牛整合素β6基因的原核表达及其抗血清的制备研究

[目的]为深入研究ITGB6亚基的基因功能奠定基础.[方法]利用基因克隆技术获得整合素β6基因(ITGB6)胞外区,然后将其重组到pET-32a(+)质粒中,经鉴定及序列分析确定获得了重组阳性克隆;转化大肠杆菌BL21感受态细胞,IFTG诱导及蛋白纯化.[结果]SDS-PAGE结果显示,在94 kD处出现特异性条带;对该融合蛋白进行分离纯化后免疫新西兰兔,制备ITGB6蛋白抗血清,Western-blot分析表明抗血清可与原核表达的ITGB6蛋白特异性结合,ELISA方法检测抗血清的效价可达到1:2560.[结论]成功表达了牛的重组整合素β6基因.     

颠茄H6H基因的克隆及高效植物表达载体的构建

[目的]克隆颠茄(Atropa belladonna)H6H基因并构建高效植物表达载体.[方法]采用RT-PCR方法从颠茄中克隆莨菪碱-6β-羟化酶和1,4-丁二胺-氮-甲基转移酶基因编码区,插入经改造后获得双元三价植物高效表达载体p2301-gus,构建植物表达载体p2301-H6H,并将该表达载体导入根癌农杆菌LBA4404和发根农杆菌C58C1.[结果]获得了可直接用于遗传改良的颠茄工程菌p2301-H6H-LBA4404和p2301-H6H-C58C1.[结论]为利用植物基因工程技术提高颠茄中生物碱含量奠定了基础.     

D-甘露糖异构酶在大肠杆菌中的表达及催化条件的研究

根据已知的放射性土壤杆菌体内的D-甘露糖异构酶(Fmi)氨基酸序列,设计适合在大肠杆菌内表达的Fmi基因序列(1245 bp),采用体外基因拼接合成.构建了表达Fmi的重组大肠杆菌BL21(DE3)/pET30-Fmi,诱导表达的Fmi蛋白经SDS-PAGE验证为可溶性蛋白,分子量44000.通过实验优化了Fmi的催化条件,结果表明Fmi催化的最适pH值为7.5,最适温度为45℃,催化1 h酶活达5.29 U/mL.以25%的果糖溶液为底物时,催化2 h果糖转化率达29.4%.     

rcanl-1l基因的克隆和表达

从小鼠脑组织中通过总mRNA的提取、RT-PCR方法,利用pEASY-T1载体克隆出了鼠源rcanl-1l基因,并构建出了重组有rcan1-1l基因的PET21a原核表达质粒,进行了表达条件的初步摸索,这为今后研究roanl-1l基因以及蛋白的结构与功能打下了前期实验基础.     

里氏木霉外源表达载体的构建

[目的]构建里氏木霉外源表达栽体.[方法]以里氏木霉40359的CBHI启动子和终止子构建了里氏木霉40359外源表达栽体,并用该表达载体成功表达了抗潮霉素B磷酸转移酶基因,得到6株可在含175 mg/L潮霉素的基础培养基上生长的抗性菌株;提取6株抗性菌株的DNA整合到里氏木霉菌株中,并对其进行潮霉素耐受性检测.[结果]6株抗性菌株的抗潮霉素能力比原始菌株提高了75%;转基因菌株的潮霉素抗性可以稳定遗传,证明表达栽体构建成功.[结论]为开展里氏木霉分子生物学研究与遗传改造奠定了基础.     

蜡梅nsLTPs基因家族4个成员的原核表达研究

[目的]利用现代分子生物学技术探索蜡梅(Chimonanthus praecax)资源的药用价值.[方法]在有关蜡梅生物学特性研究基础上,构建蜡梅非特异性脂转移蛋白(nsLTP)基因家族4个成员(GenBmnk登录号:FJ889521、FJ904082、FJ904083、FJ904084)的原核表达载体CpLTP1-pET、CpLTP2-pET、CpLTP3-pET、CpLTP4-pET,并对诱导温度、IPTG浓度和诱导表达时间等条件进行了优化.[结果]在1.0mmol/L IPTG、37℃常规诱导条件下诱导6h可获得高效表达,总表达产率可达到细菌全蛋白质总量的50%以上,包涵体表达远高于可溶性蛋白的表达;在0.5 mmoL/L IPTG、28 oC条件下诱导6h能够获得较好的可溶性表达,利用His-Bind蛋白纯化回收试剂盒获得4个纯化的重组蛋白.[结论]成功构建蜡梅nsLTP的原核表达栽体,转化大肠杆菌Origami2(DE3)获得4个纯化的重组蛋白,可为后续的抗菌抗病毒活性研究提供研究材料,并为蜡梅资源药用价值的开发提供研究思路.     

SCIRR10截短体真核表达载体的构建及其在COS-7细胞中的表达

目的:构建SCIRR 10蛋白3个截短体的哺乳动物细胞表达系统,探讨SCIRR 10蛋白的功能位点.方法:PCR扩增出SCIRR 10蛋白的3个功能截短体表达DNA序列,构建3种真核表达质粒载体pcDNA3.1/Myc-His-SCIRR 10(1-145aa)、pcDNA3.1/Myc-His-SCIRR 10(1-90aa)和pcDNA3.1/Myc-His-SCIRR 10(1-70aa).将3个表达载体质粒转染到COS-7细胞中,应用Western blotting 方法检测3个蛋白截短体的表达情况.结果:3个被截短的SCIRR 10蛋白相对分子质量分别为18 800、12 800和10 700.在COS-7细胞中可以成功表达3个SCIRR 10蛋白截短体.相对3个SCIRR 10截短体蛋白的表达量,β-tubulin蛋白无明显变化.结论:成功构建SCIRR 10蛋白3个截短体的哺乳动物细胞表达系统,通过此系统获得有生物活性的3个SCIRR 10截短体蛋白.     

外源抗菌肽在转基因鱼腥草中的表达及抗茎腐病研究

[目的]鉴定转基因鱼腥草中外源抗菌肽的表达,分析抗茎腐病的能力.[方法]通过SDS-PAG电泳和Western blot方法检测转基因鱼腥草植株中外源抗菌肽的表达,接种不同浓度的立枯丝核菌孢子,比较转基因植株与非转基因植株的染病情况.[结果]外源抗菌肽能在转基因鱼腥草中表达;立枯丝核菌的最佳侵染孢子浓度为3×105/ml,观察时间以3d为宜,转基因鱼腥草较非转基因鱼腥草的抗茎腐病能力增强.[结论]外源抗菌肽的表达能增强转基因鱼腥草植株的抗茎腐病能力.     

公农2号紫花苜蓿抗寒基因CAS19在烟草中的表达

[目的]研究公农2号紫花苜蓿抗寒基因CAS19在烟草中的表达.[方法]根据苜蓿抗寒基因(CAS19 )核酸序列设计1 对引物,用RT-PCR 扩增出分离CAS19 的蛋白基因,并克隆到pMD18-T Vector载体中,再亚克隆到表达载体PBI121,成功构建重组表达质粒 PBCAS.经农杆菌介导转基因入烟草,并通过Sounthern-blotting杂交分析检测转基因苗.[结果]CAS19抗寒基因可以在烟草中得以高效表达.[结论]为进一步探明CAS19抗寒基因在烟草中的表达机制提供了理论依据.     

川西高原黑唇鼠兔乳酸脱氢酶C基因的原核表达

[目的]研究黑唇鼠兔乳酸脱氢酶C(LDH-C)基因的原核表达及重组蛋白的纯化.[方法]采用RT-PCR方法克隆鼠兔LDH-C基因,并将其连接到表达载体pET-32a上,构建鼠兔LDH-C基因的大肠杆菌BL21(DE3)工程菌,通过IPTG诱导表达,表达产物用SDS-PAGE进行分析,使用亲和层析方法进行蛋白纯化.[结果]RT-PCR扩增出1条约1.0 kbp的条带,与预期结果相符;重组表达载体经PCR和双酶切鉴定,产生1个约1.0 kbp的目的片段,表明表达载体构建成功;表达产物经SDS-PAGE分析,得到了分子量略大于45.0kDa以包涵体形式存在的融合蛋白;通过镍亲和层析柱进行纯化,得到了较纯的融合蛋白.[结论]黑唇鼠兔LDH-C基因被成功克隆和表达.     

黑曲霉糖化酶基因的克隆及其在毕赤酵母X33中的表达

[目的]以毕赤酵母(Pichia pastoris)X33为宿主菌高效表达黑曲霉(Aspergillus niger)糖化酶,为进一步扩大糖化酶在工业上的应用奠定基础.[方法]利用RT-PCR技术,提取黑曲霉总RNA进行反转录,以得到的cDNA为模板,根据NCBI数据库中A.niger CBS513.88糖化酶的cDNA序列(glaA)设计引物,通过PCR扩增得到去除天然信号肽的糖化酶成熟肽编码基因glaAm,并将其克隆到pUC19载体中,序列分析表明,glaAm的开放阅读框由1 879个核苷酸组成,编码625个氨基酸.以此片段构建了pFLDα-glaAm重组表达载体,经Nsi I线性化后,电击转化毕赤酵母X-33.摇瓶培养中通过添加终浓度为0.5%的甲醇诱导糖化酶的分泌.[结果]SDS-PAGE和Starch-PAGE显示,糖化酶得到正确的分泌表达,且具有较高的生物活性,发酵上清液中酶活最高达到380.78 U/ml(发酵液).重组糖化酶的最适反应温度和最适pH分别为60～65℃和4.0,在pH 2.5～5.5范围内均保持90%以上的酶活力.该酶具有较高的热稳定性,pH 4.0条件下,该酶在50℃下稳定;60℃处理60 min,仍能保持90%以上的酶活力;65℃下的半衰期约为44 min.[结论]黑曲霉糖化酶基因在毕赤酵母X33中得到了异源高效表达.     

Expression of rat NK-2 (neurokinin A) receptor in E. coli

With the goal of obtaining sufficient functional protein for structural analysis, rat neurokinin-2 receptor was produced in Escherichia coli by linking it to the periplasmic maltose-binding protein. As a first step, we present a biochemical and pharmacological investigation of the recombinant receptor. Western-blots showed that the fusion protein was associated with the membranes. The agonist [4,5-3H-Leu9]neurokinin A and the NK-2 antagonist [3H]SR48,968 bound to the receptor in a highly specific manner. Saturation binding of the [3H]agonist demonstrated a single class of receptors (KD = 10.5 nM, Bmax = 2.5 pmol/mg protein). The [3H]antagonist bound with higher affinity to a larger receptor population (KD = 0.2 nM, Bmax = 7.2 pmol/mg protein). Competition of [3H]agonist binding with other agonists demonstrated a potency order of: neurokinin A > [Nle10]NKA(4-10) = [beta-Ala8]NKA(4-10) > substance P > senktide Against the [3H]antagonist, agonists were only partially inhibitory. Selective NK-2 antagonists inhibited binding of both [3H]ligands with an identical order of potency: SR48,968 > R396 > MEN10,376, which is consistent with NK-2 receptor pharmacology in rat tissue.     

Constructing mental representations of objects from successive views.

Investigated the effects of complexity on processing objects by testing 3 graduate students and 7 staff members on a 3-dimensional analog of J. Hochberg's (1968) aperture viewing paradigm, the orthogonal slices task developed by J. Metzler and R. N. Shepard (1974), and a sequence matching task. In Exp I, Ss constructed, transformed, and compared mental representations of Shepard-Metzler figures varying in the number of component parts. Findings show that processing time increased with complexity. The results of Exp II show no effects of complexity on processing time when Ss merely judged the equivalence of the patterns used in Exp I presented in sequence. Rather, constructed mental representations appeared to preserve some of the spatial character of the corresponding objects. This conclusion was strengthened by the results of recognition tasks that showed that discrimination of constructed objects from appropriate distractors was better after Ss did the 1st (orthogonal slices) task than after they did the 2nd (sequence matching) task. (25 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

R.E.A.L. classification of non-Hodgkin lymphoma from the clinico-oncologic viewpoint

In 1994 the International Lymphoma Study Group (ILSG) published the "Revised European-American Classification of Lymphoid Neoplasms" (R.E.A.L. Classification). Lymphomas were classified according to their presumed normal counterparts, to the extent possible. Within both T- and B-cell categories differentiation between lymphomas and/or leukemias of "precursor" or "peripheral" neoplasms are defined arising from antigen independend or antigen reactive cell proliferation. Lymphomas undoubtedly characterized by currently available morphologic, immunologic, and genetic technics represent "real" disease entities. Provisional categories include lymphomas that have been described in some detail, but without consensus within the ILSG. Proposed names are based predominantly on established usage. With respect to similar treatment approaches and difficulties of the ILSG members in subclassifying large cell lymphomas, centroblastic, immunoblastic and large cell anaplastic lymphomas of B-cell type were "lumped" together as large B-cell lymphomas. Within a prospective treatment trial overall survival was significantly better in centroblastic as compared to B-cell immunoblastic lymphoma diagnosed by optimal histomorphology according the criteria of the Kiel Classification. Thus the R.E.A.L. Classification fails to identify patients who may require other than standard treatment. Future studies will demonstrate whether subclassifying the proposed "peripheral" T-cell lymphomas, unspecified into T-zone lymphoma, lymphoepitheloid (Lennert's) lymphoma and pleomorphic, small, medium, and large cell lymphomas according the Kiel Classification is of clinicopathologic relevance. On the contrary the subtypes of chronic lymphocytic leukemia of T-cell type form two distinct entities within the R.E.A.L. Classification separating T-CLL/prolymphocytic leukemia from large granular lymphocyte leukemia of T- and NK-cell type. Within the R.E.A.L. Classification the lymphoplasmacytoid immunocytoma of the Kiel Classification will be subsumed together with the prognostically significantly better B-cell chronic lymphocytic leukemia. Opposite to the original intention of the ILSG two proposals are developed on clinical grouping of entities. Clinical indolent lymphoid neoplasms usually have "low grade" histologic appearances, with a predominance of small cells subsuming with the exception of the mantle cell lymphoma all of the low grade lymphomas of the Kiel classification. Aggressive lymphomas (intermediate risk) are defined as tumors whose survival if untreated is measured in months, highly or very aggressive lymphomas and/or leukemias will kill untreated patients within weeks. Unlike the Kiel Classification proposed categories subsume lymphomas irrespective of cytomorphology, thus grouping together potentially curable and uncurable diseases. Undoubtedly the R.E.A.L. Classification forms at present the best compilation of existing knowledge upon neoplasms of the immune system, enabling cooperation between clinicians and scientists all over the world. According to the ILSG this proposal should be considered a starting point for future periodic reevaluations.     

Apolipoprotein E Gene Variability and Cognitive Functions at Age 79: A Follow-Up of the Scottish Mental Survey of 1932.

Apolipoprotein E (APOE) genotype is a possible influence on nonpathological cognitive aging. The authors studied 462 community-dwelling, 79-year-old people born in 1921, whose childhood IQ had been assessed in the Scottish Mental Survey of 1932 (Scottish Council for Research in Education, 1933). Adjusting for sex, childhood IQ, and self-reported illnesses, the authors found that those with an APOE e4 allele had significantly lower Wechsler Logical Memory (D. Wechsler, 1987) scores than those without an e4 allele. Those people with APOE s2/e3 genotypes had significantly higher Wechsler Logical Memory scores than e3/s3, who were significantly higher than e3/e4. Neither nonverbal reasoning nor verbal fluency were affected. In this sample, APOE genotype contributed to verbal memory in old age. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

液压抓斗故障诊断分析

针对电炉炼钢厂废钢PEINER电动液压抓斗频繁发生机械故障，严重影响了生产的顺利进行，经跟踪、分析，初步查明工作频繁、设计缺陷及维护不周是造成液压控制块和油泵损坏的直接原因．整治后，基本未再发生故障．     

pHairyRed: A Novel Binary Vector Containing the DsRed2 Reporter Gene for Visual Selection of Transgenic Hairy Roots

T We developed a new plant transformation vector, pHairyRed, for enabling high throughput, non-destructive selection of Agrobacterium rhizogenes-mediated 'hairy-root' transformation. pHairyRed allows easy in planta visualization of transgenic tissue with minimal disturbance to the plant. The DsRed2 reporter gene, encoding a red fluorescent protein, was cloned to yield pHairyRed (harbouring a multiple cloning site), which was used with the highly efficient K599 A. rhizogenes strain to infect soybean (G/ycine max L. Merrill) plants. DsRed2 fluorescence was easily detected in planta for the duration of a 5-week study with negligible levels of background autofluorescence. This enabled visual selection of transformed roots and subsequent excission of non-transformed roots. pHairyRed-transformed roots nodulated normally when inoculated with Bradyrhizobium japonicum. Within the nodule, DsRed2 fluorescence was plant-specific, being absent in the bacteroid-dominated nodule infected zone. To test the reliability of pHairyRed as a high-fidelity binary vector reporter system, the gene encoding the soybean Nod factor receptor, GmNFR1α, was cloned into the vector for use in a complementation study with a non-nodulating nfr1α mutant of soybean. Complementation was achieved and, without exception, DsRed2 fluorescence was detected in all hairy roots that successfully formed nodules (100%, n = 34).We anticipate broad application of this reporter system for the further analysis of root-related events in soybean and related legumes.