草鱼(Ctenopharyngodon idellus)呼肠孤病毒(GCRV)VP6相互作用蛋白的筛选

为筛选与草鱼呼肠孤病毒GCRV096 VP6发生相互作用的宿主蛋白,先将VP6基因的PCR扩增产物,克隆至酵母表达载体p GBKT7以构建其诱饵质粒(p GBKT7-VP6);再将酵母菌Y2HGold(含p GBKT7-VP6)与酵母菌Y187[含草鱼肾脏细胞(CIK)c DNA文库质粒]进行人工诱导融合,然后经选择培养筛选阳性克隆。结果共筛选到4株阳性克隆,经测序、生物信息学分析,分别属于两条不同的核苷酸序列,其中之一所编码的产物与GAPDH(3-磷酸甘油醛脱氢酶)同源性较高。可见,该酶极可能在GCRV096侵染宿主的初期发挥着重要作用。     

应用酵母双杂交系统筛选虾夷扇贝FOXL2的相互作用蛋白

FOXL2是脊椎动物的雌性决定基因,在多种无脊椎动物中也被认为是雌性决定与分化的关键基因,但其在无脊椎动物中的作用机制目前尚不清晰。本文利用Clontech酵母双杂交系统筛查虾夷扇贝FOXL2的相互作用蛋白。研究首先构建诱饵质粒pGBKT7-FOXL2,并检测诱饵蛋白的毒性及自激活活性,结果显示诱饵蛋白对酵母菌株无毒,也无自激活活性。利用该诱饵质粒,本研究从虾夷扇贝酵母双杂交cDNA文库筛选得到17个阳性克隆,测序验证得到12个读码框正确的阳性克隆,它们来自4种蛋白:COP9信号复合体亚基5、钠/钾ATP酶β3、哺乳动物室管膜相关蛋白1和胰脂肪酶相关蛋白2。研究进一步将诱饵质粒与4种蛋白的猎物质粒进行共转化验证,结果均为阳性,表明这4种蛋白与虾夷扇贝FOXL2都可能存在相互作用。本文为研究FOXL2在无脊椎动物性别决定与分化中的作用提供了参考。     

利用酵母双杂交研究铜绿微囊藻生物钟蛋白KaiB的自身相互作用

从铜绿微囊藻(Microcystis aeruginosa)PCC7820中克隆了生物钟基因kaiB,并将其克隆入酵母双杂交系统的两个质粒pGADT7和pGBKT7中。经测序验证后,将重组质粒pGADT7-kaiB和pGBKT7-kaiB转化酵母菌AH109进行自激活性和自身相互作用的检测和验证。结果表明,铜绿微囊藻生物钟蛋白KaiB无自激活性,KaiB蛋白自身能发生相互作用。因此,可用KaiB蛋白为诱饵利用酵母双杂交系统筛选文库钓取与KaiB发生相互作用的蛋白。     

草鱼出血病病毒VP7蛋白基因的人工合成与原核表达

根据GenBank已发表的GCRVVP7蛋白基因序列,设计合成2条特异性引物和19条搭桥引物,通过搭桥PCR人工合成带有3处变异碱基的GCRVVP7蛋白基因整个编码框,经过酶切、连接、PCR鉴定和测序鉴定克隆到原核表达载体pColdTF中,并转化大肠杆菌BL21(DE3),进行IPTG诱导表达和表达产物的SDS-PAGE、Western-blotting分析、纯化以及反应原性的iELISA检测。结果表明,成功人工合成了长831bp的GCRV VP7蛋白基因编码框和构建了VP7蛋白基因的重组质粒pCold TF-VP7,pColdTF-VP7转化菌经IPTG(1mmol/L)诱导3—4h即可获得特异性蛋白VP7重组蛋白的高效表达,VP7重组蛋白相对分子量大小约为73.9ku,与预期大小相符,且与鼠抗GCRV血清有良好反应原性。     

酵母双杂交系统用于WSSV结构蛋白VP39相互作用的宿主蛋白基因的初步研究

为研究WSSV的结构蛋白VP39相关蛋白的基因,应用酵母双杂交系统筛选中国明对虾(Fenneropenaeus chinensis)鳃细胞全长cDNA文库。将构建好的VP39基因的重组载体pGBKT7-VP39转化酵母Y187后,与含文库质粒的酵母株AH109融合,在营养缺陷培养基上进行反复筛选,得到1个阳性克隆,测序并进行生物信息学分析。结果显示,此cDNA片段在数据库中是新序列,该蛋白在WSSV识别并结合宿主的过程中所起的作用还有待于进一步研究。本实验成功筛选了VP39相关蛋白的cDNA片段,为进一步研究WSSV的致病机理奠定了基础。     

牙鲆NPY的体外表达及体内检测

神经肽Y(Neuropeptide Y,NPY)被普遍认为是一种重要的促食因子,在调节鱼类的摄食行为方面起着至关重要的作用。为进一步研究牙鲆NPY蛋白的生物学功能,作者克隆了牙鲆NPY成熟肽序列(m-NPY)及含有信号肽的全长序列(f-NPY),利用原核表达系统分别进行体外重组表达,筛选出诱导剂IPTG最佳诱导浓度为0.8 mmol/L及最佳诱导时间3 h;此外,根据牙鲆NPY第49-64位氨基酸序列制备了多克隆抗体并通过Western blot验证该多抗能够有效检验重组NPY及牙鲆体内NPY的表达。研究结果为研究重组牙鲆NPY蛋白在牙鲆水产养殖产业中的应用及检测提供了依据。     

草鱼(Ctenopharyngodon idellus)出血病二价细胞灭活疫苗体液免疫效果及免疫保护率评价研究

草鱼出血病是由草鱼呼肠孤病毒(GCRV)引起的草鱼养殖业一种严重的病毒性出血病,给草鱼养殖产业造成重大损失。该病毒是一种双链分节段RNA病毒,根据GCRV多个病毒分离株基因序列分析及临床发病特点,共有三种基因型GCRV,其中基因Ⅰ型和Ⅱ型较为流行。通过细胞分离培养基因Ⅰ型(GCRV-ZV8909)和Ⅱ型(GCRV-HZ13) GCRV,研制出草鱼出血病二价细胞灭活疫苗,通过绝对定量的方法,测定基因Ⅰ型和Ⅱ型的病毒含量分别为2.0×10⁹及1.5×10⁶ copies/mL。用生理盐水将其分别进行50倍稀释和100倍稀释,包括原液及生理盐水对照共4组,分别腹腔注射免疫(20±5)g健康草鱼,剂量均为0.2mL/尾,每组40尾,各组于免疫后3、5、7、14、21、28、42、56、70、84 d分别取3尾采集血液制备血清。分别利用经原核表达并纯化制备两种基因型GCRV的VP7蛋白以及实验室保存的草鱼IgM单抗,建立了三抗体夹心酶联免疫方法(TAS-ELISA)分析评价疫苗体液免疫效果。结果表明,草鱼经腹腔注射不同剂量的细胞灭活疫苗后,与对照组相比,各免疫组血清抗体均有不同程度的提高,且原液组与50倍稀释组基本持平,但都高于100倍稀释组,各免疫组在免疫后5d即可检测到两种基因型GCRV抗体,且抗体水平持续到84d,但两种基因型抗体水平达到高峰的时间不一致,抗基因Ⅰ型GCRV抗体达到高峰是在28d,抗基因Ⅱ型GCRV抗体是在14d达到高峰。两种基因型抗体水平达到高峰时的血清效价分别为1︰800、1︰600。免疫保护率实验结果表明,原液组保护率最高,为67%,50倍稀释组为60%,100倍稀释组只有33%。     

锯缘青蟹(Scylla serrata)呼肠孤病毒5个结构蛋白互作分析

采用酵母双杂交系统,对青蟹呼肠孤病毒(Scylla serrata reovirus)5个结构蛋白(VP3,VP6,VP9,VP11,VP12)间的双向互作进行了分析。将5个结构蛋白对应的ORF分别亚克隆至诱饵载体p GBKT7和猎物载体p GADT7,成功构建了10个酵母双杂交重组载体。将重组诱饵载体或重组捕获载体转化至酵母菌Y2H Gold中进行自激活检测,结果显示5个结构蛋白均不能激活酵母菌报告基因HIS3的表达,表明5个病毒蛋白均不具有自激活活性。通过酵母双杂交实验,从5个结构蛋白25个可能性互作对中,共筛选出2个双向互作对(VP11VP6,VP11VP12)和3个自身互作对(VP3VP3,VP11VP11,VP12VP12)。本研究是有关Ss RV结构蛋白互作的首次报道。     

致病性鳗弧菌W-1外膜蛋白ompU基因克隆及在大肠杆菌中的表达

从致病性鳗弧菌W-1基因组DNA扩增并克隆了1 156bp的特异性片段,含有完整的外膜蛋白ompU基因阅读框,由993个核苷酸组成,编码330个氨基酸残基的蛋白质。与国外已发表的鳗弧菌外膜蛋白基因的序列同源性为100%,与创伤弧菌(Vibrio vulnificus)、霍乱弧菌(Vibrio cholerae)、费氏弧菌(Vibrio fischeri)和副溶血弧菌(Vibrio parahaemolyticus)的序列同源性分别为72%,69%,68%和64%。将该外膜蛋白基因克隆于pBV220表达质粒,在大肠杆菌中得到了表达,SDS-PAGE分析表明表达蛋白分子量约38kDa,Western blot分析发现表达蛋白能与鳗弧菌外膜蛋白抗体很好的反应。     

Expressed sequence tag analysis and cloning of trehalose-6-phosphate synthase gene from marine alga Laminaria japonica (Phaeophyta)

A high quality cDNA library was constructed from the brown alga Laminaria japonica,with the titer of 1.2×10 5 pfu/ml.The average insert size of the cDNA library is about 1.6 kb.From the cDNA library,591 cDNA clones were randomly selected and sequenced.As a result,574 EST(expressed sequence tag) sequences were generated.All of the 574 ESTs were submitted to the dbEST database section of GenBank with the accession numbers from CX942625 to CX943198.The cDNA library was screened with a α-32 p labeled 453 bp T P S gene probe,which is a partial sequence yielded from Porphyra yezoensis.Four positive cDNA clones were screened and the sequencing data showed that these four cDNA clones covered majority of L.japonica TPS cDNA sequence.After PCR amplification,sequencing and assembling,the entire ORF(open reading frame) sequence of the T P S gene was obtained,which was named LjTPS.LjTPS encodes a protein containing 908 amino acids with a calculated molecular mass of 101 674 Daltons.The LjTPS gene was successfully expressed in E.coli and rice.The LjTPS gene has potential application both in plant breeding to stress tolerance and in deciphering the T P S gene function and mechanism to stress tolerance.     

刺参肌肉组织cDNA文库的构建

用RNA提取试剂--TRIZOL Reagent提取刺参(Apostichopus japonicus)肌肉组织总RNA,用SMART cDNA Library Construction Kit构建cDNA文库.经测定原始文库滴度达到 3.2×10~6,扩增后文库滴度达到 5.1×10~9,重组率达到96.7%,从扩增文库随机挑取12 个克隆进行PCR 扩增鉴定,结果显示,插入片段大小为0.5～2.5 kb.通过各项指标验证成功构建了刺参肌肉组织cDNA 文库.     

不同引物扩增对东海陆架沉积物真核微型生物多样性的影响

采用三对通用引物(ITS1/ITS4,EF3/EF4,NS1/NS4)检测其对东海陆架DH-13站点表层沉积物真核微型生物的扩增特异性和多样性差异。对323个有效克隆子进行OTU分型和多样性分析,结果表明,NS1/NS4引物扩增所获得的文库多样性最高,EF3/EF4其次,ITS1/ITS4最后。通过NCBI比对和分类学研究,发现优势种群主要为原生生物界,包括丝足虫门(Cercozoa,占所有OTU的36.4%)、横裂甲藻纲(Dinophyceae,占14.2%)、不等鞭毛门(Stramenopiles,占7.4%)和纤毛虫门(Ciliophora,占6.3%);另含部分真菌界如子囊菌门(Ascomycota,占7.4%)和壶菌门(Chytridiomycota,占6.3%)。原生生物方面,NS1/NS4引物表现的多样性最高,适合于扩增丝足虫门、横裂甲藻纲和不等鞭毛门,并扩增到独有的眼虫门(Euglenozoa)、隐藻门(Cryptophyta)和顶复门(Apicomplexa);ITS1/ITS4适合于扩增甲藻和纤毛虫门;EF3/EF4则适合于丝足虫门。真菌方面,ITS1/ITS4适合于扩增子囊菌门;EF3/EF4适合于子囊菌门和壶菌门,而NS1/NS4适合于壶菌门。系统发育分析表明,三对引物的原生生物系统树中的相似序列多来自海洋环境样品,真菌序列则分别来自陆地土壤真菌和海洋真菌,说明三对引物更适合于海洋沉积环境微型原生生物的分子生态分析。     

刺参(Apostichopus japonicus)高温胁迫消减cDNA文库的构建与分析

采用抑制性消减杂交技术(SSH)研究刺参高温胁迫下差异表达的基因.分别以高温实验组为检测组(tester)、常温对照组为驱动组(driver),进行正向抑制性消减杂交;以常温组为tester、高温组为driver,进行反向消减杂交,成功构建了刺参正反双向差异消减文库.从两个文库随机挑选384个白斑克隆进行斑点杂交进一步...     

Isolation of CYP2L2 and two other cytochrome P450 sequences from a spiny lobster, Panulirus argus, hepatopancreas cDNA library

Cytochrome P450 monooxygenases constitute an enzyme superfamily, with at least 74 known families. Members of CYP families 1–4 are important in the phase 1 metabolism of lipophilic xenobiotics, such as those found in contaminated marine environments. Previous studies (James et al. Arch. Biochem. Biophys. (1996) 329, 31–38) showed that a major form of P450 in spiny lobster, Panulirus argus, hepatopancreas was CYP2L1, a new sub-family, and that there was evidence for other P450 forms in hepatopancreas. We now report the sequence of a second member of this subfamily, named CYP2L2, present in P. Argus hepatopancreas. The deduced amino acid sequences of CYP2L1 and CYP2L2 share 54.7% sequence identity and an additional 13.6% of the sequences show conservative substitutions. Analysis of the sequences of CYP2L1, CYP2L2 and other representative CYP2 family members (from rat and mouse sub-families 2A, 2B, 2D and 2E) showed that the crustacean sequences clustered together. In addition to CYP2L2, cDNA clones of 66 to 117 base pairs from the 5′ coding region of two more P450 isoforms were isolated from the spiny lobster cDNA library. The deduced amino acid sequence of one of these additional cDNA clones was identical to the first 22 amino acids of the N-terminal sequence of a P450 protein previously isolated from hepatopancreas microsomes. These studies confirm earlier biochemical evidence that the hepatopancreas contains multiple forms of cytochrome P450.     

Construction of full length cDNA expression library of hepatopancreas of Penaeus monodon

mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyAT-tract System 1000 Kit. By using mRNA as template, double - strand cDNA with EcoR I/Xho I ends was synthesized by using a ZAP Express cDNA Synthesis Kit. The cDNA was inserted into the lambda ZAP Express vector predigested with EcoR I/Xho I, and the recombinant DNA was in vitro packaged into lambda phage with GigapackIII Gold packaging extracts. These recombinant phages were then used to transfect E. coli XL1 - Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2 × 10~5 pfu in capacity and its recombination ratio is higher than 99 % . The size of the inserted cDNAs was determined by EcoR I/Xho I digestion of 9 phagemids prepared by in vivo excision of plaques selected randomly from amplified cDNA library . The longest inserted cDNA is about 1.6 kb in length. The complete sequence (about 1.2 kb) of actin cDNA was amplified from the library by PCR reveals that this library contains full-length