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。     

瑟氏泰勒虫感染的牛外周血单个核细胞酵母双杂交cDNA文库构建及鉴定

为了研究牛瑟氏泰勒虫与牛外周血单个核细胞之间蛋白的相互作用,以自然感染瑟氏泰勒虫的牛外周血单个核细胞为材料,纯化并提取总RNA,应用SMART技术合成双链cD NA(ds cD NA)。利用纯化柱纯化ds cD NA,并与线性化的p GADT7-Rec共转化酵母菌Y187,以同源重组的方式在酵母细胞内构建牛外周血单个细胞的酵母双杂交cD NA表达文库。结果显示,文库容量为4.0×108CFU/mL,随机挑取单克隆进行菌落PCR检测,插入的双链cD NA片段大小集中在0.4~2.0 kb,文库重组率为95%。表明此文库达到要求,可用于酵母双杂交筛选。     

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。     

有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库的构建

利用 Stratagene公司 Hybri ZAP- 2 .1建库试剂盒构建了经有丝分裂原刺激的鲤鱼外周血白细胞 c DNA文库。采取健康鲤鱼外周血 ,分离白细胞 ,经 L PS、PHA和 Con A分组作用不同时间后 ,分别提取总 RNA,将各组样品混合后 ,以 Oligo(d T)纤维素柱分离纯化 m RNA,经逆转录合成 c DNA的第 1链和第 2链 ,与 Eco R 接头连接 ,酶切后 ,用CHROMA SPIN- 4 0 0柱离心层析纯化 ,与 Hybri ZAP- 2 .1载体连接 ,体外包装后转染 E.coli XL I- Blue宿主菌 ,进行滴度测试和文库扩增。结果构建的经有丝分裂原刺激的鲤鱼外周血白细胞 c DNA文库原始库容量为 1.6 7× 10 6 pfu,插入片段长度在 0 .4× 10 3～ 3.0× 10 3bp,重组率为 98.9% ,扩增后的文库滴度为 6 .16× 10 9pfu/m L ,该文库符合 c DNA文库构建的标准 ,为克隆和研究鱼类免疫应答相关基因提供了有效的工具     

猪杂种一代与亲本肌肉组织间正反向消减cDNA文库的构建

应用抑制消减杂交技术成功构建了杂种一代梅大猪、亲代大白猪的背最长肌正反向消减cDNA文库。以β-actin为指标检测2个文库的消减效率分别为2^15和2^10倍。从相应的质粒文库中分别获取了700个和623个有效阳性克隆，PCR检测插入片段主要分布在150～600bp之间。亲本与子代肌肉组织间消减cDNA文库的构建为进一步分离、鉴定与杂种优势相关的基因奠定了基础。     

羊草叶片cDNA文库的构建及部分表达序列标签的分析

采用SMART技术,以品质优良羊草“吉生一号”叶片为材料,构建了高质量cDNA文库。原始文库滴度达到10⁶cfu/mL,扩增文库滴度接近10¹¹cfu/mL。随机抽样检查结果表明,插入片断大小在0.5~3.0kb,主要集中在1kb左右,其中检测到插入片断大于1kb的占70%,最大达到2.5kb,其重组率达到98%。同时在扩增文库中检测到了羊草维生素E合成途径中的关键酶基因α-生育酚环化酶(TC)及γ-生育酚甲基转移酶(γ-TMT)的特异信号,挑选307个筛选出了285条EST序列,将得到的117条非重复序列与GenBank中已知序列比对,获得了如3-磷酸甘油醛脱氢酶、光系统Ⅱ蛋白D1、翻译起始因子蛋白、翻译延生因子蛋白、RNaseS-likeproteinprecursor蛋白等基因。羊草高质量的cDNA文库的构建为进一步从分子水平研究羊草及开发利用这一基因资源提供了条件。     

猪外周血单个核细胞cDNA酵母表达文库的构建及与猪瘟病毒E2蛋白相互作用细胞蛋白的筛选

为研究猪瘟病-毒(CSFV)蛋白与宿主细胞蛋白之间的相互作用,本研究通过SMART技术合成猪外周血单个核细胞(PBMC)双链cDNA,以携带与载体pGADT7-Rec重组位点同源序列的特异引物经long-distance PCR扩增双链cDNA.纯化双链cDNA并与载体pGADT7-Rec共转化酵母菌株Y187,经同源重组构建PBMC cDNA酵母表达文库.以CSFV E2蛋白为诱饵进行酵母双杂交筛选,得到阳性克隆根据序列比对分析结果,进一步进行共转化验证.结果显示筛选到17个与CSFV E2蛋白相互作用的宿主细胞蛋白,基因注释(GO)分析表明这些蛋白分别参与免疫、代谢、细胞生长与增殖、生物调节等过程,为研究它们与CSFV E2的相互作用奠定了基础.     

羔羊外周血单个核细胞转化MTT比色法最佳检测条件的筛选

为了建立羔羊外周血单个核细胞转化检测的MTT比色法,对影响MTT比色法的3个主要因素(包括刺激原、细胞浓度、培养时间)进行了探索性的研究.结果表明,MTT法检测的最佳条件为:植物血凝素(PHA)浓度为10 pg/mL,细胞密度为1×107个/mL;或者刀豆蛋白A(ConA)浓度为40μg/mL,细胞密度为2×107个/...     

家蚕微孢子虫cDNA文库的构建及部分EST同源性分析

以成熟的家蚕微孢子虫 (Nosemabombycis,N .b)为实验材料 ,以λTriplEx2为载体 ,构建了滴度为 1 86× 10 6pfu/mL ,插入片段平均在 5 0 0bp以上的较高质量的家蚕微孢子虫cDNA文库。从cDNA文库中随机挑取 180个克隆进行EST测序 ,得到了 130个有效序列。经BLASTn、BLASTx同源性分析后发现 ,7个ESTs编码基因在氨基酸水平上与脑孢虫有较高的同源性 ,推定为家蚕微孢子虫基因 (putativegene) ,同时获得了 2 7个未知序列。     

ST细胞酵母双杂交cDNA文库的构建及鉴定

为了筛选与猪细小病毒（porcine parvovirus,PPV）结构蛋白VP2相互作用的宿主蛋白,深入研究病毒入侵及致病的分子机制,本试验建立了ST细胞酵母双杂交cDNA文库。采用RNeasy Min Kit提取ST细胞的总RNA,反转录合成cDNA第一链之后,通过SMART技术合成了双链cDNA,并利用同源重组的方法,构建了ST细胞的酵母cDNA文库。结果显示,文库滴度为8.1×10⁸ CFU/mL,插入的双链cDNA片段大小为250~1 000 bp,平均大小约为500 bp,文库的重组率为96%。此文库可用于筛选与病毒相互作用的ST细胞宿主蛋白,为进一步阐明病毒的致病机制奠定了基础。     

An improved method for the isolation of enriched canine peripheral blood mononuclear cell and peripheral blood lymphocyte preparations

A two-step purification method was developed for obtaining (1) peripheral blood mononuclear cell preparations of greater than 97% purity and (2) peripheral blood lymphocyte preparations of greater than 95% purity from canine whole blood with yields similar to or greater than those obtained by conventional techniques.     

Equine herpesvirus-1 infected peripheral blood mononuclear cell subpopulations during viremia

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for β-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.     

Effects of dexamethasone on peripheral blood mononuclear cell phenotype in weanling piglets

The aim of this study was to evaluate the effect of dexamethasone treatment on the immune system of weanling piglets. Piglets were administered dexamethasone (DEX; 1mg/kg, IM) every 12h for 2 consecutive days (short-term experiment) or DEX (1mg/kg, IM) daily for 2 weeks (long-term experiment). The relative percentage of CD8(+) T cells in peripheral blood mononuclear cells (PBMCs) was significantly decreased (P<0.05) in both short- and long-term DEX-treated groups compared to their control groups. The percentage of IgM(+) cells in PBMCs of the long-term DEX-treated group was greatly increased (P<0.05) in comparison to the control group. The results of this study indicate that short-term DEX-treatment increases leucocyte function; however, long-term DEX-treatment depresses leucocyte function, especially that of CD8(+) T cells.     

Principles of peripheral blood mononuclear cell apheresis in a preclinical canine model of hematopoietic cell transplantation

BACKGROUND: Preclinical studies of peripheral blood mononuclear cell (PBMC) transplantation conducted in a well-established canine hematopoietic cell transplantation (HCT) model have been successfully translated to human patients over the past 5 decades. OBJECTIVE: We retrospectively investigated the safety and feasibility of PBMC apheresis in the canine model of HCT by analyzing apheresis parameters, cell yields, and the impacts of donor-related and apheresis-related variables on collection yields and donor stability. ANIMALS: One hundred and twenty dogs that underwent PBMC aphereses were evaluated. METHODS: Aphereses were performed with a COBE Spectra blood separator and a central dual-lumen catheter, with or without recombinant canine granulocyte colony-stimulating factor (rcG-CSF) stem cell mobilization. RESULTS: Aphereses from dogs not given rcG-CSF yielded an average volume of 280 +/- 42 mL containing an average of 15,086 +/- 9,834 leukocytes/mL. Aphereses from dogs given rcG-CSF yielded an average volume of 261 +/- 55 mL containing an average of 39,711 +/- 24,488 leukocytes/mL. Higher pre-apheresis white blood cell (WBC) counts correlated with higher apheresis WBC yields (R=0.50, P<.0001). The correlations of collection time, inlet volume, and collection flow rate on WBC yields were statistically significant but only weak to moderate in magnitude (R=0.34, P=.0001; R=0.38, P=.0006; R=0.26, P=.002, respectively) as were the correlations of collection time and inlet volume on collection volumes (R=0.30, P=.002; R=0.42, P<.0001, respectively). All dogs recovered promptly after PBMC aphereses and catheter removal, without complications. CONCLUSIONS AND CLINICAL IMPORTANCE: These data may be useful for translating PBMC apheresis technology to the field of veterinary oncology for the treatment of dogs with hematologic malignancies.     

Isolation of an antigenically "null" cell line from pig peripheral blood mononuclear cells

A new pig cell line (A4) isolated from a primary culture of pig peripheral blood mononuclear cells was characterized. A4 was demonstrated to be morphologically, antigenically and functionally distinct from the more commonly isolated pig lymphoblastoid B cell lines (e.g. P-SC). When the A4 cell line and clones derived from it were tested against a panel of monoclonal antibodies, which define specific subpopulations of pig mononuclear cells, little or no reactivity was observed. The A4 cell line, unlike the P-SC cell line, was unable to induce a mixed lymphocyte reaction. The amount of immunoglobulin secreted by A4 cells as detected by an ELISA was reduced compared to that produced by P-SC cells. The P-SC cell lines produced an IL-1-like factor, whereas no IL-1-like activity was found in the A4 supernatant. The A4 cell line appeared to be a null cell in respect to the P-SC cell line properties; only the slight amount of immunoglobulin produced suggested that the A4 cell line is of the B cell lineage. An association of viral particles with cells of the A4 morphology and null antigenic characteristics was observed and may provide an explanation for the reduced B cell properties of A4 cells.     

Canine PHA-stimulated adherent cell enhance interferon-gamma production and proliferation of autologous peripheral blood mononuclear cells

Dendritic cells are specialized antigen‐presenting cells with immuno‐modulating functions that are attractive for clinical applications for cancer immunotherapy. This study examined immunostimulatory functions of phytohemagglutinin (PHA)‐stimulated adherent cells (PHA‐Ad cells) from peripheral blood mononuclear cells (PBMCs) in dogs. PHA‐Ad cells enhanced interferon‐γ from autologous PBMC in vitro. PHA‐Ad cells also stimulated antigen‐independent proliferation of peripheral blood lymphocytes. These results suggest that PHA‐Ad cells from PBMC possess a stimulatory function to evoke anti‐tumour immunity and that they demonstrate potential for therapeutic applications in dogs.     

Comparison of three mobilization protocols for peripheral blood stem cell apheresis with Spectra Optia continuous mononuclear cell protocol in healthy dogs

Peripheral blood stem cell (PBSC) transplantation following consolidation therapy is a feasible treatment option for canine haematological malignancies. In veterinary medicine, haematopoietic stem cells are generally mobilized into peripheral circulation using a granulocyte colony‐stimulating factor (G‐CSF). This pilot study aimed to evaluate the haematopoietic stem cell mobilization effect of three different regimens for PBSC apheresis with Spectra Optia continuous mononuclear cell (CMNC) protocol in healthy dogs. Stem cell mobilization was performed using high‐dose plerixafor (CXCR‐4 inhibitor) alone, a G‐CSF alone, or a combination of the low‐dose plerixafor and G‐CSF. Three dogs were assigned to each mobilization protocol. Regardless of the mobilization protocol, the total blood volume processed was uniformly set as 270 mL/kg and many PBSCs, defined as CD34+/CD45^dim cells, within the apheresis product were compared. Changes in complete blood count, PBSC counts, and blood chemistry analysis were monitored before, during, and after apheresis. All dogs tolerated the apheresis procedure using the Spectra Optia system with minimal adverse effects. The mean PBSC counts of the apheresis products for plerixafor, G‐CSF, and the combination groups were 1.3 ± 0.24, 4.2 ± 0.47, and 6.4 ± 0.9 × 10⁶ cells/kg, respectively. The apheresis procedure using Spectra Optia CMNC protocol in dogs is safe and feasible. Furthermore, PBSC mobilization with a combination of G‐CSF and plerixafor appeared more effective than either compound alone in mobilizing PBSC to the peripheral blood in dogs.     

Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides

Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species. This work examines whether oligodeoxynucleotides (ODNs) containing CpG motifs stimulate peripheral blood mononuclear cells from pigs. Results show that pigs respond to CpG ODN by proliferating and secreting IL-6, IL-12 and TNF-alpha. By screening a large panel (>100) of ODNs, the palindromic hexamer 'ATCGAT' was identified as being optimally active in all animals examined (N=10). These findings are the first to establish the immunostimulatory activity of CpG ODN in pigs, and suggest that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.     

Generation of canine dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent antigen-presenting cells that are expected to be therapeutic agents for tumor immunotherapy. In this study, we generated DCs of sufficient number for DC-based immunotherapy from peripheral blood mononuclear cells (PBMC) in dogs. PBMC were cultured in the presence of phytohemagglutinin (PHA). On day 6, large adherent cells with dendrite-like projections were seen, and the number of these large cells with projections increased on day 8. These cells were positive for esterase staining. They expressed MHC class II, CD11b, CD8 and weakly CD4 on their surface. They tended to make contact with lymphocytes under culture conditions. We obtained about 2-5 x 10(6) of DCs from 10 ml of peripheral blood. These DCs phagocytosed HEK-293 cells by overnight co-culturing. These cells generated from PBMC are possible canine DCs and are applicable to clinical trials of DC-based whole tumor cell immunotherapy in dogs.