Expressed Sequence Tags(ESTs) Analysis of Angiostrongylus cantonensis

从gEnbAnk下载1277条广州管圆线虫的表达序列标签(EST),用blASTX比对分析,并用SIgnAlPV3.0预测潜在的抗原或过敏原相关蛋白n端是否具有分泌信号肽或信号锚定肽。结果显示,得分值>100有614条,其中14条与广州管圆线虫一致,540条与其他物种的相关蛋白相匹配,60条与数据库中序列无同源性。614条序列可分为10类,抗原或过敏原相关蛋白有80条,编码22种蛋白,其中12种具有分泌信号肽,3种具有信号锚定肽。A total of 1 277 ESTs of Angiostrongylus cantonensis were downloaded from GenBank and analyzed with BlastX.SignalP V3.0 analysis was applied to predict potential putative antigen or allergen relative proteins with N-terminal secreted signal peptides or signal anchors.BlastX analysis showed that there were 614 ESTs scored more than 100, of which 14 were identical with A.cantonensis, 60 ESTs did not match any proteins in the databases.The identified 614 ESTs could be grouped into 10 categories, 80 ESTs expressed 22 antigen or allergen relative proteins, in which 12 had N-terminal secreted signal peptides and 3 had signal anchors.福建省科技计划项目(No.2008N2005);厦门市科技计划项目(No.3502Z20074036)---

Cloning and Expression of D-like Aspartic Protease of Anisakis simplex

作者单位： 厦门大学生命科学院， 厦门361005 通讯作者， E-mail:[email protected][中文文摘]目的克隆简单异尖线虫Ⅲ期幼虫(L3)的D-天冬氨酸蛋白酶基因(AsAP)全长,研究其表达蛋白的特性。方法根据GenBank中简单异尖线虫D-天冬氨酸蛋白酶基因表达序列标签的部分信息,设计特异引物并用cDNA末端快速扩增技术得到AsAP全长序列,分析推导的蛋白序列特征,并预测其三级结构。用RT-PCR扩增简单异尖线虫L3的AsAP基因编码序列,产物用EcoRⅠ和SalⅠ双酶切,连入表达载体pET32а(+),转化大肠埃希菌(E.coli)BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测。结果简单异尖线虫L3的AsAP基因全长1 753 bp,编码453个氨基酸,与锡兰钩虫(Ancylostoma ceylanicum)的D-天冬氨酸蛋白酶相似性达65%。该蛋白具有两个保守的催化域,1个活性中心翼环,S2和S3亚位点各1个;具有由20个氨基酸组成的N端信号肽,构成疏水性强的跨膜域。不同浓度的IPTG(0.2～1.6 mmol/L)诱导对AsAP表达的影响较小,1.0 mmol/LIPTG诱导2 h后表达量达到最高水平。结论克隆并表达了简单异尖线虫的D-天冬氨酸蛋白酶。[英文文摘]Objective To clone and express the full length of D-like aspartic protease gene(AsAP) of the third stage larvae of Anisakis simplex.Methods According to the partial information of D-like aspartic protease encoding gene of A.simplex from GenBank,specific primers were designed to amplify 3′end and 5′ end of AsAP gene using rapid amplification of cDNA ends(RACE),and the full length of the D-like aspartic protease gene was obtained.Using total RNA of the third-stage larvae of A.simplex,coding sequence of the AsAP gene was amplified by reverse transcription-PCR(RT-PCR).The PCR product was digested by EcoRⅠ and SalⅠ,and cloned into pET32 vector.The recombinant plasmid was checked by double enzyme digestion and sequencing,and the positive recombinant plasmid was transformed into E.coli BL21(DE3).Expression of the protein induced by IPTG under gradient concentration and different time was conducted.Result A 1 753 bp full length of AsAP was obtained,which contained 30 bp 5′UTR,361 bp 3′UTR and a 1 362 bp open reading frame(ORF) encoding 453 amino acids with a predicted molecular mass of Mr 50 726.It showed 65% identity with the D-like aspartic protease of Ancylostoma ceylanicum.The predicted amino acid sequence contains two conserved catalytic motif,an active site flap,an S2 subsite and an S3 subsite.A 20 amino acids signal peptide was found in the N-terminus,with significant hydrophobic property.Different concentration of the IPTG(0.2~1.6 mmol/L) showed little effect on the expression,and the production of the protein was up to maximum after 2 hours induction.Conclusion The AsAP gene has been cloned and expressed.国家自然科学基金(No.81171595);福建省自然科学基金(No.2010J01229

Cloning and Expression of L-like Cysteine Protease of Anisakis simplex

目的克隆简单异尖线虫l-样半胱氨酸蛋白酶基因(ASCP)全长,研究其表达特性。方法根据gEnbAnk中简单异尖线虫表达序列标签l-样半胱氨酸蛋白酶基因的部分信息,设计特异引物,用CdnA末端快速扩增技术扩增3′端部分,获得基因全长序列。根据基因全长序列设计引物,以简单异尖线虫总rnA为模板,rT-PCr扩增ASCP基因编码序列,产物经ECOrⅠ和SAlⅠ双酶切,克隆至表达载体PET32а(+),转化大肠埃希菌bl21(dE3)株,以异丙基-β-d-硫代半乳糖苷(IPTg)诱导表达,表达效果经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SdS-PAgE)检测。结果3′端扩增片段大小为1211bP,拼接完整后基因全长1462bP,编码411个氨基酸,与秀丽隐杆线虫的l-半胱氨酸蛋白酶相似性达36.4%;重组载体PET32A(+)-ASCP经ECOrⅠ和SAlⅠ双酶切后有一条约1150bP的条带,测序结果显示重组载体构建成功。SdS-PAgE结果表明,重组蛋白相对分子质量约为Mr60000(含6个组氨酸的标签),与目的蛋白相符。用不同浓度的IPTg诱导对表达量的影响很小,1MMOl/lIPTg诱导2H后表达量达到最高水平。结论成功克隆并表达了l-样半胱氨酸蛋白酶。Objective To clone and express the full lenth of L-like cysteine protease gene of Anisakis simplex (AsCP) .Methods According to L-like cysteine protease encoding gene of A.simplex from GenBank EST database, specific primers were designed to amplify 3′-end of AsCP gene using rapid-amplification of cDNA ends (RACE), and the full lenth of the L-like cysteine protease gene was obtained.Specific primers were designed according to the full length of the gene.Using total RNA of A.simplex third-stage larvae, coding sequence of the AsCP gene was amplified by RT- PCR.The PCR product was digested by EcoR I and Sal I, and cloned into pET32а(+) vector.The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into Escherichia coli BL21(DE3).Expression of the protein induced by IPTG of gradient concentration (0.2, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mmol/L) and by the same concentration (1 mmol/L) of IPTG at different time(0, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h) was conducted.The expression situation of recombinant protein was analyzed by SDS-PAGE.Results A 1 211 bp of 3′-end of AsCP gene was amplified by 3′RACE, full length of the gene was 1 462 bp and coding 411 amino acids.It showed 36.4% identity with the L-cysteine protease of Caenorhabditis elegans.Double enzyme digestion of the constructed recombinant plasimid pET32a(+)-AsCP showed that there was about 1 150 bp fragment, the constructed recom- binant plasmid was then identified by sequencing.SDS-PAGE showed that the recombinant protein (Mr 60 000) was identical with the target.IPTG showed little effect on the protein expression, and the production of protein was up to maximum after 2 hours induction.Conclusion The AsCP gene has been cloned and expressed.福建省科技计划项目(No.2008N2005)---

Detection of Anisakid Nematodes by an SYBR Green Ⅰ Real-time PCR

目的运用荧光定量PCr法检测异尖线虫类病原体。方法于鱼类内脏中检获6种异尖线虫类幼虫:抹香鲸异尖线虫、简单异尖线虫、内弯对盲囊线虫、带鱼针蛔线虫、灰海鳗对盲囊线虫和台湾海峡鱼类中一优势种对盲囊线虫。提取各虫体dnA,PCr扩增ITS-2序列,测序并进行数据库比对。依据测序结果设计特异引物,常规PCr检验引物特异性。将ITS-2序列扩增产物回收、纯化后经T克隆转入大肠埃希菌dH5α,提取重组质粒,鉴定后作为标准品模板建立荧光定量PCr标准曲线,并做敏感性和重复性试验。结果构建的荧光定量PCr标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数均在0.998以上。重复性实验中,6种虫体对应的变异系数(CV)最小值为0.18%,最大值为2.80%,试验间平均CV最小值为0.55%,最大值为1.94%,无非特异性扩增,溶解曲线的特异性和重复性良好。灵敏度实验中,可检出的最低模板浓度为1x102拷贝/μl,比常规PCr灵敏度高100倍。结论初步建立了SybrgrEEnⅠ荧光定量PCr检测异尖线虫类病原体的方法 。Objective To establish an SYBR Green Ⅰ real-time quantitative PCR method for the detection of anisakid nematodes with zoonotic potential from Taiwan Strait.Methods Anisakid larvae of six species (Anisakis simplex, A.physeteris, Raphidascaris trichiuri, Contracaecum aduncum, C.muraenesoxi, and Contracaecum sp., a predominant species in fishes in the strait) were obtained from the guts of marine fishes and identified chiefly based on the morphological features.The ITS-2 rDNA sequences from the larvae were amplified by PCR using universal primers, then cloned and bidirectionally sequenced.According to these sequences, six specific forward primers were designed and synthesized.Specificity was determined by a series of conventional PCR respectively, the ITS-2 sequences amplified above were cloned into T vector which was subsequently transformed into E.coli DH5α.Following extraction and identification, the positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve.Sensitivity and reproducibility were determined.Results All the 6 standard curves established by the recombinant plasmids showed adequate linear relationship between threshold cycle(Ct) and template concentration.Melt curves were specific and all the 6 correlation coefficients were above 0.998.In the reproducibility test, the coefficients of variation (cv) of Ct values for detection of the 6 nematodes ranged between 0.18% and 2.80%, and the cv of the inter-assay ranged between 0.55% and 1.94%.The sensitivity of the real-time PCR was 1×102 copies/μl, about 100 times higher than the conventional PCR assays.The real-time quantitative PCR detection needed only 3.5 hours from the sample treatment to result report.Conclusion An SYBR Green Ⅰ fluorescent quantitative PCR has been developed for detecting anisakid nematodes with adequate sensitivity and specificity.福建省科技计划项目(No.2008N2005)---

The Research and Development of Agriculture Multi-media Expert Consultative System

在开发龙眼裁培技术多媒体专家咨询系统和花椰菜裁培技术多媒体专家咨询系统的基础之上 ,分析了多媒体农业专家咨询系统的知识特点 ,并以花椰菜裁培技术多媒体专家咨询系统为例 ,提出了基于农业的多媒体专家咨询系统的若干见解 .Based on the development of longan cultivate technique and cauliflower cultivate technique multi-media expert consultative system, this paper analyzes the knowledge characteristics of both systems, and proposes several ideas about multi-media agriculture expert consultative system

一种抗风浪实验围隔

本发明提供了一抗风浪实验围隔，包括支撑平台、2个以上围隔主体以及石笼，支撑平台由立杆和网状支架构成，网状支架由横杆和纵杆纵横交错编织构成，立杆与网状支架榫卯连接，立杆的下端位于水底的泥土中，围隔主体通过扣带与网状支架连接、其开口位于水面上方并且与网状支架的网孔相对，相邻两个围隔主体通过连接线系紧连接，连接线的端部系紧于立柱上；围隔主体的底部设有卷边，卷边中包裹有带扣环的钢丝绳或钢筋，扣环上设有线绳，扣环与石笼通过线绳连接，石笼以及卷边均埋于水底的泥层中，扣环中设有地锚桩；围隔主体通过硬质支撑平台支撑，多个围隔主体形成围隔群，整体结构强度高，保证风浪状态下围隔主体外的水体不会进入围隔主体内。</p

Feasibility of the Application of Work Sample Tests in Open Recruitment of Doctors

招聘到优秀的医疗人员是医院人事部门最为重要和紧迫的任务。鉴于传统的招聘方式存在明显的不足,借鉴国外和企业的招聘经验,可以考虑将工作样本测验应用于公开招聘中。通过分析招聘现状、医疗人员工作的特殊性及职责,结合工作样本测验的要素,探讨工作样本测验实施的步骤和方式,并讨论其存在的优点和问题

Perceptions on Justice and Distributive Justice

文章在住户调查数据的基础上,对居民关于收入分配的公平感及其与主客观因素之间的关联性进行了描述。结论显示,对不同收入来源的公平性评价与总体收入分配公平感之间通常具有较强的关联性;而这些公平性评价与个人年龄、教育程度以及收入水平之间也存在着较强的关联性。从总体收入分配公平性评价的回归分析中可推论,总体收入分配公平感较低可能源于实际收入决定机制与人们公正观念之间的背离。Based on the household survey,the paper discusses the public opinion on current income distribution and its relevance with the subjective perception and some objective variables.The basic finding shows,the conception on justice of different income sources has impacted on the distributive justice,which are highly correlated with the age,educational attainment,and income of the respondent.The regression results about the opinion on the justice of current income distribution might indicate the injustice of current income distribution by public opinion may result from the fact that the actual income generation mechanism deviates from the perceptions of justice on income determination.国家社科基金项目:经济增长的穷人受益性统计研究(09CTJ002