日本大耳白兔TLR3部分cDNA克隆及mRNA在组织中的分布

采用RT—PCR技术从日本大耳白兔的脾脏组织克隆出兔的Toll样受体基因3（命名为。TLR3），并对其mRNA在兔的17种组织中的分布情况进行了检测。结果显示，RTLR3核苷酸序列与GenBank中登载的穴兔（Oryctolagus cuniculus）TLR3序列（登录号：NM_001082219）相似性为100％；兔的胸腺、脾脏、睾丸等14种组织中检测出TLR3 mRNA的转录产物，而在骨骼、胃和皮肤中未能发现。推导的氨基酸序列同源性比对表明，兔TLR3与人、野猪、牛、猫、褐鼠等动物的同源性最高，为80％-85％；与原鸡同源性次之，为61％；与草鱼、绿河豚等同源性在45％～48％之间；系统进化树分析表明，兔TLR3与马的亲缘关系最接近，与猪、人／绵羊、牛、猩猩／猫的亲缘关系依次渐远，与鼠的亲缘关系最远。可见，TLR3在不同种属动物及不同组织中所起的作用具有生物多样性。     

Cloning and analysis of cross-intron genomic gene encoding ACO in Paeonia suffruticosa Andr.

The last step of ethylene biosynthesis in higher plants is to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by ACC oxidase (ACO). In our investigation, a cross-introns genomic DNA gene encoding ACC oxidase, PsgACO, was isolated from Paeonia suffruticosa. It revealed that PsgACO (FJ855434) has 1281 bases, containing four exons and three introns, encoding 312 amino acids. The four exons stretched from 1 to 105, 217 to 434, 592 to 925 and 1000 to 1281 bases. A splicing junction sequence of all the exon-introns conformed to the GT-AG rule in the cross-intron genomic DNA sequence of the ACO gene. PsgACO showed high homology to many characterized ACC oxidases both at the nucleic acid and amino acid levels. As well, twelve amino acid residues were conserved among many ACOs from other species. A phylogenetic tree analysis indicated that the amino acid of ACOs is quite conserved among the different eudicots. The phylogenetic tree showed that both the tree peony and herbaceous peony are quite isolated taxa. Bioinformatic analysis showed that the molecular weight of ACO is 35.29 kD, with a theoretical pI of 5.25. It is a non-secrete, stable hydrophilic protein, located in the cytoplasm.     

鸡CD4蛋白基因的克隆及其mRNA在淋巴器官中的表达

从鸡胸腺、脾脏、哈德氏腺、外周血液和法氏囊等免疫器官中分离淋巴细胞,应用RT-PCR对这些免疫器官中鸡CD4分子mRNA的表达进行了研究。同时,用RT-PCR对鸡脾淋巴细胞CD4分子cDNA进行了克隆和序列测定。结果表明,在上述器官中,法氏囊淋巴细胞不表达鸡CD4分子mRNA,其他器官中均有表达。通过序列分析表明,本试验扩增得到的基因为编码鸡CD4分子的基因。     

O型口蹄疫病毒VP1嵌合基因的构建及原核表达

首先合成我国O型口蹄疫病毒2个疫苗毒株VP1基因的3个抗原袁位，与另外扩增的流行毒株VP1基因末端273bp片段相连,构建出O型VP1嵌合基因片段（VP1O）。然后，将VPIO基因连接到原核表达载体pET28a上，构建了重组表达质粒pET28a-VP1O，转化大肠杆菌BL21（DE3）进行诱导表达。SDS-PAGE电泳表明。VP1O基因在大肠杆菌中获得表达。Western blotting检测证实表达的VP1O蛋白具有良好的生物学活性。对表达蛋白通过包涵体洗涤的方法进行初步纯化，获得了较高纯度的VP1O蛋白。     

一株禽流感病毒全基因的序列分析

通过反转录-聚合酶链式反应(RT-PCR)技术对禽流感病毒A/Turkey/Wisconsin/1/66(H9N2)的8个基因片段即PA、PB1、PB2、NS、NP、M、HA、NA分别进行扩增,然后将其克隆到PMD18-T载体后进行序列测定和拼接;并将克隆到的8个基因片段与以下毒株各个基因的相应序列进行比较分析:Duck/HongKong/Y280/97(DHKY280/97)、Duck/HongKong/YT439/97(DHKY439/97)、Quail/HongKong/G1/97(QHKG1/97)、A/Chicken/Beijing/1/94(CBJ1/94)、Chicken/HongKong/G9/97(CHKG9/97)、A/Turkey/California1/66(TC/1/66).结果表明:我们克隆到的TW1/66株的8个基因片段均含有相应病毒基因的完整开放阅读框架:TW1/66的各基因与TC/1/66株相应各基因同源性最高(NS基因除外,同源性只有(67.4%).与其它各毒株各基因同源性均较低,但与DHKY439/97各基因同源性高于与DHKY280/97、QHKG1/97、CBJ1/94、CHKG9/97各基因同源性.     

牛分枝杆菌MPB70基因的克隆及其在大肠杆菌中的表达

本研究以牛分枝杆菌Vallee111染色体DNA为模板,以MPB70成熟蛋白基因特异性引物进行PCR扩增,获得约500bp的DNA片段.通过T-A克隆技术,将PCR产物克隆至pGEM-T Vector中,成功地构建出克隆载体pGEM-T-70.以BamH Ⅰ和EcoR Ⅰ双酶切pGEM-T-70和pET28a( ),并将纯化的MPB70基因亚克隆至pET28a( )中,构建出原核表达载体pET28a-70.将pET28a-70转化至感受态E.coli BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约25Ku外源蛋白带.Western blot分析发现,该蛋白具有牛分枝杆菌抗原性,从而为进一步研究MPB70的亚单位疫苗及DNA疫苗奠定基础.     

Identification of divergent variants of Grapevine virus A

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.     

H5亚型禽流感病毒血凝素基因的克隆及表达

研究以RT-PCR方法从H5N1亚型禽流感病毒A/DKZJ/12/00(H5N1)中获得禽流感病毒HA部分基因,将目的基因定向克隆到原核表达载体pET-32a,将测序和酶切验证正确的阳性重组质粒转化大肠杆菌BL21,经IPTG诱导表达。结果表明该蛋白得到了可溶性的表达,表达蛋白的分子质量为25ku;Western-blot分析结果表明,该蛋白可以与禽流感H5阳性血清反应(禽流感的单克隆抗体所识别),检测HA的抗体时具有良好的免疫原性。     

北极狐多巴胺受体D1基因的克隆测序

为了获得北极狐多巴胺受体D1基因序列,给北极狐自咬行为提供理论依据。采用聚合酶链式反应方法,从北极狐耳组织扩增出多巴胺受体D1基因的部分外显子序列,并对其进行克隆测序,将该序列提交到Genebank上。Genebank中的Blast分析表明,北极狐多巴胺D1受体基因与家狗(Canis familiaris)的同源性为99%,与牛(Bos taurus)的同源性为93%,与人(Homo sapiens)的同源性为92%。     

猪细胞因子cDNA表达文库的构建及其基因的克隆鉴定

为克隆和研究猪细胞因子及相关基因,应用建库试剂盒成功构建了猪细胞因子cDNA表达文库。采取健康猪外周血及淋巴结,分离单个核细胞,经LPS PHA联合刺激不同时间后,提取总RNA。将各组样品混合,分离纯化mRNA。反转录合成cDNA第一链和第二链,与EcoRI和HindⅢ接头连接。酶切和过柱分级分离后,与λScreen载体连接,经体外包装转染E.coliER1647宿主菌,进行文库容量测定和扩增。以扩增文库的DNA为模板,利用已知基因引物克隆猪IL-2和IL-4的cDNA并进行测序。结果表明,成功构建了猪细胞因子cDNA文库,文库原始库容量为8×105,插入片段在300~2 000 bp,扩增得到特定的IL-2和IL-4基因,说明文库质量高、代表性强,为进一步从文库中筛选未知细胞因子及相关基因提供了有效的工具。