猪蛔虫性别特异基因在第三、四期幼虫的表达谱研究

为研究猪蛔虫性别特异基因在第三期、第四期幼虫的表达情况，本研究采用表达谱基因芯片技术，从所构建的猪蛔虫雌、雄成虫cDNA消减文库中分别挑取克隆，制备成cDNA微阵列芯片、标记有荧光素Cy5-dUTP和Cy3-dUTP的各组探针（L3＋♀；L3＋♂；L4＋♀；L4＋♂）分别与制备好的芯片进行杂交、扫描，原始信号值经均一化处理后，获取每个点的Ratio值（Ratio=Cy5／Cy3）．根据该值筛选出表达差异最明显的841个克隆，进行测序分析，在获得的707个有效序列中有61个是猪蛔虫新的ESTs、将在第三、四期幼虫以及成虫中显著表达的克隆进行排列组合比较，获得各期特有和各期之间共有的表达克隆一在第三期幼虫中，雌、雄性别特异基因分别有21和9个，编码的主要蛋白有卵黄原前导蛋白、睾丸幼胚蛋白等；在第四期幼虫中特异表达的雌、雄性别基因分别有22和6个，其中免疫抑制卵巢信息蛋白、主要精子纤维蛋白（MFP）等表达明显本项研究结果不仅初步阐明了猪蛔虫性别特异基因在第三、四期幼虫的表达情况，而且亦为筛选控制猪蛔虫病的“关键”基因并阐明其功能奠定了基础。     

犬弓首蛔虫卵黄原蛋白基因的克隆及表达分析

为克隆犬弓首蛔虫(T.canis)的卵黄原蛋白(YP)基因,本研究根据T.canis cDNA文库中的EST数据,采用RT-PCR方法扩增T.canis的YP基因(TcF-YP),并克隆至pGEM-T载体中进行测序。测序结果表明,TcF-YP基因片段大小为492 bp,编码163个氨基酸残基。同源分析显示,TcF-YP基因与猪蛔虫的卵黄原蛋白基因同源性最高,为78%。同时对该基因在T.canis雌虫、雄虫和雌虫各组织的表达谱进行研究,结果显示TcF-YP基因只在T.canis雌虫中高量表达,是雌虫特异的基因。对雌虫各组织的基因表达谱分析显示TcF-YP基因在子宫和肠中高量表达,在卵巢中有极微量的表达。本实验为TcF-YP基因的功能研究奠定了基础。     

基于高通量测序的猪鞭虫雌、雄虫差异基因表达分析

为了探明猪鞭虫(Trichuris suis)雌、雄虫的转录组差异,丰富T.suis转录组数据信息,利用Illumina Hiseq2000对临床采集的T.suis雌、雄虫进行高通量测序,将组装得到的Unigenes在NR、GO、COG和KEGG数据库中比对注释,并进行差异表达基因分析。结果,T.suis雌、雄虫分别获得59 657 854条和48 414 184条高质量测序数据,分别组装获得21 026个和28 886个Unigenes。注释到NR、GO、COG和KEGG数据库的雌虫Unigenes数量分别为11 700、2 287、1 650和9 690个,雄虫Unigenes数量分别为12 902、2 414、1 791和10 377个,在NR数据库比对显示84.90%以上信息均来源于猪鞭虫。T.suis雌、雄虫共有4 320个差异表达基因。以雌虫为对照,雄虫中共有3 496个基因表达上调,有824个基因表达下调。共有906个差异表达基因获得160个KEGG通路富集,其中显著富集通路7个。在T.suis转录组中发现了许多重要基因的表达,包括与T.suis繁殖相关的主要精子蛋白、组蛋白H2A/H2B及卵黄蛋白原,与T.suis致病性、免疫及炎症调节相关的蛋白酶、丝氨酸蛋白酶抑制分子和半胱氨酸蛋白酶抑制分子,以及众多未知功能的基因。本研究揭示了T.suis雌、雄虫差异表达基因的数量,获得了这些差异表达基因的功能、分类和代谢通路注释,为丰富T.suis转录组信息,揭示鞭虫与宿主互作特点及抗鞭虫靶点药物研发奠定基础。     

日本血吸虫WD40信号蛋白的原核表达

本研究克隆了编码日本血吸虫WD40信号蛋白的cDNA,并分析了WD40在日本血吸虫不同发育时期的转录情况.同时还利用原核表达系统对该蛋白的部分序列进行了表达,并进行了重组蛋白的纯化和多克隆抗体的制备,Western blot分析表明本研究中获得多克隆抗体可与重组表达的蛋白和血吸虫全虫蛋白裂解物具有特异性反应.本试验结果为进一步研究WD40信号分子的功能奠定了初步基础.     

日本血吸虫ELAV-like 2基因的克隆、表达及功能分析

日本血吸虫成熟雌虫所产虫卵是造成宿主病理损害及疾病再传播的根源,因此从控制雌虫生殖发育入手对控制血吸虫病的发生具有重要意义。本研究通过5′-RACE和3′-RACE获得了在发育阻遏雌虫中高表达的SjELAV-like 2基因的全长序列,并对其进行克隆和表达,制备了相应的多克隆抗体,Western blotting分析结果表明该蛋白质具有良好的反应原性;实时定量分析发现该基因在血吸虫童虫时期高表达,成熟后趋于稳定,且在雌虫体内的表达量显著高于同时期合抱雄虫;该基因在雄虫体内的表达量基本恒定;免疫组化研究发现,在血吸虫成虫雌虫该蛋白主要定位于虫体体被。利用RNA干扰技术对该基因的生物学功能进行了初步研究,RT-qPCR结果显示,RNAi可明显抑制SjELAV-like 2基因的表达;对该基因表达的长期干扰导致受干扰虫体产卵能力及卵胚孵化能力的下降,统计分析发现,干扰组宿主每克肝荷卵减少达39.67%(P0.01),虫卵的卵胚孵化减少达75.56%(P0.01)。扫描电镜观察发现,与NC组相比,siRNA干扰组虫体体被上泡状突出物明显增多,这些结果表明SjELAV-like 2基因对日本血吸虫生长发育具有重要影响。     

猪蛔虫雄虫cDNA文库的构建

采用Tripure Isolation Reagent抽提猪蛔虫雄虫成虫的总RNA,用poly(A)PuristTM纯化试剂盒分离mR-NA。分离mRNA后,用Clontech公司的CreatorTMSMARTTMcDNA文库构建试剂盒构建了猪蛔虫雄虫成虫的cDNA文库。结果获得了7.26×105独立克隆,重组率达96.7%,插入片段的平均长度约为1 kb。猪蛔虫雄虫cDNA文库的成功构建为利用文库筛选雄虫差异表达基因提供了材料来源,为研究猪蛔虫性别发育的分子机制奠定了基础。     

人瘦素基因的克隆及原核表达

为了获得大量的、可供试验研究和制作生物反应器的人瘦素蛋白,从人脂肪组织中提取总RNA,用RT-PCR法获得了编码人瘦素(leptin)167个氨基酸残基的cDNA序列,并克隆至载体pMD19-TSimple上进行序列分析。以克隆的人瘦素cDNA为模板,用特异引物扩增瘦素基因,经EcoRI和BamHI双酶切,定向插入高效原核细胞表达载体pGEX-6P-1中,构建重组质粒并转化大肠杆菌BL21(DE3),通过IPTG诱导表达,进行SDS-PAGE检测。结果:用RT-PCR法扩增得到的cDNA片断序列分析显示,克隆的基因序列和GenBank公布的人瘦素基因序列一致;SDS-PAGE分析基因表达产物,在20 ku处有一特异条带,说明重组质粒在大肠杆菌BL21(DE3)中可表达人瘦素蛋白。     

亚洲璃眼蜱唾液腺差异表达基因文库的构建及分析

从单雌蜱克隆群中挑选未吸血雌蜱60只，随机分成两组。未吸血组直接剖取唾液腺．半饱血组于蜱吸血第5d采集，分离唾液腺。Trizol法提取总RNA，经第一链合成、LD—PCR、RasⅠ酶切、接头连接和抑制消减杂交（SSH）等步骤，获得差异表达基因的cDNA片段。将纯化的cDNA片段与pGEMT—Easv我体连接，转化DH5a，获得204个白色菌落。扩增检查表明，136个克隆含有插入片段，片段大小为250bp-850bm，测出有效序列120个。由10个cDNA片段的RT—PCR检查结果初步断定，本研究消减效果良好。由网上资源分析得：21个片段与其他蜱的基因，19个片段与按蚊、库蚊、钩虫、奥斯特线虫等其他吸血寄生虫的基因，9个与果蝇的基因具有同源性。     

镰形扇头蜱组胺结合蛋白全长基因的克隆与分析

从本实验室构建的镰形扇头蜱（Rhipicephalus haemaphysaloides）抑制消减杂交cDNA文库中选择了1条可能编码组胺结合蛋白（HBP）的EST序列（RhHBP）,以5′末端快速扩增的方法（5′RACE）扩增获得5′末端序列,拼接获得全长基因。DNAMAN（v4.0）、DNAstar（v4.0）和Genetyx（v4.0）软件分析阅读框、编码产物、序列同源性和系统进化,同时也分析了该基因在吸血前后雌雄蜱体内的表达情况。5′RACE法获得1条约400 bp的DNA片段,克隆测序,拼接获得1条长803 bp的全长基因,编码219个氨基酸残基。该基因在吸血雌蜱唾液腺内特异表达,初步分析该基因是组胺结合蛋白基因。     

环形泰勒虫3-磷酸甘油醛脱氢酶的原核表达、多克隆抗体制备及亚细胞定位

为了研究环形泰勒虫3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)的功能,本试验利用PCR技术从环形泰勒虫裂殖体cDNA中扩增环形泰勒虫GAPDH (TaGAPDH)基因,将扩增产物克隆到原核表达载体pET-30a(+)中,并转入大肠杆菌BL21(DE3)中进行融合蛋白诱导表达,融合蛋白纯化后免疫家兔,制备多克隆抗体,分别用间接ELISA和Western blotting检测抗体的效价和特异性;利用激光共聚焦荧光显微镜观察TaGAPDH蛋白在环形泰勒虫裂殖体中的亚细胞定位.结果显示,克隆得到了TaGAPDH全长基因,大小为1 020 bp;SDS-PAGE分析结果显示,融合蛋白大小约44 ku,且以包涵体形式存在.制备的多克隆抗体效价高达1:12 800,具有很高的特异性;亚细胞定位显示TaGAPDH蛋白主要分布于环形泰勒虫裂殖体细胞质内.以上结果表明本试验成功克隆表达了TaGAPDH基因,并制备了针对TaGAPDH蛋白的兔多克隆抗体,为筛选环形泰勒虫病疫苗、药物靶点及研究环形泰勒虫能量代谢奠定了基础.     

猪蛔虫感染期幼虫抑制消减cDNA文库的构建

为筛选与线虫感染性相关的基因,本研究以猪蛔虫为对象,构建猪蛔虫感染期幼虫差异表达消减cDNA文库,为研究线虫期特异性发育的分子机制奠定基础。分别提取感染期幼虫和其它各期幼虫及成虫的总RNA,纯化mRNA后,采用Clontech公司PCR-selectTM试剂盒进行反转录合成cDNA并进行抑制消减杂交(SSH),构建猪蛔虫感染期幼虫差异表达的消减cDNA文库,并采用Southern斑点杂交进行消减效率的检测。随机从文库中抽取45个克隆进行测序及在线BLAST分析。试验结果表明,感染期幼虫差异表达的消减cDNA文库具有较强的特异性;在得到的41个表达序列标签(ESTs)中,有40个ESTs与已报道的基因有较高的相似性,主要代表猪蛔虫第三期幼虫基因和成虫头部基因,有1个cDNA片段可能代表新基因。猪蛔虫感染期幼虫差异表达消减cDNA文库的成功构建,为进一步研究幼虫发育差异表达基因的功能奠定了基础。     

犬弓首蛔虫雄虫cDNA文库构建及EST分析

为构建T.canis雄虫cDNA文库,采用Trizol法提取T.canis雄虫的总RNA,合成cDNA,连接到λTripEx2载体上,通过包装蛋白对连接产物的包装,接种到大肠杆菌XL-1-Blue中进行原始文库和扩增文库的滴度测定.经质量鉴定表明:初始文库的滴度为5.25×106 pfu·mL-1,扩增后文库的滴度为6.90×109 pfu ·mL-1.文库的插入片段大小在500～2 000 bp,平均片段大小为1 000 bp,重组率为99.47％.所有指标均显示已成功构建了T.canis雄虫的cDNA文库.利用该文库获得了189条5'有效表达序列标签(EST).对ESTs拼接后代表了101个Unigenes,含有27个Contigs和74个Singletons.其Unigenes在GenBank中的序列号为HO348195～HO348295.同源性分析检索到有56个Unigenes与已知基因同源,其中具有已知或推测功能的基因有40个,未知功能基因有16个,未比对上的基因45个.未比对上的基因与NR数据库中的蛋白序列没有任何意义的匹配,为研究中发现的新基因.这些结果为进一步开展犬弓首蛔虫功能基因及分子机制研究奠定了基础.     

Identification and expression profiles of multiple genes in Nile tilapia in response to bacterial infections

To understand the molecular mechanisms involved in response of Nile tilapia (Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6h post infection with Aeromonas hydrophila. A total of 31 unique expressed sequence tags (ESTs) were identified from 192 clones of the subtractive cDNA library. Quantitative PCR revealed that nine of the 31 ESTs were significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(5)CFU per fish (≈ 20% mortality). Of the nine upregulated genes, four were also significantly (p<0.05) induced in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(6)CFU per fish (≈ 60% mortality). Of the four genes induced by A. hydrophila at both injection doses, three were also significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with Streptococcus iniae at doses of 10(6) and at 10(5)CFU per fish (≈ 70% and ≈ 30% mortality, respectively). The three genes induced by both bacteria included EST 2A05 (similar to adenylate kinase domain containing protein 1), EST 2G11 (unknown protein, shared similarity with Salmo salar IgH locus B genomic sequence with e value of 0.02), and EST 2H04 (unknown protein). Significant upregulation of these genes in Nile tilapia following bacterial infections suggested that they might play important roles in host response to infections of A. hydrophila and S. iniae.     

西农萨能羊泌乳高峰期和初期乳腺组织差异表达基因研究

利用抑制性削减杂交和实时定量PCR研究西农萨能羊泌乳高峰期差异表达基因,结果表明成功构建泌乳高峰期和泌乳初期乳腺组织差异表达削减eDNA文库,以GAPDH为指标检测文库削减效率为2^5倍,共获得78个阳性克隆,PCR检测插入片段主要分布在150～1000bp,挑选插入片段不等的30个克隆测序,获得25个有效序列,代表18个基因。对文库中所包含的血清淀粉样蛋白A3（SAA3）,ATP结合盒亚家族G成员2（ABCG2）,心脏型脂肪酸结合蛋白（H-FABP）和黄嘌呤脱氢酶（XDH）进行实时定量PCR检测,发现上述4个基因在泌乳高峰期乳腺组织中的表达水平分别是泌乳初期的17．0,7．7,16．3和1．7倍。结论：构建的消减文库可用于筛选泌乳高峰期差异基因,已鉴定的4个基因很可能是调控产奶量和乳成分变化的候选基因。     

Efficacy of QCDCR formulated CpG ODN 2007 in Nile tilapia against Streptococcus iniae and identification of upregulated genes

The potential of using a QCDCR (quilA:cholesterol:dimethyl dioctadecyl ammonium bromide:carbopol:R1005 glycolipid) formulated CpG oligodeoxynucleotide (ODN), ODN 2007, to confer protection in Nile tilapia against Streptococcus iniae infection was evaluated in this study. At two days post treatment, QCDCR formulated ODN 2007 elicited significant (P<0.05) protection to Nile tilapia, with relative percent survival of 63% compared to fish treated by QCDCR alone. To understand the molecular mechanisms involved in the protective immunity elicited by ODN 2007, suppression subtractive cDNA hybridization technique was used to identify upregulated genes induced by ODN 2007. A total of 69 expressed sequence tags (ESTs) were identified from the subtractive cDNA library. Quantitative PCR revealed that 44 ESTs were significantly (P<0.05) upregulated by ODN 2007, including 29 highly (>10-fold) and 15 moderately (<10-fold) upregulated ESTs. Of all ESTs, putative peroxisomal sarcosine oxidase was upregulated the highest. The 69 ESTs only included six genes that had putative functions related to immunity, of which only two (putative glutaredoxin-1 and carboxypeptidase N catalytic chain) were confirmed to be significantly upregulated. Our results suggest that the protection elicited by ODN 2007 is mainly through innate immune responses directly or indirectly related to immunity.     

利用抑制消减杂交技术筛选鹅就巢行为相关基因

旨在利用抑制消减杂交技术构建鹅就巢与产蛋cDNA基因文库,筛选就巢母鹅上调与下调表达的基因.以就巢期和产蛋期鹅卵巢组织互为检测子和驱动子,进行正反双向消减杂交,获得鹅卵巢差异表达基因文库.从双向文库随机挑选单菌落进行PCR验证,结果表明文库质量良好.选取654个阳性克隆进行测序分析,获得641条表达序列标签(ESTs).对ESTs进行去除载体序列、质量检测、聚类及拼接,经BLASTn比对,有166条ESTs找到与之匹配的同源序列,其中92个是功能基因.比较就巢与产蛋SSH文库,发现就巢SSH库中参与细胞凋亡、信号转导及细胞结构的基因出现频率较高;产蛋特异性基因文库中参与物质能量代谢、细胞防御的基因较多;尚有许多差异片段属于未知功能蛋白或理论推定蛋白.结果提示,催乳素受体、抗苗勒管激素以及雌激素受体基因在就巢期上调表达可能与鹅就巢行为的启动与维持有关.该结果为进一步研究鹅就巢行为分子调控机制奠定基础.     

鸡脾脏组织抑制消减cDNA文库构建及其差异表达基因的分析

为分离与鸡抗病性密切相关的差异表达基因,并进行功能分析,利用抑制性消减杂交技术,以大骨鸡和海兰褐商品代蛋鸡20周龄时的脾脏组织为试验材料构建消减cDNA文库。文库的插入片段集中在600 bp左右,挑取760个克隆进行PCR筛选获得663个阳性克隆,经过点杂交筛选后选择了531个阳性克隆,从中随机挑取100个阳性克隆进行测序。经过同源性比对归并后得到37个差异表达基因或ESTs序列,其中,32个是已知基因,包括一般抗病性或免疫性能、特异抗病相关基因、细胞信号分子、膜蛋白质、转录因子等差异表达基因;5个为功能尚未确定的基因。并对4个可能影响鸡抗病性能的差异表达基因进行了RT-PCR半定量检测鉴定。该试验结果为进一步研究这些差异表达基因在抗病过程中的重要功能及其调控作用机理奠定了基础。     

Validation and application of a high fidelity mRNA linear amplification procedure for profiling gene expression

The need for microgram quantities of RNA for microarray experiments has hindered application of this novel technology in cell types/tissue samples with limited abundance of RNA. In this study, potential application of T7-based linear RNA amplification was investigated for use in gene expression profiling experiments where starting material is limited. Yield and integrity of amplified antisense RNA (aaRNA), microarray hybridization intensities, and fidelity of differential gene expression detected were determined for arrays generated for unamplified versus amplified RNA from the same homogenous starting pools. Total RNA was extracted from bovine spleen and fetal ovary, serially diluted to concentrations ranging from 2 microg to 500 pg and amplified. Quality and quantity of total input RNA and aaRNA were assessed by spectrophotometry, gel electrophoresis and bioanalyzer. In experiment 1, we determined the optimal amounts of aaRNA generated from 20, 40, 200 ng and 2 microg input total RNA for use in cDNA synthesis, labeling and array hybridization that would yield robust and consistent hybridization signals on a bovine oocyte cDNA microarray. In experiment 2, comparison of microarray hybridization intensities and fidelity of differential gene expression between aaRNA generated from 2, 20 and 40 ng input total RNA versus unamplified RNA (uRNA) were conducted. The hybridization intensities for each of the 7000 spots per slide for microarrays conducted using aaRNA versus uRNA were highly correlated (2 ng = 0.84, 20 ng = 0.88, 40 ng = 0.90; P < 0.01). The false positive rate was low and similar (4.0% versus 4.4%) for arrays done with uRNA and aaRNA. Ninety-seven ESTs were detected as differentially expressed in the fetal ovary versus spleen at > 1.5- or < 0.5-fold using uRNA (P < 0.05). However, the number of genes detected in arrays using aaRNA was approximately 1.5-2.5 times greater than with uRNA. Approximately, 65-70% of differentially expressed genes were common between uRNA and aaRNA arrays. Relative fold-expression (Cy3/Cy5 ratios) for 25 overlapping abundant genes was comparable for uRNA versus aaRNA arrays with 2 and 20 ng total RNA as input. Results demonstrate that T7-based linear amplification of small amounts of input RNA and use of aaRNA in microarray experiments retains fidelity of detection of differential gene expression that is relatively comparable to experiments done with uRNA and provides a potentially viable approach to facilitate gene expression profiling using limited amounts of starting material.