水稻新基因OsRwp34在大肠杆菌中表达的毒害作用

在水稻高温诱导cDNA文库中克隆了一个新的水稻基因OsRwp34,同源性分析表明OsRwp34是一典型的孤儿基因。为了研究OsRwp34的功能,构建了OsRwp34的表达载体pET-28(a)OsRwp34,并转化大肠杆菌菌株BL21(DE3),用IPTG诱导并定时从培养液中取样以检测OD600值和用平板记数法测定活菌数,结果发现在诱导30min后宿主菌开始大量死亡,表明OsRwp34的表达产物使宿主菌死亡。随后构建了1个N末端缺失15个氨基酸残基和2个C末端分别缺失12和47个氨基酸残基的OsRwp34缺失体,IPTG诱导后都没有出现毒性作用,推测N和C末端氨基酸残基的同时存在是OsRwp34的毒性作用所必须的。其详细机理有待进一步研究。     

重组链激酶及其三种突变体的活性研究

利用PCR技术从Streptococucspyogenes的基因组DNA中扩增了链激酶的编码基因ska,并进行了序列分析 ,利用基因删除及定点位变技术获得了删除了C-末端 42个氨基酸残基编码区的突变链激酶基因skaΔC42 ,第 5 9位Lys残基突变为Glu的突变链激酶基因skaK5 9E以及删除C-末端 42个氨基酸且第 5 9位Lys残基突变为Glu的突变链激酶基因skaΔC42K5 9E ,将ska及其三种突变体分别克隆到表达载体pET 1 5b上 ,构建分别表达野生型链激酶 (SK)、C-末端缺失 42个氨基酸残基的突变体 (SKΔC42 )、第 5 9位Lys残基突变为Glu的突变体 (SKK5 9E)及C-末端缺失 42个氨基酸且第 5 9位Lys残基突变为Glu突变体 (SKΔC42K5 9E)的表达载体pSK ,pSKΔC42 ,pSK K5 9E ,pSKΔC42K5 9E ,分别转化E .coliBL2 1 (DE3) ,IPTG诱导后在大肠杆菌中实现了高效表达 ,经亲和层析、离子交换层析及分子筛层析 ,获得了rSK、rSKΔC42、rSKK5 9E及rSKΔC42K5 9E ,活性分析表明rSK与其三种突变体具有相同的比活性。     

利用大肠杆菌表达禽流感病毒聚合酶酸性蛋白

目的：通过在大肠杆菌中分段表达禽流感病毒聚合酶酸性蛋白（PA蛋白）,探索PA基因中可能影响表达的区域。方法：构建分段缺失的PA蛋白突变体,用IPTG在大肠杆菌RosettaGamiB（DE3）中诱导表达,比较各突变体的表达效率。结果：N端缺失长度在143～408个氨基酸残基之间的9个突变体在大肠杆菌中的表达水平较高;而突变体PA/K（Δ1-40aa）、PA/M（Δ1-56aa）、PA/N（Δ41-56aa）和PA/P（Δ57-75aa）的表达水平很低;全长PA蛋白和缺失N端20个氨基酸残基的突变体PA/L则检测不到表达。结论：PA基因的61～225bp和325～426bp可能是影响PA蛋白表达的2个重要区域,为下一步表达全长PA蛋白奠定了基础。     

大肠杆菌CMP-唾液酸合成酶最小活性域的研究

比较大肠杆菌与脑膜炎奈瑟氏球菌的CMP－唾液酸合成酶的氨基酸序列,发现大肠杆菌CMP－唾液酸合成酶的保守区域主要位于N－端,其C－末端似乎对其催化活性没有作用。通过PCR方法,对大肠杆菌CMP－唾液酸合成酶的C－末端进行了一系列截短,将得到的产物连接至表达载体pET-15b中,在大肠杆菌BL21（DE3）pLysS中表达。经IPTG诱导,发现从C－末端截去189个氨基酸酶仍有催化活性,说明大肠杆菌CMP－唾液酸合成酶的最小活性域主要集中在N－不端的229个氨基酸。在催化活性的C－端缺失突变合成酶的比活,最适pH及热稳定性发生变化,提示被截去的C－端氨基酸残基虽不直接参与构成酶的催化活性中心,但可影响催化活性域的构象,从而对酶的催化活性与稳定性产生影响。     

蜘蛛大壶状腺丝蛋白基因的克隆和原核表达

以悦目金蛛(Argiope amoena)丝腺SMARTRACEcDNA文库为模板进行RT-PCR,克隆了1条大壶状腺丝蛋白(major ampullate spidroin,MaSp)基因cDNA序列。该条cDNA序列编码的氨基酸序列可区分为两部分(1)富含丙氨酸的片段和富含甘氨酸的片段相间排列构成的重复氨基酸序列区,并且富含甘氨酸的片段中有脯氨酸分布;(2)约100个氨基酸残基组成的C末端非重复氨基酸序列区。把MaSp基因cDNA序列亚克隆到质粒pET28b( )中,构建原核表达质粒pET28b( )-MaSp,表达质粒转化大肠杆菌BL21(DE3),用IPTG诱导表达。SDS-PAGE、氨基酸组成测定和N末端氨基酸序列测定的结果表明,表达产物为重组MaSp,表达量约为40mg/L。还对C末端非重复氨基酸序列对重组MaSp在水媒介中溶解性的影响进行了探讨。     

猪丹毒丝菌C43065株表面保护性抗原AN端保护区在大肠杆菌中的表达

目的：在大肠杆菌中表达猪丹毒丝菌C43065株表面保护性抗原A（SpaA）N端保护区（SpaA-N）,并检测其抗原性。方法：利用PCR方法从猪丹毒丝菌C43065株基因组中扩增出spaA基因片段,构建pMD18-spaA重组质粒并对插入片段进行测序;以pMD18-spaA重组质粒为模板,PCR扩增得到spaA-N基因片段,构建重组表达质粒pGEX-spaA-N,经序列测定证实正确后转化大肠杆菌BL21（DE3）,再经IPTG诱导表达GST-SpaA-N融合蛋白并纯化。结果：扩增得到的spaA基因长1881bp,编码由626个氨基酸残基构成的多肽;SDS-PAGE和Western印迹检测结果表明,诱导表达获得相对分子质量约64000的GST-SpaA-N融合蛋白,该融合蛋白能与相应抗体发生特异性反应。结论：获得了在大肠杆菌中可溶性表达的GST-SpaA-N融合蛋白,为进一步研究猪丹毒丝菌免疫保护性抗原奠定了基础。     

大肠杆菌BL21(DE3)中DnaE intein介导ABCA1蛋白的连接

摘 要：【目的】利用Ssp DnaE intein的蛋白质反式剪接技术研究在大肠杆菌中对ABCA1基因表达产物的连接作用。【方法】将ABCA1的cDNA于满足剪接所需的保守性氨基酸Cys978密码子前断裂为N端和C端两部分,分别与天然存在的反式作用Ssp DnaE intein的123个氨基酸的N端和36个氨基酸的C端编码序列融合,构建到原核表达载体pET-28a(+)。转化感受态大肠杆菌BL21(DE3)细胞,诱导表达后观察重组蛋白的表达和ABCA1的连接。【结果】转化菌经IPTG诱导表达,SDS-PA     

狂犬病病毒CVS株核蛋白基因的原核表达、纯化及鉴定

用KT-PCR技术对狂犬病病毒(RV)CVS株核蛋白(N)基因进行扩增,经克隆与序列分析,该基因编码450个氨基酸残基.将目的基因克隆到原核表达载体pET28a( )中,构建了重组质粒pET-N,将其转化表达菌BL21(DE3),于37℃1.0mmol/L IPTG条件下诱导表达.大肠杆菌菌体裂解产物经SDS-PAGE电泳分析,在分子量约为54kD处出现一新蛋白带,和预期的目的蛋白的分子量相符.质谱鉴定的结果表明成功表达了RV N蛋白,为狂犬病的进一步研究奠定了基础.     

金黄色葡萄球菌Cna基因的生物信息学分析及原核表达

[目的]对金黄色葡萄球菌的Cna基因进行生物信息学分析,并进行克隆和原核表达,为金黄色葡萄球菌重组疫苗的研发提供参考数据。[方法]使用Protparam、PSORT、Signal P-5.0、TMpred、Net Phos 3.1 Server、PSIPRED、SWISSMODEL等在线生物信息软件,分析Cna蛋白的理化性质、亚细胞定位、跨膜区域、磷酸化修饰位点、二级结构和三维结构。以基因组DNA为模板扩增Cna基因编码N1-N2子域的片段,扩增产物经酶切回收后与载体p ET-32a(+)相连接,构建重组表达质粒,将该重组质粒转化E.coli TG1,经菌落PCR、测序鉴定后,增菌培养并抽提质粒,再转化E.coli BL21(DE3),经IPTG诱导表达后进行SDS-PAGE分析。[结果]Cna蛋白由1 183个氨基酸残基组成,相对分子质量为133 006.82 Da,等电点为5.89,负电荷氨基酸残基数为180个,正电荷氨基酸残基数为167个,分子式:C5863H9241-N1569O1936S10,原子总数18 619个,是一种稳定的亲水蛋白质,定位于细胞膜上,第4～23、1 158～1 177位氨基酸残基分别形成一个典型的跨膜区,具有148个潜在的磷酸化修饰位点,二级结构中无规则卷曲和β折叠占较大比例,三维结构与二级结构预测结果相符。成功构建了重组表达质粒p ET-32a(+)-Cna,经IPTG诱导后重组蛋白(N1-N2子域)获得了表达,重组蛋白相对分子质量为52.1 k Da。[结论]Cna基因的生物信息学分析及该基因的成功表达,为研究Cna蛋白在免疫保护中的作用奠定了一定的基础。     

小鼠PC-1基因在大肠杆菌中的表达和纯化

利用PCR和基因重组技术构建了小嫌PC－1基因全长cDNA及其N端45个氨基酸残基的表达质粒pGEX-4T-1-mPC-1和pGEX-4T-1-mPC-1-45。经IPTG诱导后,在大肠杆菌DH5α中,GST－mPC-1和GST－mPC-1-45两个融合蛋白都获得了可溶性高表达。经谷胱甘肽Sepharose-4B亲和柱层析纯化后,获得了纯的GST－mPC-1和GST－mPC-1-45蛋白。     

Geminiviruses: a tale of a plasmid becoming a virus

Background  

Geminiviruses (family Geminiviridae) are small single-stranded (ss) DNA viruses infecting plants. Their virion morphology is unique in the known viral world – two incomplete T = 1 icosahedra are joined together to form twinned particles. Geminiviruses utilize a rolling-circle mode to replicate their genomes. A limited sequence similarity between the three conserved motifs of the rolling-circle replication initiation proteins (RCR Reps) of geminiviruses and plasmids of Gram-positive bacteria allowed Koonin and Ilyina to propose that geminiviruses descend from bacterial replicons.     

Vegetation as a component of a non-nested hierarchy: a conceptual model

Abstract. A general conceptual model of vegetation based on hierarchy theory is presented. The model emphasizes that prediction of vegetation requires consideration of both mechanisms of vegetation change and the constraints within which it occurs. The mechanisms of vegetation change are the responses to and effects upon their surroundings of individual plants. The most general constraints upon vegetation are aspects of the environment not affected by vegetation over successional time, and the pool of species within dispersal range. Examples of such environmental factors include macroclimate and soil parent material. In some cases, vegetation may alter important labile environmental factors such as soil nutrient and water availability. Some vegetation compositions appear to be resistant to changes in the general constraints. Due to both sources, there are multiple possible vegetation compositions given the same general constraints. Disturbance is defined as an abrupt change in the constraints on the vegetation resulting in a change in the vegetation's state or dynamics. Both the recognition of disturbance and the distinction between independent and labile environmental factors depend on the spatial and temporal scale of observation. For example, a particular wildfire at a given stand may be a disturbance, whereas at a larger scale of observation the same event may contribute to the wildfire regime, part of the constraints at that scale. Similarly, levels of soil organic matter may constrain vegetation over short time scales, due to influencing availability of water and nutrients. Over long time scales, the vegetation itself is a primary determinant of soil organic matter content. This model contains elements of both the initial, holistic theory of vegetation and recent, reductionistic approaches. It reiterates the need to considerboth mechanisms and constraints, stressed by contemporary and earlier workers. Hierarchy theory provides new insights concerning sufficient conditions for prediction, possible limits on predictability, and appropriate research strategy.     

Testing a Primary and a Secondary Endpoint in a Group Sequential Design

Summary We consider a clinical trial with a primary and a secondary endpoint where the secondary endpoint is tested only if the primary endpoint is significant. The trial uses a group sequential procedure with two stages. The familywise error rate (FWER) of falsely concluding significance on either endpoint is to be controlled at a nominal level α. The type I error rate for the primary endpoint is controlled by choosing any α‐level stopping boundary, e.g., the standard O'Brien–Fleming or the Pocock boundary. Given any particular α‐level boundary for the primary endpoint, we study the problem of determining the boundary for the secondary endpoint to control the FWER. We study this FWER analytically and numerically and find that it is maximized when the correlation coefficient ρ between the two endpoints equals 1. For the four combinations consisting of O'Brien–Fleming and Pocock boundaries for the primary and secondary endpoints, the critical constants required to control the FWER are computed for different values of ρ. An ad hoc boundary is proposed for the secondary endpoint to address a practical concern that may be at issue in some applications. Numerical studies indicate that the O'Brien–Fleming boundary for the primary endpoint and the Pocock boundary for the secondary endpoint generally gives the best primary as well as secondary power performance. The Pocock boundary may be replaced by the ad hoc boundary for the secondary endpoint with a very little loss of secondary power if the practical concern is at issue. A clinical trial example is given to illustrate the methods.     

Heating of a Local Region of a Branching Streamer as a Starting Point of a Space Leader and a Negative-Leader Step

Plasma Physics Reports - Localized plasma formations called space leaders are observed in streamer coronas of negative leaders of long laboratory sparks. The main leader completes a step when the...     

Transformation of a psychrotrophic bacterium with a plasmid from a mesophile

The psychrotrophic bacteriumBacillus psychrophilus was transformed with the broadhost-range plasmid pC194. The ability of the transformant to express chloramphenicol (CAM) resistance and the possible effects of such expression on the physiology of the psychrotroph were examined. The transformant exhibited growth rates, filament formation at elevated temperatures, synthesis of cold shock proteins and cold acclimation proteins, similar to the parentalB. psychrophilus.