罗非鱼病毒病防控技术研究提供了重要的实验材料。     

Purification and characterization of two novel cysteine protease inhibitors, Eel-CPI-2 and Eel-CPI-3, in the skin mucus of the Japanese eel Anguilla japonica

We have discovered multiple acidic cysteine protease inhibitors, in addition to the known Eel-CPI-1, in the skin mucus extract of the Japanese eel Anguilla japonica by using the two-dimensional gel system of gelatin reverse zymography. Two of the acidic inhibitors, which we have named Eel-CPI-2 and Eel-CPI-3, were purified to homogeneity by anion exchange chromatography on a column of DEAE-Sepharose CL-6B, followed by fast protein liquid chromatography on Superdex 75 10/300 GL and HiTrap Q HP columns. The amino acid compositions of Eel-CPI-2 and Eel-CPI-3 were found to be almost identical and closely similar to that of the eel galectin AJL1. The molecular masses of Eel-CPI-2 and Eel-CPI-3 were elucidated to be 16,089.080 and 16,089.137 Da, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The apparent dissociation constant of Eel-CPI-2 and Eel-CPI-3 for cysteine protease papain was determined to be 1.79 × 10⁻⁷ and 1.05 × 10⁻⁷ M, respectively, by a quartz crystal microbalance technique.     

罗非鱼无乳链球菌LrrG-Sip融合蛋白免疫原性研究

为探究无乳链球菌LrrG(Leucine-rich repeat protein from GBS)和表面免疫原性蛋白Sip(surface immunogenic protein)串联表达的LrrG-Sip重组融合蛋白的免疫原性,该研究将原核表达的LrrG-Sip重组融合蛋白分别以0.5μg·g~(-1)(R1组)、1.0μg·g~(-1)(R2组)和1.5μg·g-1(R3组)每尾200μL腹腔注射免疫尼罗罗非鱼(Oreochromis niloticus),同时以Sip蛋白(S组)、LrrG蛋白(L组)以及PBS(P组)作为对照。2周后对所有免疫鱼体进行无乳链球菌(Streptococcus agalactiae)人工攻毒,攻毒剂量为其半致死浓度(LD_(50):4.0×108CFU·mL~(-1))。结果显示LrrG-Sip重组融合蛋白R1组对尼罗罗非鱼的相对免疫保护率最高,达89.14%;且免疫后第14和第28天,该组鱼体血清抗体OD_(450nm)值分别达0.63和0.64,均显著高于单一蛋白对照组(S和L组)和PBS组(P0.05);R1组鱼体血清过氧化物酶(POD)和碱性磷酸酶(AKP)活性在上述2个时间点也显著高于其他组(P0.05);但溶菌酶(LZM)和总超氧化物歧化酶(T-SOD)活性与其他组之间差异不显著(P0.05)。初步表明LrrG-Sip重组融合蛋白具有良好的免疫原性,其免疫原性明显优于单个蛋白,且能有效减少免疫剂量。     

低温驯化下4种不同品系罗非鱼血清皮质醇与免疫相关指标的变化

选取4种不同品系罗非鱼, 分别为吉富罗非鱼(Oreochromis niloticus)、奥尼罗非鱼(O. niloticus×O. aureus)、红罗非鱼(O. nilotica♂× O. mossambica♀)和奥利亚罗非鱼(O. aureaus)。在26℃水温下饲养3周后, 选取规格基本一致的罗非鱼(体质量50.73 g±4.23 g)进行低温驯化实验。水温以3℃/d的速度从26℃降至8℃, 分别于水温为26℃、20℃、14℃和8℃时进行采样, 比较不同驯化阶段4种不同品系罗非鱼血清皮质醇和免疫相关指标的变化规律。结果表明, 与26℃时的免疫指标相比, 水温降至8℃时, 吉富与红罗非鱼血清皮质醇水平显著升高; 然而血清C3、C4、IgM以及头肾C型溶菌酶mRNA水平显著下降(P<0.05), 血清中高皮质醇水平对鱼体的免疫产生了抑制作用。水温为8℃时, 奥尼罗非鱼血清皮质醇、IgM和补体C3以及头肾抗菌肽mRNA水平显著升高; 奥利亚血清皮质醇、C4和IgM以及头肾C型溶菌酶mRNA水平与26℃时相比无显著差异, 然而血清溶菌酶与C3水平降低。驯化实验结束后, 比较了4种不同品系罗非鱼在8℃水温下48 h内的累积死亡率。吉富与红罗非鱼组累积死亡率较高, 分别达到43.3%和40.0%; 奥尼罗非鱼其次, 为23.3%; 奥利亚罗非鱼最低, 为20.0%。较高的血清IgM和头肾溶菌酶和抗菌肽mRNA水平可能有助于提高奥尼和奥利亚罗非鱼的抗低温应激能力, 增加低温时的成活率。本研究通过分析4种品系罗非鱼不同驯化阶段血清皮质醇和免疫相关指标, 探讨不同品系罗非鱼在低温驯化过程中的免疫保护机制, 旨在为下一步抗低温新品系罗非鱼的选育提供理论依据。

     

盐酸小檗碱对草鱼补体系统及补体c3作用的研究

为了探究盐酸小檗碱(berberine hydrochloride)对草鱼(Ctenopharyngodon idellus)补体c3的作用机理,用含0、0.06%、0.12%、0.24%盐酸小檗碱饲料投喂草鱼,在第7 d、14 d检测血清中补体c3含量,在第14 d检测肝脏中补体c3 mRNA表达量及急性感染嗜水气单胞菌(Aeromonas hydrophila)后存活情况。并进一步对盐酸小檗碱体内药代动力学和补体消耗试验进行了研究。结果显示;摄食盐酸小檗碱的草鱼血清中补体c3含量和肝脏补体c3 mRNA表达量均显著性增加(P0.05/P0.05),草鱼的存活率与对照组相比也有显著性提高(P0.01)。补体消耗试验结果显示,当盐酸小檗碱在血清中浓度小于5 mg/L时与对照组相比补体消耗不显著,当盐酸小檗碱浓度大于5 mg/L时,补体消耗达到显著性差异(P0.05),表明此时盐酸小檗碱与补体c3直接结合,激活补体。药代动力学实验发现,灌服盐酸小檗碱后,出现双峰现象,两峰值分别是0.243 mg/L和0.117 mg/L,此浓度远远低于与补体c3直接结合量最低量(5 mg/L)。结果表明,盐酸小檗碱对草鱼补体c3的作用机理可能是通过上调补体c3mRNA的表达增加补体c3数量,而非通过直接结合补体c3来改变其活性。     

Attractability and palatability of liquid protein hydrolysates for Nile tilapia juveniles

The experiment was conducted with the aim of determining the attractability and palatability of liquid protein hydrolysates for Nile tilapia. Five experimental diets were formulated containing 5% inclusion of fishmeal (FPE; positive control), 5% inclusion of liquid protein hydrolysate of porcine mucosa (PHM), 5% inclusion of liquid protein hydrolysate of poultry (PHF), 5% inclusion of liquid fish oil (OPE) and a diet without fishmeal (negative control). Five juveniles (2.51 ± 0.13 g) were distributed in 10 L tanks, and the animals were fed five times a day, with a previous draw being made of the offered diets. The same amount of pellets was offered and the following feeding behaviours were evaluated with the aid of three‐minute footages per feeding: time to capture first pellet, number of rejections, number of approaches without capturing the pellet and number of consumed pellets. None of the observed parameters displayed significant differences (p > .05), and all diets presented negative palatability indexes in comparison to FPE. The SPE diet provided a attractability and palatability index of 6.17%, while OPE index was 2.87%, PHM 2.82% and PHF 2.74%. Both PHF and PHM reduced the final consumption of pellets in comparison to FPE in approximately 28% and 20%, respectively, although it did not present significant values.     

Purification and characterization of transglutaminase from squid gill

ABSTRACT: The present study used squid gill as a source of transglutaminase (TGase) because it has extremely high TGase activity compared with other tissues. The enzyme was purified using successive chromatographies of Sephacryl S-300 and hydroxyapatite columns. The yield and purification-fold of the enzymatic activity was 12.6% and 14.1-fold, respectively. The molecular mass of the purified enzyme was estimated to be 94 kDa by using sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis. Enzyme activity was enhanced 15-fold with an increase in NaCl concentration. Although the activity was dependent on Ca²⁺ concentration, it was not sufficiently activated even by 50 mM CaCl₂ in the absence of NaCl, but could be fully activated with 10 mM CaCl₂ in 0.7 M NaCl. However, in the absence of substrates, the enzyme was rapidly inactivated. The pH and temperature optima of the enzyme were approximately pH 8.0 and 20°C, respectively. It was stable in the absence of Ca²⁺ at pH 7.5–9.0 and had a rate constant (K _D ) of 1.6 × 10^–5 s^–1 for thermal inactivation at 50°C. These results in which squid gill TGase could be activated at higher concentrations of Ca²⁺ and NaCl than at a physiological concentration, suggest that contact with seawater or body fluid seems to activate the enzyme if the tissue is disrupted.