人呼吸道合胞病毒微型基因组拯救系统的建立及功能鉴定

目的建立人呼吸道合胞病毒(human respiratory syncytial virus,hRSV)微型基因组拯救系统,并鉴定其功能。方法利用包含T7启动子、锤头状核酶、hRSV的前导序列、基因起始信号、非编码区、多克隆位点、基因终止信号、尾随序列、丁肝核酶、T7终止子的基本载体pRSV1和pRSV2,与增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)基因构建微型基因组质粒pRSV1-eGFP和微型反基因组质粒pRSV2-eGFP。以pcDNA3. 1(+)为表达载体,与优化后的RSV的核蛋白(N)、磷蛋白(P)、聚合酶大片段(L)和转录延长/终止抑制因子(M2-1)基因序列构建辅助质粒pcDNA-N、pcDNA-P、pcDNA-L和pcDNA-M2-1。将微型基因组质粒或微型反基因组质粒通过单转染、与RSV共感染或与辅助质粒共转染至表达T7 RNA聚合酶(T7 RNP)的BSR-T7/5细胞,荧光显微镜或流式细胞仪观察eGFP的表达,并鉴定微型基因组的功能及辅助蛋白的生物学活性。结果微型基因组质粒、微型反基因组质粒和4个辅助质粒经双酶切及测序证明构建正确;微型基因组质粒通过先感染后转染或与辅助质粒共转染,观察到eGFP的表达,证实了微型基因组的功能活性;缺乏N、P或L蛋白,检测不到eGFP的表达;缺乏M2-1蛋白,eGFP的表达量降低。结论成功建立了RSV微型基因组拯救系统,并证实了其拯救功能,为利用反向遗传学手段拯救重组RSV,研发RSV减毒活疫苗奠定了基础。     

呼吸道合胞病毒F1蛋白截短体(F_(212-489))的原核表达及纯化

目的原核表达并纯化呼吸道合胞病毒(respiratory syncytial virus,RSV)F1蛋白截短体(F212-489)。方法从质粒p MD-18T-f中PCR扩增截短f1基因(f212-489),经TA克隆测序正确后,将目的片段插入原核表达载体p ET-28a,构建重组表达质粒p ET-28a-f212-489,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经镍离子亲和层析纯化后,进行Western blot鉴定。结果重组表达质粒p ET-28a-f212-489经双酶切鉴定构建正确;表达的重组蛋白相对分子质量约为30 000,主要以包涵体形式表达,表达量约占菌体总蛋白的10%;纯化的重组蛋白纯度为85%,可与羊抗RSV多克隆抗体特异性结合。结论成功表达了RSV F1蛋白截短体(F212-489),纯化的重组蛋白反应原性良好,为更好地研究RSV的免疫机理及通过基因工程方法研制特定部位的亚单位或多肽疫苗奠定了基础。     

甲型流感病毒HA基因核酸疫苗表达载体的构建及表达

目的构建含有甲型流感病毒HA基因的核酸疫苗表达载体,转染COS-7细胞,并检测目的蛋白的表达。方法将甲型流感病毒New Caledonia/20/99(H1N1)接种鸡胚后,收获尿囊液,提取流感病毒总RNA,RT-PCR法扩增HA基因。经亚克隆后获得质粒pMD-T/HA,酶切鉴定后测序,将其与质粒pVAX1分别双酶切后连接,构建甲型流感病毒表达载体pVAHA。脂质体法转染COS-7细胞,并检测目的蛋白的表达。结果扩增出1700 bp片段,与预期的HA基因大小相符,测序结果与GenBank报道一致,酶切鉴定表明甲型流感病毒表达载体pVAHA构建正确,Western blot检测证明了流感病毒HA蛋白的表达。结论已成功构建了甲型流感病毒HA基因核酸疫苗表达载体pVAHA。     

分泌型rhGM-CSF/IL-2融合蛋白表达载体的构建

目的 将GM-CSF基因与IL-2基因通过一中间接头G-G-G-S-G-G-S-G连接到一起,并在E.coli中分泌表达具有更高GM-CSF和IL-2生物活性的融合蛋白分子。方法 在引物设计中,通过选择密码子,在Linker的中前部设计了一个BamHI酶切位点,GM-CSF的下游引物和IL-2的上游引物均起始于该位点。然后通过PCR、酶切、连接等一系列分子克隆的方法,构建融合蛋白的克隆及表达载体。结果 经EcoRI/SaII双酶切鉴定,克隆载体pUC19GM-CSF/L/IL-2及表达载体pBVGMCSF/L/IL-2中均插入了正确的外源片段。结论 克隆及表达载体的成功构建,为进一步表达融合蛋白及活性研究打下了基础。     

柯萨奇B4病毒非结构蛋白P_2C基因的克隆与表达

目的克隆并表达柯萨奇B4病毒(CVB4)非结构蛋白P2C基因。方法提取CVB4总RNA,RT-PCR扩增P2C基因,克隆入pUCm-T载体中,进行酶切和测序鉴定。双酶切重组质粒pUCm-T-P2C,将P2C基因片段定向亚克隆至原核表达载体pMAL-C2中,转化大肠杆菌JM109,IPTG诱导表达,SDS-PAGE和Western blot鉴定表达产物。结果RT-PCR扩增得到987bp的基因片段,测序结果与GenBank公布的P2C基因序列一致。pMAL-C2-P2C经双酶切鉴定,证明构建正确。表达的融合蛋白相对分子质量约78000,表达量约占菌体总蛋白的25%,且具有良好的反应原性。结论已成功克隆并表达了CVB4P2C基因,为进一步研究其生物学活性奠定了基础。     

Asia 1型口蹄疫病毒P1-2A-3C基因真核表达质粒的构建及鉴定

目的构建Asia 1型口蹄疫病毒(FMDV)P1-2A-3C基因真核表达质粒。方法采集疫区牛的水疱液及水疱皮,经RT-PCR法扩增出Asia 1型FMDV的前体蛋白基因P1-2A片段和蛋白酶基因3C片段,分别克隆至pMD18-T载体上,通过酶切、连接,获得质粒pMD18-T-P1-2A-3C。经酶切获得P1-2A-3C片段,插入真核表达载体pVAXⅠpCMV启动子下游,构建pVAXⅠ-P1-2A-3C真核表达载体,用脂质体法转染HeLa细胞,进行IFA检测。结果Asia 1型FMDV真核表达载体pVAX1PⅠ-2A-3C,经酶切鉴定证明构建正确,转染HeLa细胞后,可见明显的黄绿色荧光,表明P1-2A-3C基因得到了表达。结论pVAXⅠ-P1-2A-3C可作为Asia1型FMD核酸疫苗的候选疫苗。     

结核分枝杆菌TB10.4与呼吸道合胞病毒F1蛋白的融合表达

目的在大肠杆菌中融合表达结核分枝杆菌(Mycobacterium tuberculosis,MTB)TB10.4蛋白与呼吸道合胞病毒(Respiratory syncytial virus,RSV)F1蛋白。方法从MTB标准菌株H37Rv全基因组中扩增TB10.4(N/X)和TB10.4(N/B)基因,分别插入原核表达载体pET-28a和pET-30a中,构建重组表达质粒TB10.4(N/X)/pET-28a和TB10.4(N/B)/pET-30a;从F/18T/DH5α菌株中扩增F1(B/X)基因,与TB10.4(N/B)/pET-30a质粒连接,构建重组表达质粒TB10.4-F1/pET-30a;将质粒TB10.4(N/X)/pET-28a和TB10.4-F1/pET-30a分别转化感受态E.coliBL21(DE3),IPTG诱导表达;表达的重组蛋白进行Western blot鉴定。结果重组表达质粒TB10.4(N/X)/pET-28a和TB10.4-F1/pET-30a经双酶切及测序证明构建正确;表达的重组TB10.4和TB10.4-F1蛋白相对分子质量约为13 000和59 000,可与鼠抗His单抗特异性结合。结论成功在大肠杆菌中表达了重组融合蛋白TB10.4-F1,为进一步研究其免疫原性及保护性奠定了基础。     

结核分枝杆菌热休克蛋白基因真核表达载体的构建及表达

目的构建结核分枝杆菌热休克蛋白X(heat shock protein X,HspX)基因与EGFP融合基因的真核表达载体p EGFP-N1-HspX,并在小鼠单核巨噬细胞RAW 264.7中表达。方法以结核分枝杆菌(H37Rv)基因组DNA为模板,PCR扩增HspX基因,定向插入至质粒p EGFP-N1的多克隆位点,经酶切及测序鉴定正确后,将质粒p EGFP-N1-HspX转染RAW264.7细胞,荧光显微镜下观察GFP的表达,Real-time PCR及Western blot法鉴定HspX基因的表达。结果质粒p EGFP-N1-HspX经酶切及测序鉴定,证明构建正确;质粒p EGFP-N1-HspX转染24 h后,荧光显微镜下可见GFP表达,表达的重组融合蛋白Flag-HspX相对分子质量约43 000;转染组细胞中HspX基因m RNA水平明显高于空载体组(P0.01)。结论成功构建了p EGFP-N1-HspX融合基因真核表达载体,并在RAW264.7细胞中获得表达。     

呼吸道合胞病毒表位重组蛋白F1-F/M2的原核表达、纯化及其免疫原性

目的原核表达、纯化呼吸道合胞病毒(respiratory syncytial virus,RSV)表位重组蛋白F1-F/M2,并检测其在小鼠体内的免疫原性,为RSV亚单位疫苗的研制、疫苗所致免疫病理反应机理的研究提供参考。方法利用定点突变技术对原核表达载体pGEX-6p-1进行改造,在其GST标签前制造一个HindⅢ酶切位点;从RSV long株中扩增F1片段,从质粒pET28a-G1-F/M2中扩增F/M2基因片段,并在克隆载体pUC19的辅助下,将F1-F/M2基因连接至改造后的pGEX-6p-1载体中,构建重组表达质粒pGEX-6p-1-F1-F/M2,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经6×His-tag标签蛋白纯化试剂盒纯化后,经尿素浓度梯度透析复性,SDS-PAGE分析其纯度,BCA法检测其浓度,Western blot法分析其反应原性。将纯化的重组蛋白F1-F/M2经铝佐剂乳化后,经腹腔免疫BALB/c小鼠,共3次,间隔10 d,末次免疫后10 d,采用空斑减少试验检测小鼠血清、肺组织匀浆和鼻腔冲洗液中抗RSV中和抗体水平;取小鼠脾脏,分离脾单个淋巴细胞,采用ELISAPOT技术检测重组蛋白诱导产生IFNγ的细胞免疫水平。结果重组表达质粒pGEX-6p-1-F1-F/M2经双酶切和测序证实构建正确;表达的重组蛋白F1-F/M2相对分子质量约为20 800,表达量约占菌体总蛋白的20%,几乎全部以包涵体形式表达;纯化的重组蛋白的纯度约为90%,浓度为0.42 mg/ml,可与小鼠抗RSV血清发生特异性反应;重组蛋白F1-F/M2能诱导小鼠血清中产生较高滴度的中和抗体(1∶289),肺部组织匀浆中略弱(1∶242),鼻腔冲洗液中未检测到中和抗体;重组蛋白F1-F/M2免疫小鼠的脾淋巴细胞经体外抗原刺激能产生特异性IFNγ斑点。结论成功原核表达了表位重组蛋白F1-F/M2,纯化后的重组蛋白能诱导产生较高滴度的中和抗体及CTL应答,有望开发成RSV亚单位疫苗。     

梅毒螺旋体-15脂蛋白的原核表达及其纯化

目的原核表达梅毒螺旋体-15(Treponema pallidum-15,TP-15)脂蛋白基因,并进行纯化及鉴定。方法人工合成455 bp的TP-15基因序列,克隆至原核表达载体p ETDuet-1,构建原核表达质粒p ETDuet-1/TP-15,经双酶切及测序鉴定后,将鉴定正确的重组质粒转化至E.coli BL21(DE3),经IPTG诱导表达;表达产物经Ni2+亲和层析纯化,纯化产物进行SDS-PAGE和Western blot分析。结果重组质粒p ETDuet-1/TP-15经双酶切及测序鉴定证明构建正确;表达产物相对分子质量约27 000,主要以包涵体形式存在,表达量约占菌体总蛋白的2.34%,纯化后蛋白纯度达99%以上,可与HRP标记的梅毒螺旋体多克隆抗体发生特异性结合。结论构建了原核表达质粒p ETDuet-1/TP-15,并于E.coli中成功表达,纯化后获得了高纯度的TP-15蛋白,为进一步研究建立梅毒病人血清检测试剂盒奠定了基础。     

乙苯,苯乙烯的生产及合成乙苯,苯乙烯催化剂的研究进展

苯乙烯是重要的石油化工原料。本文介绍了国内外苯乙烯生产的发展概况，合成乙苯，苯乙烯所用的催化剂种类，并就苯乙烯生产及催化剂的发展趋势提出了自己的观点。     

Synthesis and characterization of homopolymers and copolymers of various acrylates and acrylonitrile

Various homopolymers and copolymers of methyl acrylate, ethyl acrylate, butyl acrylate, and acrylonitrile in different feed ratios were synthesized. These were characterized by IR, ¹³C-NMR, DSC, DTA, and TGA. Spectroscopic characterization helped in differentiating copolymers of different mol ratios. Thermal analysis revealed different degradation patterns for homopolymers and copolymers. The temperature and energy changes associated with various phase transitions were dependent on the chemical composition of homo- and copolymers, as expected. © 1993 John Wiley & Sons, Inc.     

生物质气化及生物质与煤共气化技术的研发与应用

总结了生物质原料的特点及生物质单独气化的缺点;介绍了国内外生物质气化技术及生物质与煤共气化技术的研发与应用现状;分析了在此领域国内外的发展趋势与前景;概括了开展生物质与煤共气化技术研发的意义。     

Electronic structures and reactivities of monometallic and bimetallic clusters of Au and Cu

The variation of the Au 4f binding energy of Au clusters with the cluster size has been established by measuring the binding energies of clusters whose size distributions were independently determined by HREM and STM. The binding energy increases significantly when the cluster size is less than 2 nm. Au-Cu bimetallic clusters of the composition Cu₃Au have been deposited for the first time on carbon substrates. The shifts in the core level binding energies of the bimetallic clusters show the effect of alloying in the case of large clusters, but show effects of both alloying and cluster size in the case of the small clusters. The interaction of CO with Cu₃Au clusters is stronger than with a bulk Cu metal. The interaction of CO with small Cu clusters also seems to be stronger than with bulk Cu or with large Cu clusters.     

钾盐资源及钾肥供需情况分析及预测

阐述了国内外钾盐资源及钾肥生产现状,对国内外钾盐的供需形势进行了分析及预测,从资源、原材料、国际市场三方面提出了解决我国钾盐短缺的措施。     

Kinetics and Mechanism of Oxidation of Mono- and Polyacetals

Abstract

Kinetics and mechanisms of oxidation of 6 acetals by molecular oxygen and ozone in liquid phase have been studied. Reaction with molecular oxygen (70°C, 15–16 hr) leads to the formation monoethers of the corresponding glycols with 68–90% selectivity. Salts of metals and complexes with crown-ethers have increased the reaction rate significally. Ozone have reacted with acetals with formation similar products. The mechanisms of intermediate stages have been proposed.     

Comparison of the Adhesion and Cohesion of Triblock and Random Copolymers of Styrene and Butadiene

A substantially greater detachment energy is required to strip a polyethylene tereph-thalate (Mylar) film from a styrene-butadiene-styrene (SBS) triblock copolymer compared to that for peeling from a random styrene-butadiene (SBR) copolymer. This is true even though the intrinsic interaction between the Mylar and each elastomer is expected to be similar because of their virtually identical chemical composition. It is proposed that this difference in peel strength (between the SBS and SBR) is a consequence of the much higher dissipative capacity of the former elastomer. Another manifestation of this is the higher cohesive tear strength of the SBS compared to the SBR. Extents of energy dissipation within each elastomer during detachment of the Mylar adherend are consistent with the hypothesis that the average maximum stress experience before detachment is some similar fraction of each elastomer's tensile strength.     

责任和责任心的涵义与结构

责任是人应主动承担的角色义务和对其因过失所造成后果应承担的责罚.有两层涵义:一是义务;二是后果.责任心是个体自觉做好分内事务和履行道德义务的心理倾向,是个性心理品质成分中自我特征维度上的重要内容.责任心具有两个方面的涵义:一是角色分内职责;二是角色道德义务.责任心是一种通过责任认知、责任个性和责任适应的动态形式表现出来的静态品质,责任心是责任心过程结构与责任心关系结构相互制约、相互影响的统一体.     

二甲基二烯丙基氯化铵和丙烯酰胺的合成及应用

用复合引发体系(过硫酸盐-偶氮类引发剂)和脂肪胺类氧化还原体系引发,在实验室获得了单体转化率≥98%,特性粘数高于13.6 dL/g的阳离子共聚物PDA,探讨了控制聚合物分子量的影响因素.     

Wetting properties of homopolymers and copolymers of pentafluorostyrene and methylacrylate and homopolymer blends

Polypentafluorostyrene (PPFS), polymethylacrylate (PMA), and poly(pentafluorostyrene-co-methylacrylate), poly(PFS-co-MA) were prepared and the wetting characteristics of polymer blends of PPFS and PMA were compared with that of poly(PFS-co-MA) via contact angle measurements. The critical surface tension of polypentafluorostyrene was found to be 22.6 dyne/cm, which is comparable to the value reported for polytrifluoroethylene (22 dyne/cm). The critical surface tension of poly(PFS-co-MA) is not linearly related to its composition. The polymer blends of PPFS and PMA exhibit significant surface enrichment of the fluoropolymer. The harmonic-mean method¹ was employed to determine surface tensions of these polymers and many known polymers. It is found that the method produces useful surface tension data provided the contact angle values are derived from testing liquids of dissimilar polarity.