丹参C4H基因pET32a原核表达载体的构建

【目的】克隆丹参中肉桂酸-4-羟化酶基因(SmC4H)的核心区,并构建SmC4H基因的原核表达载体。【方法】设计合成SmC4H基因全长引物,应用PCR克隆SmC4H基因的编码区并添加酶切位点,应用EcoRⅤ、NotⅠ双酶切SmC4H-pGM-T后与原核表达载体pET32a连接,转化到大肠杆菌BL21(DE3)中进行双酶切鉴定,诱导表达重组蛋白并进行SDS-PAGE电泳以及Western Blot分析。【结果】克隆得到了1 200 bp大小的基因片段,经测序鉴定其为SmC4H基因片段;连接表达载体测序结果表明,SmC4H-pET32a构建成功,其诱导表达蛋白经SDS-PAGE检测及Western Blot分析发现,重组蛋白表达效果较好,且主要以包涵体形式存在。【结论】成功构建了SmC4H-pET32a原核表达载体,并成功诱导了SmC4H-pET32a重组蛋白的表达。     

卵形鲳鲹 JAK3 蛋白的原核表达与纯化

【目的】纯化获取卵形鲳鲹(Trachinotus ovatus)JAK3(TroJAK3)重组蛋白,为了解TroJAK3蛋白功能、相互作用及抗体制备奠定基础。【方法】应用分子生物学技术构建重组质粒pET-32a-TroJAK3,转化大肠杆菌BL21感受态细胞,经IPTG诱导表达,SDS-PAGE和Western blot检测TroJAK3重组蛋白表达情况。【结果】PCR扩增片段长度为3 333 bp,经双酶切鉴定、测序确认序列和开放阅读框正确,结果显示成功构建重组质粒pET-32a-TroJAK3。经终浓度为1 mmol/L IPTG 37℃诱导4 h后进行SDS-PAGE检测,结果表明TroJAK3重组蛋白以包涵体形式大量表达,分子量约为140 ku。经Ni-IDA树脂柱纯化,获得纯化的重组蛋白。Western blot检测结果显示有140 ku的条带,表明TroJAK3重组蛋白能被抗His抗体识别。【结论】成功构建了重组质粒pET-32a-TroJAK3,纯化得到高纯度的TroJAK3融合蛋白。     

弓形虫棒状体蛋白15的原核表达、纯化及多克隆抗体的制备

【目的】克隆弓形虫（Toxoplasma gondii）RH虫株速殖子的棒状体蛋白15(ROP15)基因,原核表达、纯化重组蛋白,并制备多克隆抗体。【方法】根据GenBank登录的弓形虫RH株ROP15(DQ096561.1)基因序列设计了1对引物,以提取的弓形虫RH虫株速殖子RNA为模板,通过RT-PCR获得该虫株的ROP15基因,将该基因连接至原核表达载体pGEX-4T-1上,获得重组质粒pGEX-4T-ROP15,测序结果与预期结果一致;转化BL21(DE3)感受态细胞,经IPTG诱导获得重组融合蛋白,运用亲和层析法纯化可溶性ROP15重组蛋白,SDS-PAGE对表达和纯化的目的蛋白进行分析。用纯化的ROP15重组蛋白免疫新西兰大白兔,制备多克隆抗体,运用Western blot分析特异性。【结果】成功克隆了弓形虫RH虫株速殖子ROP15基因,大小为924 bp;菌液PCR和酶切鉴定表明重组质粒pGEX-4T-ROP15构建成功,获得了约59 ku的可溶性融合蛋白表达产物;以纯化的可溶性ROP15重组表达蛋白为抗原制备兔源性抗血清,Western blot显示该多克隆抗体与ROP...     

鸡VEGF基因的克隆及融合蛋白的表达与分离纯化

为获得鸡VEGF基因片段,原核表达GST-cVEG基因,并对融合蛋白进行分离纯化,应用RT-PCR从鸡睾丸中扩增出VEGF片段,将PCR产物克隆至pMD 18-T载体.随后,将cVEGF片段亚克隆至带有GST的原核表达载体pGEX-4T-2中,测序鉴定.将重组质粒pGEX4T2-cVEGF转化大肠杆菌BL21(DE3)菌株,IPTG诱导,并进一步通过Ni-NTA Agarose亲合层析纯化,纯化产物以SDS-PAGE和Western blot进行鉴定,结果表明,成功地构建了原核表达质粒pGEX4T2-cVEGF.GST-cVEGF融合蛋白在大肠杆菌中得到了表达,Ni-NTA Agarose纯化后得到较纯的蛋白.Western blot分析证实,纯化后蛋白是GST-cVEGF融合蛋白,为进一步研究CST-cVEGF融合蛋白疫苗抗小鼠肿瘤模型奠定了基础.     

牛布鲁氏菌Wbkc基因原核表达及抗原性分析

克隆牛布鲁氏菌的甲酰基转移酶基因（Wbkc）,在大肠杆菌中表达/纯化,并对甲酰基转移酶的抗原性进行分析。以牛布鲁氏菌的染色体DNA为模板,扩增Wbkc基因,双酶切后克隆至pET-28a上,在大肠杆菌BL21中诱导表达,His Trap FF层析柱纯化,Western blot鉴定甲酰基转移酶的抗原性。提取的重组质粒经PCR鉴定、双酶切鉴定和测序分析确定目的基因成功插入到了克隆载体中。SDS-PAGE分析证明,表达产物为相对分子质量29 000的融合蛋白。Western blot结果表明,表达的蛋白具有特异的免疫反应原性。     

鸡干扰素α受体Ⅱ胞外域的克隆及GST融合蛋白原核表达

应用RT-PCR技术扩增鸡干扰素α受体Ⅱ胞外域（CHIFNAR2 EC）基因片段,将扩增产物克隆至pMD-18T,转化J M109感受态。重组质粒与表达载体pGEX-6P-1经双酶切后构建重组表达质粒pGEX-6P-CHIF-NAR2EC,转入BL21,提取质粒酶切和测序鉴定。测序结果与参考序列比较,核苷酸同源性为99%。用IPTG诱导表达,对诱导条件进行初步优化,表达蛋白主要以包涵体的形式存在。通过切胶的方法进行纯化,获取具有较高纯度的融合蛋白。表达产物经SDS-PAGE和Western Blot检测显示融合蛋白分子量大小约为50 KD,采用抗GST抗体进行Western Blot,成功检测到特异性目的条带。     

牛妊娠相关糖蛋白9(bPAG9)的真核表达及纯化

【目的】构建proEM-bPAG9重组表达载体,并在HEK293细胞中转染表达。为进一步抗体的制备和奶牛早孕诊断技术的研究奠定基础。【方法】通过基因优化和PCR法扩增得到bPAG9全基因序列,经双酶切法将bPAG9基因插入到表达载体proEM中,构建proEM-bPAG9重组载体后转到DH5α感受态细胞中,重组质粒转染至哺乳动物细胞HEK293中进行瞬时表达,再通过亲和层析纯化bPAG9重组蛋白(rbPAG9),经SDS-PAGE 和 Western Blot 检测rbPAG的表达效果。【结果】通过全基因合成法得到1 182 bp的bPAG9基因片段,构建的重组质粒proEM-bPAG9经双酶切获得约4 369 bp和1 176 bp的两条片段,与预期值相符;对重组载体测序,与优化后的基因序列完全一致,氨基酸未发生突变;SDS-PAGE 和 Western Blot 鉴定显示,获得相对分子质量约为68 kDa 的bPAG9重组蛋白,通过Ni2+亲和层析纯化后,rbPAG9纯度可达90%。【结论】通过基因优化及真核表达获得分子质量为68 kDa 的rbPAG9重组蛋白。     

丹参C4H基因pET32a原核表达载体的构建

【目的】克隆丹参中肉桂酸-4-羟化酶基因（SmC4H）的核心区,并构建SmC4H基因的原核表达载体。【方法】设计合成SmC4H基因全长引物,应用PCR克隆SmC4H基因的编码区并添加酶切位点,应用EcoRⅤ、NotⅠ双酶切SmC4H-pGM-T后与原核表达载体pET32a连接,转化到大肠杆菌BL21(DE3)中进行双酶切鉴定,诱导表达重组蛋白并进行SDS-PAGE电泳以及Western Blot分析。【结果】克隆得到了1 200 bp大小的基因片段,经测序鉴定其为SmC4H基因片段;连接表达载体测序结果表明,SmC4H-pET32a构建成功,其诱导表达蛋白经SDS-PAGE检测及Western Blot分析发现,重组蛋白表达效果较好,且主要以包涵体形式存在。【结论】成功构建了SmC4H-pET32a原核表达载体,并成功诱导了SmC4H-pET32a重组蛋白的表达。     

猪BMP4基因原核表达及优化

构建猪BMP4基因pET32a-BMP4原核表达质粒,在E.coli中表达并分离纯化目的蛋白。采用PCR技术以pMD18-T-BMP4重组质粒为模板扩增获得BMP4基因编码成熟肽片段,并插入到原核表达载体pET32a中,构建原核表达重组质粒pET32a-BMP4。将阳性重组质粒转化到表达宿主菌中,在不同的温度、IPTG浓度和时间下进行诱导表达,SDS-PAGE分析鉴定。构建的重组表达质粒经PCR、酶切和DNA测序鉴定与预期的结果一致,表达宿主菌经IPTG诱导表达分子量约为31 ku的融合蛋白,表达产物占菌体总蛋白的30%以上。成功构建了pET32a-BMP4原核表达质粒,并经纯化得到BMP4目的蛋白,为进一步研究该蛋白的结构和功能奠定基础。     

猪CCL17基因的克隆与原核和真核表达

【目的】克隆猪胸腺活化调节趋化因子CCL17基因,研究其在原核及真核系统中的表达。【方法】提取仔猪脾脏组织总RNA,利用RT-PCR技术扩增CCL17,并将其与已知序列的CCL17进行同源性比较;CCL17基因经测序验证后,将其分别克隆入表达载体pET-32a和pEGFP-C1,构建该基因的重组原核表达载体pET-CCL17和真核表达载体pEGFP-CCL17,分别进行双酶切和测序鉴定;前者在大肠杆菌BL21(DE3)中诱导表达目的蛋白,后者以脂质体介导法转染至猪血管内皮细胞(SUVECs),通过绿色荧光标记和Western blot检测细胞中CCL17基因的表达水平。【结果】PCR扩增得到CCL17309bp的DNA片段;重组表达质粒pET-CCL17和pEGFP-CCL17经双酶切鉴定和测序分析证明,载体构建成功。pET-CCL17经IPTG诱导表达后,SDS-PAGE分析表明,目的蛋白在宿主大肠杆菌BL21(DE3)中高效表达CCL17融合蛋白,分子质量约32ku,与其理论分子质量基本相符;pEGFP-CCL17转染SUVECs,经Westernblot检测,证实导入的CCL17基因得到了表达。【结论】克隆了猪的CCL17基因,利用大肠杆菌成功表达了CCL17重组蛋白,真核重组质粒在SUVECs中也得到了表达。     

穴盘规格和苗龄对塑料大棚菜豆性状及产量的影响

本试验以菜豆品种先行者为试材,采用32孔、50孔两种规格穴盘,设置10、15、20、25 d等4个苗龄段,通过研究不同穴盘规格和苗龄对菜豆性状及产量的影响,筛选出适合菜豆育苗的穴盘规格和适宜的苗龄。结果表明：穴盘育苗的营养面积增大,菜豆产量随之增加,25 d苗龄条件下,32孔穴盘的前期产量和总产量均显著高于50孔穴盘;随着苗龄的增长,菜豆的前期产量和总产量呈增加趋势,其中25 d苗龄的产量显著高于其他苗龄。综合上述各项指标,菜豆育苗阶段,10 d苗龄可选用32孔、50孔穴盘,15 d以上秧苗选用32孔穴盘为宜。     

不同基因型小麦苗期耐低氮性评价及筛选

氮素作为重要的营养元素,限制着小麦的生长发育和经济产量,筛选和培育耐低氮小麦品种是提高氮素利用率、降低生产成本的有效途径。以118份不同基因型小麦为材料,在低氮(0.1 mmol·L^-1)和正常氮(5 mmol·L^-1)条件下苗期水培,测定根干重、茎叶干重、根冠比、植株干重、最大根长、初生根数和二级初生根数等相关指标,采用模糊隶属函数法、主成分分析以及聚类分析法综合评价小麦品种的耐低氮性。结果表明,在低氮胁迫下小麦幼苗的根干重、根冠比和初生根数目显著提高,茎叶干重、植株干重和最大根长不同程度的降低,7个苗期性状指标在两个氮水平下均存在显著性差异。主成分分析提取3个主成分,贡献率分别为 43.575%、22.904%和17.873%,累积贡献率达 84.351%。以耐低氮性综合评价D值进行聚类分析,将118份小麦品种划分为强耐低氮型、耐低氮型、中间型、较敏感型和敏感型5类。筛选出3份耐低氮型小麦（齐大195、金丰7183和天民198）和2份强耐低氮型小麦（山农0917和鲁麦8号）。不同小麦品种的耐低氮机制不同,研究结果为小麦耐低氮品种的选育提供理论依据和材料基础。     

一串红N、P、K、Ca、Mg、Fe缺素症状研究

以‘展望红’为材料,研究N、P、K、Ca、Mg、Fe缺乏对其生长发育的影响,并总结缺素症状,研究结果表明:N缺乏时植株矮小,叶发黄;P缺乏时叶色暗绿,且影响开花;K缺乏时基部叶枯黄,并易落叶;缺Fe症状较为明显,但能正常开花;Ca、Mg未表现出缺素症状,且各项指标要优于全素处理;N、P显著影响一串红的生长发育,K对开花品质的影响较大。     

Segregation of form, color, movement, and depth: anatomy, physiology, and perception

Anatomical and physiological observations in monkeys indicate that the primate visual system consists of several separate and independent subdivisions that analyze different aspects of the same retinal image: cells in cortical visual areas 1 and 2 and higher visual areas are segregated into three interdigitating subdivisions that differ in their selectivity for color, stereopsis, movement, and orientation. The pathways selective for form and color seem to be derived mainly from the parvocellular geniculate subdivisions, the depth- and movement-selective components from the magnocellular. At lower levels, in the retina and in the geniculate, cells in these two subdivisions differ in their color selectivity, contrast sensitivity, temporal properties, and spatial resolution. These major differences in the properties of cells at lower levels in each of the subdivisions led to the prediction that different visual functions, such as color, depth, movement, and form perception, should exhibit corresponding differences. Human perceptual experiments are remarkably consistent with these predictions. Moreover, perceptual experiments can be designed to ask which subdivisions of the system are responsible for particular visual abilities, such as figure/ground discrimination or perception of depth from perspective or relative movement--functions that might be difficult to deduce from single-cell response properties.     

ICP-OES法同时测定果蔬中铅、砷、镉、铬、铜、锡含量

果蔬样品经混酸消化后,控制一定的酸度,定容后应用等离子体发射光谱法(ICP-OES)对果蔬中铅、砷、镉、铬、铜、锡六种有害重金属进行测定,研究了分析测定条件,方法简单快速。测定结果表明,五种元素的加标平均回收率在91.0%~107%之间。其RSD均小于3.5%。按该方法进行处理及测定铅、砷、镉、铬、铜、锡,在选择的测定条件下最低检出限分别为0.0006 mg/kg、0.0003 mg/kg、0.00003 mg/kg、0.00005 mg/kg、0.00003 mg/kg、0.0006 mg/kg。     

Space, stars, c60, and soot

Although carbon has been subjected to far more study than all other elements put together, the buckminsterfullerene hollow-cage structure, recently proposed to account for the exceptional stability of the C(60) cluster, has shed a totally new and revealing light on several important aspects of carbon's chemical and physical properties that were quite unsuspected and others that were not previously well understood. Most significant is the discovery that C(60) appears to form spontaneously, and this has particularly important implications for particle formation in combustion and in space as well as for the chemistry of polyaromatic compounds. The intriguing revelation that 12 pentagonal "defects" convert a planar hexagonal array of any size into a quasi-icosahedral cage explains why some intrinsically planar materials form quasi-crystalline particles, as appears to occur in the case of soot. Although the novel structural proposal has still to be unequivocally confirmed, this article pays particular attention to the way in which it provides convincing explanations of puzzling observations in several fields, so lending credence to the structure proposed for C(60).     

Strategic command, control, communications, and intelligence

Command, control, communications, and intelligence (C(3)l) for nuclear forces are essential elements in the deterrence of nuclear war. The present C(3)l) system has vulnerabilities associated with its reliability, survivability, and endurance under attack, thereby weakening deterrence by increasing the ambiguity in our capabilities. Development of a reliable and enduring C(3)l) system would reduce this ambiguity. Its reliable, positive control of nuclear forces would give the national leadership more time to assess situations, ensure discriminate retaliation, and improve our ability to manage crises in general. These capabilities could help to stop a war rapidly should one start. A reliable and enduring C(3)1) system will be needed for a long time to come, even if a freeze on strategic nuclear forces is accomplished or other arms control successes achieved. Indeed, C(3)l) may be the best source today of confidence-building measures to reduce tensions and the threat of nuclear catastrophe.