Sequence anlysis of rDNA of Anisakid nematodes with zoonotic potential from Taiwan strait

目的分析台湾海峡异尖线虫病潜在病原线虫的rdnA序列,为建立病原分子生物学鉴别提供依据。方法剖检台湾海峡常见鱼类的寄生线虫幼虫,镜检分类。提取虫体dnA、用通用引物对rdnA进行PCr扩增、克隆、双向测序及blAST比对。结果获得6条来自6种线虫幼虫的rdnA序列,其中的3种在gEnbAnk数据库中没有发现匹配的序列;证实在台湾海峡分布的灰海鳗对盲囊线虫成虫和疑似其第三期幼虫实属同种;构建了基于ITS-2的序列间内部相似性程度关系树。结论异尖线虫病原幼虫所具有的分子生物学鉴别信息位点主要集中在ITS-2间隔区。To analyze the rDNA sequence of the Anisakid nematodes with zoonotic potential from Taiwan straits,Anisakid nematodes were obtained from the gut of marine fish and identified chiefly based on the morphological characteristics.The genomic DNA of nematodes were extracted,and the sequence of rDNA ITS were amplified by PCR using universal primers,then cloned and sequenced bidirectionally.Sequences analysis was conducted by blasting database and software DNAMAN.Results of the blasting database showed that no match could be demonstrated in 3 of 6 rDNA sequences obtained from 6 different nematode species;the new sequences were submitted to the database and the accession numbers in GenBank were obtained.The relationship between adult Contracaecum muraenesoxi and its putative third stage larva was validated by molecular evidences.Sequences relationship based on the inner similarity of the internal transcribed spacer sequences was constructed.It was found that the phylogenetic informative loci were mainly gathered in the second internal transcribed spacer(ITS2) for these anisakid nematodes because of the ITS2 has higher transition ratio than that of the ITS1.It is evident that the sequence in this region could provide valuable information for the molecular distinction of anisakid nematodes with zoonotic potential from Taiwan straits.福建省科技计划项目(2008N2005);厦门市科技计划项目(3502Z20074036

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)---

Expression Analysis of a Stress Repressed Gene OsDSR4 from DUF966 Family and Generation of OsDSR4- overexpressing Transgenic Rice(Oryza sativa L. ssp. japonica)

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