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)---

Y型聚乙二醇干扰素琢-2b注射液治疗HCV基因2/3型慢性丙型肝炎患者疗效和安全性的多中心随机对照试验研究

目的以标准剂量的聚乙二醇干扰素(Peg IFN)α-2a联合利巴韦林作为阳性对照,评价新型试验药物Y型Peg IFNα-2b注射液联合利巴韦林治疗2型/3型慢性丙型肝炎(CHC)患者的疗效和安全性。方法采用多中心、随机开放、阳性药对照的Ⅲ期临床试验,筛选符合要求的2型/3型CHC患者,按照2:1的比例随机分配到Y型Peg IFNα-2b组和Peg IFNα-2a组,同时口服利巴韦林,疗程24 w,停药随访24 w。采用Abbott Real Time HCV Genotype II检测HCV基因型,采用Cobas Taq Man实时定量PCR法检测血清HCV RNA水平。详细记录不良事件。主要疗效指标为持续病毒学应答(SVR),并进行非劣效检验。结果本试验实际入组2型/3型CHC患者255例,实际治疗241例。全分析集(FAS)数据显示,158例试验组和83例对照组患者SVR分别为85.4%(95%CI 79.94%~90.94%)和79.5%(95%CI 70.84%~88.20%,P=0.2402);对符合方案分析集(PPS)人群分析显示,试验组和对照组患者SVR分别为87.9%(95%CI 82.45%~93.27%)和85.9%(95%CI 77.82%~94.01%,P=0.7060),率差的95%可置信区间均符合非劣效标准;对PPS人群分析显示,85.8%受试者获得了早期病毒学应答(RVR),RVR的阳性预测值为90.1%;试验组和对照组不良事件发生率相似,分别为95.6%和95.2%,严重不良事件发生率分别为3.8%和3.6%。结论应用Peg IFNα联合利巴韦林治疗2型/3型CHC患者,新型试验药物Y型Peg IFNα-2b具有与对照药物Peg IFNα-2a相似的疗效和安全性。国家科技部“十二五”重大专项(编号:2012ZX10002-003)；“重大新药创制”十二五科技重大专项(编号:2012ZX09303019)

鲢鳙对鱼粪消化利用的研究

以鱼粪代表天然水体中有机碎屑,研究鲢、鳙对鱼粪的消化利用,并评价有机碎屑在鲢、鳙营养中的作用。在实验室条件下(水温30℃,溶氧6毫克/升以上,pH7—8,光照度2800lx),收集鲢、鳙摄食微囊藻后排出的粪作为试验用饲料。在限制摄食量的条件下,测定鱼对鱼粪的消化率及研究鱼粪对鲢、鳙生长的影响。研究结果是:(1)鲢、鳙对鱼粪中干物质消化率分别为58.54±9.53％及68.57±8.98％。其中蛋白质消化率分别为74.49±7.96％及80.41±9.02％,脂肪消化率分别为90.23±15.33％及84

白鱀豚肌肉生化成分分析初报

<正> 白鱀豚(Lipotes vextllifer Miller)是我国特有的一种珍稀淡水鲸类。在学术上有特殊的重要价值。在五十年至七十年代,周开亚等曾作过白鱀豚的形态解剖及地理分布的一些零星报道。近十年来中国科学院水生生物研究所对白鱀豚的形态解剖、地理分布、生活习性、生物学特征以及组织学进行了较全面系统的研究,并发表了有关研究报告(陈佩薰等1980,1985;刘仁俊等1980;林克杰等1985;李钟杰1985)。但至今还未见到

我国淡水优质草食性鱼类的营养和能量学的研究——Ⅲ.草鱼、团头鲂对食物选择性的初步研究

<正> 本研究在特定的实验条件下,以不同种类食物等量混合饲养草鱼(Ctenopharyngodon idella)和团头鲂(Megalobrama amblycephala),根据各类食物的投饵量和摄饵量之间的百分比,求得各类食物选食指数,并以鱼日增重率和饵料系数探讨这两种鱼对动物和植物性食物的营养要求。为研究草食性鱼类的营养和能量学提供基本参数

鲢、鳙在东湖生态系统的氮、磷循环中的作用

我们研究了鲢、鳙在停食状况下氮、磷的排泄量及在有鱼及无鱼的水环境中鱼类及微囊藻的氮、磷释放率。结合有关参数进行换算,从量的方面评价了鲢、鳙在东湖生态系统物质循环中所起作用:①鲢、鳙摄食过程加速了水体氮、磷释放进程(有鱼水体氮、磷释放率分别为无鱼水体的1.88和1.41倍),但其释放量(粪便的氮、磷释放量分别为水体氮、磷总含量的11.45％和3.4％)不足以左右东湖水体初级生产量的变动;②鲢、鳙摄食过程一方面提高了对初级生产量的利用率,而另一方面却通过鱼体积贮从水体中移出大量氮(52.20吨)、磷(11.