利用巢式PCR快速鉴定牛传染性鼻气管炎

为了快速检测牛传染性鼻气管炎,采用SDS-蛋白酶K法,提取病毒模板DNA。根据IBRV gB基因序列设计了1对特异引物,在其上下游引物的内侧又分别设计了1对引物,以这4条引物对IBRV模板进行扩增。结果成功扩增出预期目的片段,建立了巢式PCR检测方法。敏感性、特异性等检测试验结果表明该方法能特异检测IBR病毒。本方法具有快速、灵敏、特异的优点,适用于在牛及其遗传物质的进出口检疫中进行牛传染性鼻气管炎快速病原鉴定。     

马传染性贫血病毒驴白细胞弱毒疫苗株囊膜基因的优化及其DNA疫苗体外瞬时表达研究

马传染性贫血病毒(Equine infectious anemia virus,EIAV)的密码子使用频率与哺乳动物间存在着明显差异。为此,对马传染性贫血病毒驴白细胞弱毒疫苗株(DLA-EIAV)囊膜全长基因按照哺乳动物优势密码子的使用原则进行了重新设计和合成,并以此为基础通过重叠延伸PCR、限制酶切等方法得到结合型和分泌型囊膜基因,将其插入含有鸡beta-actin/兔beta-globin复合启动子(AG)的高效表达载体pCAGGS中,构建了EIAV驴白细胞弱毒疫苗株结合型和分泌型囊膜基因的DNA疫苗质粒pCAGGS-opti-bou-env、pCAGGS- opti-sec-env。将构建的质粒纯化后分别转染293T细胞,以间接免疫荧光和Western blot方法检测转染48 h后细胞及上清中囊膜蛋白的表达。结果显示,两种表达质粒均可正确表达EIAV囊膜蛋白,与相对应未优化的表达载体pCAGGS-wt-bou-env和pCAGGS-wt-sec—env相比,密码子优化的基因体外瞬时表达水平有极为显著的提高,而且蛋白表达部位也与预期的结果符合。这一结果为EIAV囊膜蛋白的单抗制备、表位鉴定、免疫试验、新疫苗的开发等奠定了基础。     

Construction and expression analysis of idiotype TCR Vβ2 DNA vaccine in vitro

AIM:To develop an anti-lymphoblastic leukemia TCR idiotypic DNA vaccine, analyze its transfer activity into K562 cells and to detect its expression in vitro. METHODS:The TCR Vβ2 gene segment, which was identified from an idiotypic TCR Vβ2 clone-Molt4 cell line, was amplified using RT-PCR, and the PCR products were then cloned into pIRES vector. The recombinant plasmids were transferred into K562 cells. The condition of idiotypic protein expression was tested by indirect immunophenotyping fluorescein dyeing, SDS-PAGE and Western blotting. RESULTS:The recombinant DNA plasmid, pIRES-TCR Vβ2, was developed successfully. The expression of TCR Vβ2 was identified on the surface of K562 cells. A 15 kD protein, which bound to TCR Vβ2 antibody specifically, were identified from pIRES-TCR Vβ2 transfected K562 cells by Western blotting, indicating that TCR Vβ2 protein was expressed in vitro. CONCLUSION:The recombinant plasmid pIRES-TCR Vβ2 DNA vaccine was developed successfully, which was expressed TCR Vβ2 protein specifically in transfected K562 cells.     

Chronic cigarette smoking induces alteration of FIZZ1/RELMα expression in rat lung

AIM: To investigate the expression of FIZZ1/RELMα in lung tissue of chronic cigarette smoking rat, and to determine the relationship between airway inflammation and airway hyperresponsiveness. METHODS: Made rat model of chronic cigarette smoking was used. The expression of FIZZ1/RELMα in lung tissue was determined by immuno-histochemistry and in situ hybridization. RESULTS: In control rats, FIZZ1/RELMα protein and mRNA expressions were observed at low levels. In cigarette smoking rats, FIZZ1/RELMα expression increased in all the cells especially in bronchial smooth muscle cells, vascular wall cells and alveolar epithelial cells. CONCLUSION: FIZZ1/RELMα is a secreted peptide specifically expressed in lung. Cigarette smoking induces its upregulation, which possibly contributes to cigarette smoking-induced airway hyperresponsiveness.     

秋季叶面喷施IAA、6-BA或GA_3对草莓植株的影响

为了探讨植物生长调节剂对秋季草莓苗植株质量的调控,以法国3号草莓(Fragaria×ananassa Duch cv.French3)为材料,研究秋季叶面喷施50mg/L IAA、50mg/L6-BA或25mg/L GA3对草莓苗叶片光合作用、活性氧代谢和植株质量的影响。结果表明,晚秋叶面喷施这3种植物生长调节剂显著提高了叶片的净光合速率和叶绿素a、b含量,提高了SOD和CAT酶活性,同时降低了MDA和活性氧含量。另外,这3种植物生长调节剂处理显著增加草莓茎和根系的干重,使根冠比增加,IAA和6-BA处理还显著提高平均单株花量。因此,秋季叶面喷施50mg/LIAA和50mg/L6-BA可提高草莓苗植株质量。     

蜡样芽孢杆菌M22Fe-SOD基因克隆及其分析

 通过筛选蜡样芽孢杆菌M 22基因组文库, 得到约3.9 kb的基因组片段。BLAST结果显示:其中包含1条长度为915 bp、编码304个氨基酸的超氧化物歧化酶(SOD)基因, 其与Bacillus thuringiensis和Bacillus anthracis中的sodF基因同源性高达95%。此sodF基因能互补恢复大肠杆菌SOD缺陷型菌株QC871在10 μmol/L paraquat中的生长能力。对重组表达蛋白的抑制实验表明, 此SOD基因为Fe-SOD。利用pET-30b (+)构建表达载体pET-sodF并转化到大肠杆菌BL21(DE3)中, 经IPTG诱导后, SOD融合蛋白表达量显著增加。构建自杀载体p299-8, 同源重组突变蜡样芽孢杆菌M22 sodF基因后, 突变体抗氧化能力未显著降低。     

优质晚熟制汁葡萄新品种‘北丰’

 ‘北丰’由‘蘡薁葡萄’与‘玫瑰香’杂交育成。浆果在北京地区9月下旬成熟。果粒椭圆形, 紫黑色, 果粒质量2.9 g, 果穗质量38916 g, 可溶性固形物含量为19.1% ～23.1% , 含酸量0175% ,出汁率81.9% , 汁多, 紫红色, 味酸甜, 品质上等。早果, 丰产, 抗寒性、抗旱性及抗病虫能力均强。     

用表达人溶菌酶基因的重组质粒防治干乳期奶牛乳腺炎

将人溶菌酶（human lysozyme，hLYZ）cDNA插入由pcDNA3改造而成的pcDNAK表达载体。用获得的重组载体pcDNAKLYZ转染COS-1细胞，经免疫荧光试验证明能进行正确表达。将重组载体注射于哺乳母鼠，取其乳汁进行溶菌酶活性测定，结果显示，分泌在乳汁中的重组hLYZ高达139mg／L。根据乳汁体细胞检测结果，选择健康奶牛和乳腺炎阳性奶牛，分别在干奶时和产犊前2周2次注射重组质粒pcDNAKLYZ，注射途径为乳腺基部穿刺，注射剂量为300μg／乳区,于产犊后1个月采集奶样进行体细胞检测，结果显示，对前一泌乳期发生的奶牛乳腺炎的治愈率为91．5％，对下一泌乳期奶牛孔腺炎的预防有效率王少为96．97％。由此认为。构建的表达hLYZ基因的重组表达质粒，可以替代抗菌素类油乳剂用于干乳期乳腺炎的防治。     

Distribution and identification of immunocytes in humanized SCID mouse model

AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34⁺ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human β₂M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34⁺ cells were higher than those in mice transplanted with CB MNC. The mRNA of human β₂M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34⁺ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.     

实验性鸡马立克氏病羽髓病变研究

通过光镜1电镜等方法对鸡马立克氏病羽髓组织进行了形态学研究。结果显示，羽髓远端坏死，羽髓中有多量肿瘤细胞浸润，肿瘤细胞呈弥漫性或局部性浸润，电镜观察，肿瘤细胞核异型性明显，核膜凹陷，核内出现假核内包含物，在一些羽髓中，巨噬细胞吞噬肿瘤细胞较多见，而且观察到一些肿瘤细胞发生调亡，结果表明，鸡马立克氏病羽髓病变十分明显，羽髓的形态学变化可用于鸡马立克氏病的诊断。