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1.
鲫(Carassius carassius)卵黄原蛋白(vitellogenin,Vtg)是检测水体环境雌激素活性常用的生物标志物。本研究利用凝胶过滤结合离子交换层析与选择性沉淀结合离子交换层析2种方法,从鲫卵巢匀浆液中纯化得到了卵黄脂磷蛋白(lipovitellin,Lv),经鉴定该蛋白含有糖、磷、脂基团,天然分子量约为521 kD,SDS变性电泳显示分子量为117 kD和103 kD的2个亚基。Western blot结果显示,金鱼(Carassius auratus)Lv抗体和斑马鱼(Danio rerio)Lv抗体都能与鲫Lv发生很好的交叉反应。利用纯化的鲫Lv与金鱼Lv抗体和斑马鱼Lv抗体建立了2种夹心ELISA,发现金鱼Lv和鲫Lv的结合曲线基本重合,并且利用鲫Lv与金鱼Lv抗体建立的夹心ELISA工作范围为15.6~1000 ng/mL,检出限约为6.8 ng/mL,显著低于利用斑马鱼Lv抗体建立的夹心ELISA,结合此前研究者建立的鲫Vtg竞争ELISA,为鲫Vtg指标的测定提供了可靠方法。  相似文献   

2.
尼罗罗非鱼卵黄脂磷蛋白的分离纯化与性质鉴定   总被引：1，自引：1，他引：0  
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。  相似文献   

3.
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4.
ABSTRACT:   Lipovitellin (Lv), the major yolk protein derived from vitellogenin (Vg), was purified from vitellogenic ovaries of Pacific saury Cololabis saira using hydroxylapatite column chromatography followed by gel filtration. The apparent native mass of purified Lv was approximately 420 kDa, while the tertiary structure of Lv revealed by sodium dodecylsulfate–polyacrylamide gel electrophoresis was typical of teleost Lvs, consisting of a heavy chain (∼99 kDa) and a light chain (∼34 kDa). Western blot analysis using rabbit antiserum raised against Pacific saury Lv revealed a specific reaction with a polypeptide (∼194 kDa) that is present in serum from female Pacific saury but not in male serum, suggesting the approximately 194-kDa polypeptide to be the Vg monomer. This study describes the first step toward the development of specific immunoassays for Pacific saury Vg, which will be an effective tool for monitoring the reproductive development of this species.  相似文献   

5.
Vitellogenin was purified from the serum of 17‐β estradiol (E2)‐induced juvenile Catla catla using a simple two‐step purification procedure i.e. selective chemical precipitation followed by gel filtration chromatography. Purified protein migrated as single band in a native gradient PAGE which indicated the purity of the sample. The molecular weight of the native catla vitellogenin (~440 kDa) was determined using gel filtration chromatography. In SDS‐PAGE under reducing conditions catla vitellogenin dissociated into three major sub units at 115 kDa, 102 kDa and 73 kDa along with a few faint bands. Confirmation of purified protein as catla vitellogenin was supported by multiple physiological evidences, e.g. absence in male as well as juvenile sera and presence in matured female fish, ability to be synthesized upon estradiol injection in immature fish and certain unique biochemical properties like high molecular weight, phospholipoglycoprotein nature of the molecule. Western blot analysis showed that polyclonal antibody raised against purified protein detected vitellogenin in the sera of catla and in a few species selected from Cyprinidae family. These antisera were used to detect vitellogenin in liver tissue of hormone‐induced catla using immunohistochemistry and its applicability in other immunoassays is discussed.  相似文献   

6.
采用凝胶过滤和离子交换两种层析技术,从瓦氏黄颡鱼Ⅳ期卵巢粗提液中分离、纯化出卵黄脂磷蛋白(Lv),采用糖、磷、脂蛋白染色技术验证分离、纯化的蛋白为Lv,该Lv在非变性条件下分子量约为230ku,在SDS变性条件下分子量约为106ku。纯化的瓦氏黄颡鱼Lv经检测显示含有类胡萝卜素,但没有二硫键,对热相对稳定。利用纯化的瓦氏黄颡鱼Lv,制备了兔抗瓦氏黄颡鱼Lv多克隆抗血清。通过双向免疫扩散法测得Lv抗血清的效价为1∶32,还发现瓦氏黄颡鱼卵黄蛋白原(Vtg)和Lv之间有明显的免疫交叉反应性,说明两者具有相同的免疫原性;Western-blotting检测显示抗血清的特异性较好,并能特异性地识别Vtg。  相似文献   

7.
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8.
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.  相似文献   

9.
Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and Hypophthalmichthys molitrix, silver carp were studied. Total protease activity was significantly (P < 0.05) higher in rohu (1.219 ± 0.059 U mg protein−1 min−1) followed by silver carp (1.084 ± 0.061 U mg  protein−1 min−1), and catla (0.193 ± 0.006 U mg  protein−1 min−1). Trypsin activity of silver carp and rohu was 89–91% higher than catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell‐shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N‐α‐p‐tosyl‐L‐lysine‐chloromethyl ketone (TLCK) and N‐tosyl‐L‐phenylalanychloromethane (TPCK) indicated the presence of trypsin‐like and chymotrypsin‐like enzymes in all these three carps. SDS‐PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS‐PAGE evidenced the presence of various protease activity bands ranging from 21.6–93.7, 21.6–63.8 and 26.7–98.5 kDa for catla, rohu and silver carp respectively. In pH‐stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared with others.  相似文献   

10.
Technical characteristics and detergent compatibility of visceral alkaline proteases of three freshwater fish, namely Labeo rohita, Pangasianodon hypophthalmus and Cyprinus carpio of different feeding habits, were studied. Crude enzyme extract was partially purified by (NH4)2SO4 precipitation and dialysis. The molecular weight was in the range of 20–63 kDa. The enzyme purification folds post‐dialysis were found to be 1.55, 1.81 and 2.17 in case of Rohu, Pangas and Common carp respectively. The alkaline protease from Rohu, Pangas and Common carp exhibited maximum activity at pH 10.0, 9.0 and 11.0 respectively. The enzyme temperature optima observed were 60°C (Rohu and Pangas) and 70°C (Common carp). SBTI and EDTA inhibited more than 90% of the activity at conc. of 50 mM. Exposure of the proteases to non‐ionic surfactants like Tween 20–80 retained about 92%–100% and 76%–100% of their activity at conc. (v/v) of 1% and 5% respectively. Proteases were found less stable in the presence of SDS. There was moderate to lesser influence of H2O2 and sodium perborate on the proteolytic activity. The alkaline protease from omnivorous fish was found superior compared to the herbivore and carnivore in respect of pH and temperature optima and stability with detergents and oxidizing agents.  相似文献   

11.
A series of studies based on biochemical assays and electrophoretical observations has been conducted in order to investigate activity distributions and partially characterize various types of proteinases in the digestive tract of grass carp, Ctenopharyngodon idella (Val.). The casein digestion assays revealed that the presence of acidic proteinase had the highest activity at pH 2.5–3.0 and the alkaline proteinases at pH 10.0. The acidic proteinase activity distribution was found to decrease gradually from the oesophagus to the anus. Pepstatin A and EDTA inhibited the acidic proteinases activity. The SDS‐substrate‐PAGE showed that crude extraction of grass carp intestine contained an acidic proteinase active component with molecular mass of 28.5 ku. The substrate‐PAGE at neutral pH condition showed the presence of two acidic proteinase active components. The activity distribution of alkaline proteinase was found to slightly fluctuate along the intestine. And the whole intestine had very high activity. The inhibition assays and substrate specificity assays showed that trypsin was the main active component of the alkaline proteinases. The SDS‐substrate‐PAGE further showed that the crude extraction of grass carp intestine had four types of alkaline proteinase with molecular mass of 26.4, 30.8, 43.0 and 105.0 ku respectively. They were characterized to be trypsin (26.4, 30.8 and 43.0 ku) and un‐serine proteinase (105.0 ku) respectively. No chymotrypsin was detected.  相似文献   

12.
To produce a thermostable and neutral phytase (phy) of Bacillus subtilis E20 in Escherichia coli HMS174 and evaluate its efficiency in improving growth performance. The phy C of B. subtilis E20 was expressed in E. coli HMS 174, and then the 42‐kDa recombinant phy C was purified by Ni‐NAT and analysed by SDS–PAGE. The recombinant phy C had optimal ranges of pH of 6 ~ 7 and temperature of 50 ~ 60 °C. A thermostability analysis showed that the enzyme is a thermostable phytase, and around 33% of residual activity was detected after being incubated at 90 ~ 100 °C for 10 min. The recombinant phy C‐pretreated soybean meal for feed preparation improved white shrimp, Litopenaeus vannamei, growth and feed efficiency. Overall, the neutral and thermostable phy C is suitable for aquafeed, and it is able to improve the nutritional utilization, resulting in enhanced shrimp growth and reduced feed costs.  相似文献   

13.
Aminopeptidases play important roles in turnover of proteins, metabolism of hormones and neurotransmission, cell maturation and immunological regulations. In the present study, an aminopeptidase was purified to homogeneity from the skeletal muscle of grass carp by ammonium sulfate fractionation and sequential chromatographic steps, including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite and Phenyl-Sepharose. The purified enzyme revealed a molecular mass of approximately 105 kDa both on SDS–PAGE and on gel filtration of Superdex 200. The enzymatic activity toward synthetic substrates was optimal at 40°C and pH 7.0–7.5. Metal-chelating agents such as EDTA and EGTA effectively inhibited the enzyme activity while inhibitors to serine, asparatic and cysteine proteinases did not show much effect, suggesting its belonging to metalloproteinase family. A specific aminopeptidase inhibitor bestatin was most effective in suppressing the enzymatic activity and performed in a competitive fashion. The enzymatic activity was slightly enhanced by metal ions of Mg2+ and Mn2+ while inhibited to different extents by Co2+, Cu2+, Zn2+ and Ca2+. Sulfhydryl reagent was necessary to maintain its activity. Purified enzyme demonstrated amidolytic activity most effectively against synthetic aminopeptidase substrate Leu-methylcoumarylamide (MCA) while N-terminal-blocked substrates and myofibrillar proteins were not hydrolyzed. The enzyme purified in the present study was quite possibly a leucine aminopeptidase (LAP) and functions during muscular protein metabolism.  相似文献   

14.
Vitellogenin (Vtg) is an important precursor yolk protein in egg‐laying vertebrates, including fish. The 17β‐oestradiol (E2) plays a crucial role in the Vtg synthesis; moreover, certain hormones can stimulate Vtg synthesis. We investigated the possible role of E2, carp recombinant growth hormone (crGH), insulin (Ins), progesterone (P4) and 11‐deoxycortisol (11‐DOC) hormones in Vtg synthesis on primary juvenile Chalcalburnus tarichi hepatocyte culture. The amount of Vtg in the medium was measured at 2‐day intervals using enzyme‐linked immunosorbent assay (ELISA). The hepatocytes were maintained in culture for more than 2 weeks without the addition of serum components. Vitellogenin localization was visualized with the immunofluorescence method in E2‐supplemented hepatocytes. Among hormones applied to the culture, only E2 had an influence on Vtg synthesis in a time‐dependent manner, while crGH, Ins, P4 and 11‐DOC had no effect. However, in hepatocytes stimulated with E2 in combination with P4, a lower Vtg production was seen compared with Vtg produced when hepatocytes were stimulated with E2 alone. P4 proved to have potentiating effects on co‐treatment with E2‐induced Vtg production. As a result, E2 and P4 are the most important hormones for Vtg synthesis in juvenile C. tarichi hepatocyte culture.  相似文献   

15.
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.  相似文献   

16.
Streptococcus iniae is a significant pathogen impacting aquaculture production worldwide. The objectives of this study were to determine whether a developed modified S. iniae (ARS-98-60) bacterin vaccine is efficacious in Nile tilapia, Oreochromis niloticus (L.), against challenge with heterologous isolates from diverse geographical locations and to evaluate protein and antigenic variability among the isolates tested. Two groups of tilapia (approximately 5 g) were intraperitoneally (IP) vaccinated with 100 μL of the vaccine or sham vaccinated with 100 μL of sterile tryptic soy broth and held for 28 days. Fish were challenged with each isolate by IP injection of 2–3 × 107 CFU per fish using calcein to mark fish prior to cohabitation for challenge. The results demonstrated significant protection against all challenge isolates, and relative percent survivals ranged from 79% to 100%. SDS–PAGE analysis of whole-cell lysate proteins from the S. iniae isolates demonstrated similar protein profiles between 10 and 31 kDa and variation in profiles between 35 and 100 kDa. Western blot analysis using antiserum from vaccinated fish (ARS-98-60) demonstrated shared immunogenic proteins among all isolates in the molecular mass range of 22–35 kDa and high molecular mass material >150 kDa. The results suggest that the developed S. iniae vaccine has broad ranging protection among isolates exhibiting different protein profiles.  相似文献   

17.
Vitellogenin (Vtg) has proven to be a sensitive and simple biomarker in determining sex, sexual maturity, and xenoestrogenic effects in fish. Thus, our investigation has been focused on identification, partial characterization, and quantification of grey mullet (Mugil cephalus) Vtg through the use of a variety of biochemical and immunological analytical techniques. Mullet is considered both a promising aquaculture candidate and an important species for improving sediment quality in polyculture systems. In the first part of this work, grey mullet Vtg was purified from plasma of 17β-estradiol (E2)-induced male fish by a one-step chromatographic protocol, and partially characterized. Specific polyclonal antibodies were then raised against the mullet Vtg, and both an indirect ELISA and an optical immunosensor were set up and validated to quantify plasma Vtg. The indirect ELISA and the optical immunosensor assay developed showed linear measuring in the range 56.8–1047.1 ng mL−1 and 70–739 ng mL−1 Vtg concentrations in standard solutions, respectively. The results obtained suggest that the indirect ELISA allows Vtg detection over a wide dynamic range, thus resulting more suitable for rapid and sensitive sample screening. Therefore, we suggest that the direct immunosensor is a promising tool which needs more investigation to improve the sensitivity.  相似文献   

18.
雌核发育彭泽鲫后代理论上应为全雌群体,前期研究发现实验室养殖彭泽鲫F1代(相比池塘养殖)和实验室高密度养殖F2代(1.28尾/L,相比低密度0.64尾/L)组中出现了高比例的雄鱼。之前研究认为Vtg B和ZP2基因是雌性特异性表达的基因,本试验对F1、F2代雌雄鱼Vtg B和ZP2表达及卵黄蛋白原含量差异进行研究,结果发现,实验室养殖F1代雄鱼性腺中Vtg B和ZP2的表达分别极显著和显著高于雌鱼(P0.01,P0.05);池塘养殖F1代雄鱼性腺中Vtg B和ZP2的表达分别极显著高于和低于雌鱼(P0.01);F2代低密度养殖组中雄鱼性腺中ZP2的表达极显著高于雌鱼(P0.01)。实验室养殖F1代雄鱼肝胰脏中Vtg B和ZP2的表达分别极显著高于和低于雌鱼(P0.01);池塘养殖F1代雄鱼肝胰脏Vtg B和ZP2的表达均极显著低于雌鱼(P0.01);F2代雄鱼肝胰脏中Vtg B和ZP2的表达极显著高于雌鱼(P0.01)。F1代实验室养殖雄鱼全鱼卵黄蛋白原含量极显著低于雌鱼(P0.01);F1代池塘养殖雄鱼性腺、肝胰脏中卵黄蛋白原含量分别极显著高于和低于雌鱼(P0.01);F2代雄鱼性腺、肝胰脏中卵黄蛋白原含量极显著高于雌鱼(P0.01)。本研究表明,彭泽鲫Vtg B和ZP2基因并非雌性特异性表达,且不同的养殖方式、密度影响雌雄鱼Vtg B和ZP2表达及卵黄蛋白原含量。  相似文献   

19.
The sea cucumber (Stichopus japonicus) is able to undergo autolysis in response to a variety of environmental and mechanical cues. Within the framework of a long-term study of this phenomenon we have purified a protease from the body wall of the sea cucumber by means of ion-exchange chromatography with DE-52 cellulose and gel filtration chromatography with Sephadex G-100. The final enzyme preparation was nearly homogeneous on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 35.5 kDa. The purified enzyme exhibited a maximum activity for the hydrolysis of casein at pH 7.0 and 50°C and a remarkable stability at pH 4.0–7.0 and 40–60°C. Based on the inhibition and activation profiles obtained with numerous specific protease inhibitors and an activator, the protease purified from the body wall of the sea cucumber was defined to be a cysteine-like protease.  相似文献   

20.
A new proteomics technology has been implemented to study the protein repertoires of developing oocytes of giant grouper (Epinephelus lanceolatus). Knowledge of the chemical composition and physiochemical properties of vitellogenin (Vtg) is necessary to interpret the functional and biological properties attributed during ovulation. Vtg, as a biomarker indicator in sex determination, has been analyzed to determine the sex and maturational status of fish in the absence of the gonad tissue. A male giant grouper was induced by 2 mg/kg of 17ß-estradiol (E2), and blood was sampled at days 0, 1, 3, 5, and 10. SDS-PAGE 1D electrophoresis was used to analyze Vtg protein, and Vtg identification was done with 4800 Plus MALDI TOF/TOF? mass spectrophotometer (Applied Biosystems/MDS SCIEX, USA). Meanwhile, MS/MS de novo sequencing identified the proteins by matching sequences of tryptic peptides to the known sequences of other species. Vtg was confirmed by MASCOT at 95 % significant level, and molecular mass was 187 kDa. Protein resolved on SDS-PAGE as a double band of approximately the same mass as determined with MALDI-TOF. The N-terminal sequences and identification of Vtg were also determined. The potential of using MS methods to understand the structure and function of Vtg is discussed.  相似文献   

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