Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China （LCDV-cn） genome

An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene. Supported by High Technology Research and Development Program of China (863 Program, No. 2006AA100309)     

红笛鲷重组激活基因rag1原核表达条件的优化及纯化

克隆编码红笛鲷(Lutjanus sanguineus)RAG1蛋白(recombination activating protein1)活性核心区的基因序列,并与pET-28a(+)载体连接,构建原核表达载体pET-28a-RAG1,将其转入大肠杆菌BL21(DE3)菌株,利用IPTG进行诱导表达。为提高融合蛋白的表达效率,运用传统的实验方法对诱导条件进行优化。SDS-PAGE分析表明,在37℃条件下,利用0.1 mmol/L IPTG诱导8 h后,RAG1重组融合蛋白的表达量最大,相对分子质量与预测值相符,该蛋白主要以包涵体形式高效表达,利用His Trap HP亲和柱使其得到进一步纯化;Western blot分析显示,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性结合,说明表达蛋白为目的蛋白。     

新加坡石斑鱼虹彩病毒ORF162蛋白的表达、纯化及抗体制备

将新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)的ORF162的开放式阅读框插入pET-32a表达载体T7启动子控制下的6-His·Tag编码基因上游,构建SGIVORF162原核表达质粒pET-ORF162。表达质粒转化入大肠杆菌BL21(DE3)菌株,经IPTG诱导,成功表达SGIV ORF162融合蛋白。对IPTG浓度、诱导温度、诱导时间等诱导表达条件进行优化后,确定在0.7mmol/LIPTG、16℃条件下诱导14h时可溶性SGIV ORF162重组蛋白占重组蛋白总量的95%。经镍琼脂糖凝胶纯化,获得纯度为90%以上的SGIV ORF162蛋白。用纯化的SGIV ORF162蛋白免疫小鼠,获得高效特异的SGIV ORF162多克隆抗体。     

Expression of the Phycoerythrin Gene of Gracilaria lemaneiformis (Rhodophyta) in E. coli and Evaluation of the Bioactivity of Recombinant PE

Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E.coli BL21 to make two recombinant strains BEX (pGEX-PE) and BGL (pBGL). PE expressing in BEX (pGEX-PE) was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bod- ies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX (pGEX-PE) cells were col- lected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indi- cated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein con- centration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were significantly higher than the negative control BL21(pGEX-4T) (P<0.02), and the scavenging rates increased slowly following the increase of the protein content. For the heating samples, except for the 0.2 mg mL-1 BGL (pBGL) products, the scavenging effects of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were stronger than that of negative control BL21(pGEX-4T). However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially de- creased hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This re- search showed that phycoerythrin protein expressing in E. coli has the potential medical and sanitarian value.     

溶藻弧菌HY9901血红素结合蛋白HutB的原核表达及纯化

为探究溶藻弧菌(Vibrio alginolyticus)HY9901血红素结合蛋白HutB作为疫苗候选抗原的可能性,采用PCR方法扩增V.alginolyticus HY9901 hutB基因全长序列,克隆到pMD18-T载体,经双酶切、连接后,定向插入到pET-28a(+)中,构建原核表达载体pET-HutB,转入大肠杆菌BL21(DE3)中进行IPTG诱导表达。SDS-PAGE验证后,优化诱导表达条件和纯化条件,并进行Western-blot分析。结果表明,HutB蛋白在E.coli中诱导表达的最优条件:0.4 mmol/L IPTG,37℃诱导4h,表达的蛋白分子量与预期大小相符,主要以包涵体的形式存在,300 mmol/L咪唑洗脱时效果最佳,能与鼠抗His-tag单抗特异性结合。     

Purification and Refolding of a Novel β-Agarase from Inclusion Body of E. coli

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body.After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units/mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.     

红笛鲷NCCRP-1基因原核表达条件的优化及纯化

通过克隆编码红笛鲷(Lutjanus sanguineus)非特异性毒性细胞受体蛋白-1(nonspecific cytotoxic cell receptor protein-1,NCCRP-1)成熟肽基因序列,然后与载体连接构建pET21a-NCCRP融合蛋白表达载体,将其转入大肠杆菌BL21进行诱导表达并优化条件。SDS-PAGE分析表明,在异丙基-β-D-硫代半乳糖苷(IPTG)浓度0.8 mmol/L、37℃条件下培养3 h后表达量最大,分子大小与预期值相符,融合蛋白主要以包涵体形式高效表达,通过HisTrap HP柱子使其得到进一步纯化;Western blot分析表明,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性结合,说明获得该表达产物。     

复方中草药对吉富罗非鱼生长及肠道菌群的影响

将A、B、C三种复方中草药分别按质量分数1.5%的比例添加到罗非鱼商品饲料中,喂养初始体重约16.58±0.48g的吉富罗非罗28d,通过测定罗非鱼的增重率、饲料系数、肠道菌群等指标,研究三种复方中草药制剂对罗非鱼生长性能及肠道菌群的影响。结果显示:三种复方中草药制剂对罗非鱼的增重率、成活率、饲料系数等生长性能指标虽无显著影响(P>0.05),但与对照组相比,添加1.5%的复方中草药制剂C可使吉富罗非鱼的增重率提高10.82%,饲料系数降低4.71%,蛋白质效率提高15.68%;三种复方中草药制剂均可显著促进罗非鱼肠道细菌、双歧杆菌、乳酸杆菌的生长(P<0.05),C方中草药制剂还可显著抑制大肠杆菌生长(P<0.05)。结果表明,C方中草药制剂可显著促进罗非鱼肠道中乳酸杆菌等有益菌群的增殖,抑制大肠杆菌生长,从而有效促进罗非鱼生长。     

EFFECTS OF LOW MOLECULAR WEIGHT CHITOSAN (LMC-1) ON SHRIMP PRESERVATION

This study on the effects of low molecular weight chitosan (LMC- 1) and shrimp preserving agentssuch as phytic acid (PA), sodium bisulfite (SB), and crustacean preservative (CP) on the preservation ofshrimp (Trachypenaeus curvirostris) and the bacteriostasis of LMC-1 showed that: (1) Different LMC-1conontration has different bacteriostasis on E. coIi, B. subtilis and S. aureou: (2) LMC-1 and CP arebetter than PA and SB for preserving the freshness of shrimp stored at 4℃.     

溶藻弧菌Ⅲ型分泌系统注射装置蛋白VscO的原核表达及免疫原性

为探究溶藻弧菌(Vibrio alginolyticus)ZJ03株Ⅲ型分泌系统(Type III secretion system,T3SS)注射装置蛋白VscO作为疫苗候选抗原的可能性,根据GeneBank上登陆的溶藻弧菌VscO序列(NO.KJ179947),设计1对带酶切位点的特异性引物,PCR扩增vscO基因,序列分析结果显示,该基因全长462 bp,理论分子质量为18.430ku。将vscO基因定向插入原核表达载体pET-28a(+),构建重组表达质粒pET-vscO。用异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,可在大肠杆菌(Escherichia coli)BL21(DE3)中表达分子质量约为22 ku的VscO融合蛋白,且该蛋白主要以包涵体形式存在。VscO蛋白表达和纯化的最优条件为:0.1 mmol/L IPTG、37℃条件下诱导4 h,咪唑洗脱浓度为400 mmol/L。用纯化后的融合蛋白免疫SPF级小鼠,获得高效多克隆抗体。Western-blotting结果表明,鼠抗VscO血清既能与重组VscO蛋白发生反应,也能与分离自溶藻弧菌约22 ku的天然蛋白发生反应,提示T3SS注射装置蛋白VscO可能是溶藻弧菌的重要保护性抗原之一。     

H_2 PRODUCTION BY ANABAENA VARIABILIS MUTANT IN COMPUTER CONTROLLED TWO-STAGE AIR-LIFT TUBULAR PHOTOBIOREACTOR

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地胆头注射液对鸡大肠杆菌感染的治疗效果

给香鸡皮下接种大肠埃希氏菌ATCC259220.5mL/只(含菌量为4×109～5×109mL-1), 复制了鸡大肠埃希氏菌ATCC25922感染病例。用自制的地胆头注射液(每安瓿5mL,相当于生 药2g)通过胸肌多点注射,进行治疗试验。结果发现,用药后第2天,供试鸡只的发病率明显下 降,效果和环丙沙星注射液相当。在试验结束时,各试验组鸡只均被治愈。在各组供试鸡的体重、 死亡率及治疗总有效率方面,地胆头注射液均表现出明显疗效,高、中、低各剂量组鸡只的总有效 率分别达到95.29%、88.24%和75.29%。     

繁茂膜海绵对工厂化养鱼水体微生物净化作用的初步研究

在大菱鲆(Scophthalmus maximus)工厂化养殖废水中接种繁茂膜海绵(Hymeniacidon perleve),并将繁茂膜海绵与大菱鲆同池养殖,研究繁茂膜海绵对大菱鲆工厂化养鱼水体微生物的净化作用。结果表明,接种量为7 mg/L繁茂膜海绵经过8、16、24、32、40、48 h对养鱼废水中细菌总数的清除率分别为23.18%、47.65%、66.09%、81.57%、93.80%、98.96%;对弧菌的清除率分别为48.61%、56.44%、78.13%、92.88%、97.10%、98.56%;对大肠杆菌的清除率分别为39.51%、41.90%、73.24%、92.29%、97.69%、99.14%,将繁茂膜海绵以7 mg/L的生物量与大菱鲆同池养殖24 h,水体中细菌总数、弧菌和大肠杆菌的清除率分别为61.38%、50.34%、93.47%。     

Cloning, Expression and Characterization of Translationally Controlled Tumor Protein （TCTP） Gene from Flatfish Turbot （Scophthalmus maximus）

A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 451 bp and an open reading flame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5'UTR of SmTCTP started with a 5'-terminal oligopyrimidine tract (5'-TOP), a typical feature for translationaily controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known verte-brate TCTPs in a range of 58.8% to 64.1%. The length offish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch (Lateolabrax japonicus) is 170 aa in length, while that of zebrafish (Danio rer/o) and rohu (Labeo rohita) is 171 aa in length. North-ern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further inves-tigation of the biological function of TCTP in fish.     

Preparation by enzymolysis and bioactivity of iron complex of fish protein hydrolysate （Fe-FPH）from low value fish

Preparation of Fe^2＋ chelate of fish protein hydrolysate （Fe-FPH） obtained from low value fish proteins was introduced and its bioactivity was studied by compound enzymolysis. The optimum conditions for hydrolysate chelating Fe^2＋ are DH （degree of hydrolysis） at 5%, pH 7.0, 20℃ and 15 min chelating time for FM （material not being defatted）. Four types of Fe-FPH including CA （deposit after chelating）, CB （deposit in 50% of absolute ethanol solution）, CC （suspended deposit in 80% of absolute ethanol solution）, and CD （bottom deposit in 80% of absolute ethanol solution） were fractionated with absolute ethanol from FM. Structural analysis through infra-red spectrum revealed that Fe^2＋ was combined strongly with amino-group and carboxyl-group in each chelate and each Fe^2＋ could form two five-member ring structures. All of the four chelates were shown more significant antioxidative activity and can be used as natural hydrophobic and hydrophilic antioxidant. Among all the chelates, the CB possesses the most effective antioxidative activity at 92% as high as that of a-tocopherol. Among all Fe-FPHs, only CD showed the most effective antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella typhi, and Bacillus subtilis and can be used as natural antibacterial. It provides a more effective way for utilization of low value fish proteins and key information of Fe-FPH as additive in food industry.     

Extraction of astaxanthin from Euphausia pacific using subcritical 1, 1, 1, 2-tetrafluoroethane

Euphausia pacific is an important source of natural astaxanthin.Studies were carried out to assess the extractability of astaxanthin from E.pacific using subcritical 1,1,1,2-tetrafluoroethane(R134a).To examine the effects of multiple process variables on the extraction yield,astaxanthin was extracted under various conditions of pressure(30-150 bar),temperature(303-343 K),time(10-50 min),flow rate(2-10 g min-1),moisture content(5.5%-63.61%),and particle size(0.25-0.109 mm).The results showed that the extraction yield increased with temperature,pressure,time and flow rate,but decreased with moisture content and particle size.A maximum yield of 87.74% was obtained under conditions of 100 bar,333 K,and 30 min with a flow rate of 6 g min-1 and a moisture content of 5.5%.The substantial astaxanthin yield obtained under low-pressure conditions demonstrates that subcritical R134a is a good alternative to CO 2 for extraction of astaxanthin from E.pacific.     

Fatty Acid Composition of Euphausia superba,Thysanoessa macrura and Euphausia crystallorophias Collected from Prydz Bay,Antarctica

The information of trophic relationship is important for studying the Southern Ocean ecosystems.In this study,three dominant krill species,Euphausia superba,Thysanoessa macrura and Euphausia crystallorophias,were collected from Prydz Bay,Antarctica,during austral summer of 2009/2010.The composition of fatty acids in these species was studied.E.superba and T.macrura showed a similar fatty acid composition which was dominated by C14:0,C16:0,EPA(eicosapentenoic acid) and DHA(decosahexenoic acid) while E.crystallorophias showed higher contents of C18:1(n-9),C18:1(n-7),DHA and EPA than the former two.Higher fatty acid ratios of C18:1(n-9)/18:1(n-7),PUFA(polyunsaturated fatty acid)/SFA(saturated fatty acid),and 18PUFA/16 PUFA indicated that E.crystallorophias should be classified as a typical omnivore with a higher trophic position compared with E.superba and T.macrura.     

螺旋藻对乌鬃鹅生长性能、屠宰性能和肌肉营养的影响

选取1 日龄乌鬃鹅300 只,随机分成5 组,对照组（A）用基础饲粮饲养,实验组B～E 组分别在基础饲粮中添加质量分数1%（B 组）、2%（C 组）、3%（D 组）、4%（E 组）的螺旋藻,实验期63 d.结果表明：与对照组相比,添加螺旋藻对乌鬃鹅的屠宰率、半净膛率、腹脂率和肌肉灰分无显著影响（P〉0.05）,对其它生长指标和全净膛率均有显著性影响（P〈0.05）,其中2%C 组和3% D 组的料重比显著下降（P〈0.05）, E 组全净膛率显著提高（P〈0.05）;肌肉的粗蛋白质（CP）含量E〉D〉C〉B〉A,粗脂肪（EE）含量E〉A〉B〉C〉D;所测得的17 种氨基酸中,包括含有人体必需的7 种氨基酸（EAA）和2 种半必需氨基酸（HEAA）;氨基酸评分（AAS）和化学评分（CS）最高的是赖氨酸（Lys）,实验组的必需氨基酸指数（EAAI）均比对照组有所提高.添加2%～3%（w）的螺旋藻对生长性能有显著差异（P〈0.05）,对粗脂肪、粗蛋白的含量有所改善;从肌肉EAAI 来看,添加螺旋藻后的肌肉蛋白质品质优于不添加螺旋藻的对照组.建议饲粮中螺旋藻的添加量为质量分数2%.     

Expression of the Phycoerythrin Gene of Gracilaria lemaneiformis （Rhodophyta） in E. coil and Evaluation of the Bioactivity of Recombinant PE

Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E. coli BL21 to make two recombinant strains BEX (pGEX-PE) and BGL (pBGL). PE expressing in BEX (pGEX-PE) was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX (pGEX-PE) cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were significantly higher than the negative control BL21(pGEX-4T) (P<0.02), and the scavenging rates increased slowly following the increase of the protein content. For the heating samples, except for the 0.2 mg mL⁻¹ BGL (pBGL) products, the scavenging effects of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were stronger than that of negative control BL21(pGEX-4T). However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This research showed that phycoerythrin protein expressing in E. coli has the potential medical and sanitarian value.     

Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli

Anti-lipopolysaccharide factors (ALFs) are important antimicrobial peptides that are isolated from some aquatic species. In a previous study, we isolated ALF genes from Chinese mitten crab, Eriocheir sinensis. In this study, we optimized the production of a recombinant ALF by expressing E. sinensis ALF genes in Escherichia coli maintained in shake-flasks. In particular, we focused on optimization of both the medium composition and the culture condition. Various medium components were analyzed by the Plackett-Burman design, and two significant screened factors, (NH4)2SO4 and KH2PO4, were further optimized via the central composite design (CCD). Based on the CCD analysis, we investigated the induction start-up time, the isopropylthio-D-galactoside (IPTG) concentration, the post-induction time, and the temperature by response surface methodology. We found that the highest level of ALF fusion protein was achieved in the medium containing 1.89 g/L (NH4)2SO4 and 3.18 g/L KH2PO4, with a cell optical density of 0.8 at 600 nm before induction, an IPTG concentration of 0.5 mmol/L, a post-induction temperature of 32.7°C, and a post-induction time of 4 h. Applying the whole optimization strategy using all optimal factors improved the target protein content from 6.1% (without optimization) to 13.2%. We further applied the optimized medium and conditions in high cell density cultivation, and determined that the soluble target protein constituted 10.5% of the total protein. Our identification of the economic medium composition, optimal culture conditions, and details of the fermentation process should facilitate the potential application of ALF for further research.