Efficient Process Development of Recombinant Human Granulocyte Colony-Stimulating Factor (rh-GCSF) Production in Escherichia coli

Background:

The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients.

Methods:

An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h^-1 at the beginning of feeding to 0.4 h^-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography.

Results:

Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l^-1 and 32.1 g.l^-1. Also, the final specific yield (Y_P/X) and total productivity of rh-GCSF were obtained 254 mg.g^-1 DCW and 1.83 g.l^-1.h^-1, respectively. According to the available data, this is one of the highest Y_P/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen® and PD-Grastim showed that all proteins have a similar secondary structure.

Conclusion:

Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity. Key Words: Escherichia coli, Granulocyte colony-stimulating factor (GCSF), Process development     

Effect of Combination Therapy Using Hypothermia and Granulocyte Colony-Stimulating Factor in a Rat Transient Middle Cerebral Artery Occlusion Model

Background: Stroke is the third leading cause of death. Hypothermia has been recognized as an effective method in reducing brain injury. In this study, we assessed the effects of granulocyte colony-stimulating factor (G-CSF) as a neuroprotective agent and mild hypothermia on mortality, behavioral function, infarct volume, and brain edema in Wistar rats. Methods: Forty male rats were used in five groups (eight rats in each group): control, hypothermy, G-CSF, combination hypothermy + CSF, and sham. Rats were anesthetized by injection of chloral hydrate (400 mg/kg) intraperitoneally. Transient cerebral ischemia was induced by 60-min intraluminal occlusion of left middle cerebral artery. Hypothermia, initiated at the time of reperfusion and G-CSF was started one hour after reperfusion at a dose of 15 mg/kg subcutaneously. The motor behavior was measured using Garcia’s index and animals were assigned for the assessments of infarction, brain swelling, and mortality rate. Results: The mortality was 38.46% (control group) and reduced in other groups. Neurological deficit score of control group (40.31 ± 1.56) was significantly lower than in treatment groups. The total cerebral infarct volume of treatment group was significantly lower than control group (43.96 ± 44.05 mm³). Treatment with hypothermy plus G-CSF (2.69 ± 0.24%) could significantly reduce brain swelling volume than other treatment groups. Conclusion: Our major finding is that mild hypothermic treatment plus G-CSF significantly reduced mortality rate and edema and improved neurological function. The results suggest that the combination of hypothermia and G-CSF is more effectively than other treatment groups being used alone. Key Words: Granulocyte colony-stimulating factor (G-CSF), Rats, Brain ischemia, Hypothermia     

Transgenic Expression of the Recombinant Phytase in Rice （Oryza sativa）

In many plants, phytic acid (phytate, 1, 2, 3, 4, 5, 6-hexakisphosphate) is one of the main storage forms of phosphate. About 80% of phosphorus (P) in cereal plants, including rice is stored as phytic acid [1-2]. P in phytic acid can’t be utilized by monogastric animals including human, while it was estimated that only 1/3 of the total P in most of the vegetal feedstuff could be efficiently utilized by the livestock. Therefore, for animal feed with P supplementation is expected to meet the d…     

人粒细胞巨噬细胞集落刺激因子在大豆中表达的初步研究

构建了人粒细胞巨噬细胞集落刺激因子（hGM-CSF）的植物表达载体pCAMBIA3300-CSF-KD，转化大豆，获得转hGM-CSF基因的植株。hGM-CSF的植物表达载体pCAMBIA3300-CSF-KD，该因子末端被加上内质网驻留信号（KDEL），适合在植物中表达。用大豆合丰47的茎尖作为外植体，通过农杆菌介导的转化，获得17株T1转化株。PCR扩增验证转基因植株，检测到11株阳性植株。     

Preliminary studies on growth and fresh weight of lettuce (Lactuca sativa) as affected by clay pot irrigation and spacing

An experiment (Completely Randomized Design) was set up to determine the effects of Clay Pot Sub-surface Irrigation (CPSI) and spacing on the growth and fresh weight of lettuce (Lactuca sativa). The treatments were: CPSI with spacing; 15 x 15 cm, 20 x 20 cm and 30 x 30 cm. Control treatments were Watering Can Irrigation (WCI) with the same spacing as above. Treatments were replicated three times given a total of 18 experimental units. Eighteen large enamel basins of 50/20 cm (diameter/height) were filled with good topsoil and a clay pot buried neck deep in each of the basins. Seedlings were planted in all the eighteen basins. Five Hundred mL of wastewater was applied daily to plants in each container having either clay pot or watering can treatment. Plant height increased from 2.50 to 4.25 cm within 6 Weeks after Transplanting (WAT) under CPSI and only increased from 2.14 to 2.99 cm under WCI. The CPSI also supported better leave growth and fresh weight. The fresh weight of lettuce increased almost two fold under 15 x 15 cm spacing compared to 20 x 20 and 30 x 30 cm.     

The content of nitrate and protein in lettuce (Lactuca sativa var,capita (Butterhead salad)) grown under different conditions

Summary An examination of lettuce, grown under various conditions in the open air and in the greenhouse, has shown that the content of nitrate and protein vary greatly.These variations can not be explained by one single factor e.g. fertilization, time of year etc., but are caused by an interaction between several factors.A high content of nitrate in lettuce does not imply that the technique of growing is at fault. It may imply that the lettuce was cut at a time, when this appearance was most attractive to the consumer. The origin of a high nitrate content may also be that all the growth factors including the climatic factors were nonoptimal for the complex physiological processes involved in growth and protein-synthesis.Future work to improve lettuce quality should include a reduction in nitrate content by appropriate balance of all growth factors without impairing optimal yield.

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Zusammenfassung Versuche mit kopfsalat, die unter verschiedenen Bedingungen im Freiland und im Gewächshaus durchgeführt wurden, zeigten, daß der Gehalt an Nitrat und an Rohprotein stark variierte.Diese Variationen können nicht durch einen einzigen Faktor, z.B. Düngung oder Jahreszeit usw. erklärt werden, sondern haben ihre Ursache in einer Interaktion verschiedener Faktoren.Ein hoher Nitrategehalt beim Kopfsalat ist keinesfalls immer auf eine falsche Kulturmaßnahme zurückzuführen. Es kann möglich sein, daß dieser Kopfsalat zu einer Zeit geerntet wurde, als sein Erscheinungsbild für den Verbraucher äußerst attraktiv war. Andererseits kann die Ursache eines hohen Nitratgehalts auch sein, daß alle Wachstumsfaktoren — eingeschlossen der klimatischen — nicht für die komplexen physiologischen Prozesse optimal waren, die für Wachstum und Proteinsynthese wichtig sind.Für die Zukunft gilt es, die Qualität des Kopfsalates zu verbessern, indem man eine Reduktion des Nitratgehalts durch ein Gleichgewicht aller Wachstumsfaktoren anstrebt, ohne aber die Erzielung eines optimalen Ertrags außer acht zu lassen.

     

茶树CsCDF1基因克隆及表达分析

采用SMART-RACE技术克隆了茶树Dof（DNA binding with one finger）基因CsCDF1的全长cDNA序列,并利用在线生物信息学软件对其进行了分析。采用实时荧光定量PCR分析该Dof基因在茶树不同组织间的表达差异和昼夜表达规律,及其在氮饥饿处理2周后对不同浓度氮素诱导的响应。该cDNA序列全长1β606βbp,包含1个编码464个氨基酸的完整开放阅读框,含有高度保守的DOF结构域,推导的蛋白分子量为50.8βkDa,理论等电点（PI）为5.52;其编码的蛋白序列与茸毛烟草、马铃薯和美花烟草的CDF蛋白（Cycling dof factor）相似性分别为69%、67%、68%。系统发育分析结果表明,该基因编码的氨基酸序列与拟南芥CDF蛋白聚为一类,因此将其命名为CsCDF1;CsCDF1在3个不同茶树品种根系中的表达量均高于一芽二叶和成熟叶;在一天中,该基因的表达呈现出昼夜节律变化;在氮饥饿后重新供氮,不同组织中该基因对不同浓度氮素的响应均为上调。     

不同处理方式的妃子笑荔枝花穗ARF基因家族的表达分析

生长素反应因子(ARF)是一类可以结合在生长素应答基因启动子部位的转录因子,在植物的生长发育中起着至关重要的作用。为了研究荔枝ARF基因在不同花穗处理方式的表达情况,进而挖掘花穗发育过程中起主要作用的ARF关键基因,本研究利用课题前期基于转录组数据库鉴定出的LcARF基因家族,通过对妃子笑荔枝花穗分别进行疏花和喷施烯效唑处理,利用荧光定量PCR(qRT-PCR)技术进一步研究LcARF基因家族在不同处理下的表达情况。结果表明：21 个荔枝ARF基因在疏花和烯效唑处理花穗发育过程中有明显不同的表达规律,且不同基因表达量有较大差异。因此推测,ARF家族成员在花穗发育过程中特定阶段起一定的作用,同时本研究也为深入探究ARF基因家族的功能奠定理论基础。     

一个水稻小热休克蛋白的异源表达及寡聚特性分析

【目的】在前期研究中,本实验室已克隆了一个水稻小热休克蛋白基因(OsSHSP17.6),并发现该基因的表达明显受到热激和病毒侵染调控,表明该蛋白可能在逆境胁迫过程中起重要作用。本研究的目的在于进一步明确OsSHSP17.6的特性。【方法】在本研究中,进一步将该基因亚克隆至原核表达载体p ET-32a并导入大肠杆菌E.coli BL21(DE3)pLysS诱导表达,通过亲和层析的方法纯化了该重组蛋白,进一步用于非变性聚丙烯酰胺凝胶电泳和Western blotting分析。【结果】异源表达的重组OsSHSP17.6能减轻IPTG对宿主菌的毒害。非变性聚丙烯酰胺凝胶电泳和Western blotting分析显示纯化的重组OsSHSP17.6蛋白在体外能形成同源二聚体和寡聚体。【结论】这些结果支持OsSHSP17.6是一个有功能的分子伴侣蛋白并表明该蛋白可能通过形成同源寡聚体的方式参与逆境胁迫反应,这为进一步明确OsSHSP17.6的功能机制奠定基础。     

Spotted-Leaf Mutants of Rice(Oryza sativa)

Many rice spotted-leaf(spl) mutants are ideal sources for understanding the mechanisms involved in blast resistance,bacterial blight resistance and programmed cell death in plants.The genetic controls of 50 spotted-leaf mutants in rice have been characterized and a few spotted-leaf genes have been isolated as well.This article reviews the origin,genetic modes,isolation and characterization of spotted-leaf genes responsible for their phenotypes,and their resistance responses to main rice diseases.     

测定水稻硅含量的一种简易方法

 介绍了一种简易的测量水稻硅含量的方法，其主要特点为准确、方便、能批量化测定水稻硅含量。以珍汕97B/密阳46重组自交系群体，两次重复，共498个株系的谷壳、剑叶和茎秆为材料，验证了该方法的可靠性。每份材料测定2份样品，两次测定结果达极显著正相关（P<0.01），线性相关系数分别为0.97954、0.97026和0.98848。取10份代表性样品进行了高温碱熔解法的测定，两种方法测定结果高度一致， r = 0.9993。     

芸芥种质资源的遗传多样性研究

用100个随机引物对18份芸芥材料的基因组DNA进行扩增,结果有10个随机引物得到了稳定一致的RAPD谱带,每个引物扩增出2～17条谱带,共扩增出91条谱带.采用UPGMA法对扩增出的谱带进行遗传聚类分析,在遗传距离(D)=0.1333处,将18个芸芥材料分为2类8组,揭示了芸芥种质资源的遗传多样性.     

水稻亚种间的亲和性研究

对160个亚种间F_1结实率的分析表明,籼稻 IR58和二九丰、粳稻秀水117和T8340具有较好的亚种间亲和性。43个粳稻品种分别与 IR58和二九丰测交,其成对 F_1的结实率随粳稻品种不同而出现 IR58/粳高于二九丰/粳、二九丰/粳高于 IR58/粳或两者相似的情形,其最大差值可达72.6个百分点。10个籼稻品种分别与秀水117和 T8340杂交,其F_1结实率均以秀水117/籼较高,成对F_1结实率的最大差值可达39.6个百分点。这表明不存在对亚种间品种均表现亲和性的“广亲和”品种,亲和性的广与狭只是相对的,既有一般亲和性,又有特殊亲和性。本研究发现4个受测品种的亲和性与色素基因之间不存在连锁或相关关系。     

一个新的水稻液泡膜Na+/H+逆向转运蛋白基因的克隆及表达特征

通过RT PCR技术从水稻幼苗组织中克隆了一个新的Na+/H+逆向转运蛋白基因OsNHX2,它编码一条长544氨基酸的多肽。OsNHX2与水稻和拟南芥的液泡膜Na+/H+逆向转运蛋白OsNHX1、AtNHX1氨基酸序列一致性分别为78%和77%。基因结构分析表明OsNHX2与OsNHX1具有类似的外显子和内含子构成,但不同于拟南芥AtNHX1,表明OsNHX2与OsNHX1可能起源于单双子叶植物分化后的水稻染色体复制。半定量RT PCR方法检测了OsNHX2与OsNHX1在水稻耐盐品种韭菜青与盐敏感品种IR28的地上部与根部的表达。结果表明：耐盐品种韭菜青的OsNHX2与OsNHX1基因在盐胁迫下表达持续增强,而在盐敏感品种IR28的地上部,OsNHX2基因在盐胁迫处理后1 h表达增强后立即减弱,根部的表达则基本不变,而IR28地上部与根部OsNHX1则在盐胁迫处理初期表达增强,随后即减弱。研究结果表明水稻两个液泡膜Na+/H+逆向转运蛋白基因OsNHX2、OsNHX1在盐敏感程度不同的水稻品种中表达有所不同,液泡膜Na+/H+逆向转运蛋白基因的转录调控可能是决定水稻耐盐能力的一个重要因素。     

水稻根系活力的遗传分析

 以典型籼粳交(窄叶青8号/京系17) F1代花培加倍的DH群体为材料，在抽穗期用TTC法考察根系活力。利用已构建的分子连锁图谱，采用区间作图法对根系活力进行QTL分析，在第4染色体的RG449和RG809之间检测到一个QTL。用Epistat软件分析影响根系活力的单个位点和位点间的互作，结果在RG809附近检测到一个单基因，并检测到2个条件型互作和4对互适型互作，这些位点分布在第1、2、4、7、8、9和11染色体上。     

茶树WRKY转录因子基因CsWRKY57的克隆及表达分析

WRKY转录因子是植物特有的一类转录因子,在植物生长发育及胁迫应答过程中均发挥重要的调控作用。为探究WRKY转录因子与茶树抗旱及耐盐性的关系,本研究基于茶树转录组数据库中的检索结果,以陕茶1号1年生茶树为试验材料,克隆获得了1个WRKY转录因子基因,命名为CsWRKY57。生物信息学分析表明,CsWRKY57基因cDNA全长为1 222 bp,编码303个氨基酸,预测分子量为33.5 kD,理论等电点为5.49;另外,蛋白比对分析显示,CsWRKY57包含1个典型的WRKY核心序列和1个C2H2型锌指结构,属于WRKYIIc家族。实时荧光定量PCR分析结果显示,CsWRKY57基因在高盐、干旱、ABA胁迫下均被诱导表达,且表现出先增加后降低的趋势,表明CsWRKY57基因参与了茶树体内干旱、高盐和ABA的调控途径。转录激活活性试验表明,CsWRKY57无转录激活活性,意味着CsWRKY57可能需要与其他元件结合才能启动基因的表达。     

冻融法纯化水稻胚乳RNA及稻米品质重要相关基因表达谱研究

 提出了一种从水稻胚乳组织中快速分离总RNA的方法，可以简便地去除RNA中存在的淀粉等多糖类杂质，所获的胚乳总RNA其质量可满足Northern印迹、微阵列制备等分子生物学实验要求。将从966个水稻品种的胚乳中分离所得的RNA制备RNA阵列，用于稻米品质重要基因Wx基因的表达谱分析，结果证实：水稻Wx基因表达丰度总体上与水稻胚乳的直链淀粉含量呈正相关；应用于Northern印迹实验，表明在水稻胚乳组织中，淀粉分支酶Rbe1具有两个长度不同的同源基因，其中一个分子量大的为胚乳特异表达，分子量小的则在水稻各组织中共表达。     

大麻RAPD分子标记的引物筛选

利用RAPD分析技术对野生型大麻Ym43及栽培品种大麻Ym36的基因组DNA进行分析,优化了大麻总DNA的提取方法和PCR扩增条件,从Operon公司的OP系列280个随机引物中筛选出42个扩增效果良好的引物,为以后RAPD技术应用于大麻的遗传研究奠定了基础。     

大麻RAPD分子标记的引物筛选

利用RAPD分析技术对野生型大麻Ym43及栽培品种大麻Ym36的基因组DNA进行分析,优化了大麻总DNA的提取方法和PCR扩增条件,从Operon公司的OP系列280个随机引物中筛选出42个扩增效果良好的引物,为以后RAPD技术应用于大麻的遗传研究奠定了基础.     

杂交水稻种子穗萌遗传效应的研究

 以穗萌抗性水平不一的6个不育系和7个恢复系为亲本材料，采用不完全双列杂交的试验设计，并运用包括胚乳、细胞质和母体植株三套遗传体系的种子遗传模型及估算遗传方差分量和预测遗传效应值的统计分析方法，对杂交水稻种子穗萌特性的遗传效应进行了研究。结果表明，杂交水稻种子的穗发芽率同时受种子直接加性、种子直接显性和母体加性效应的影响，且以种子直接效应为主，不受细胞质和母体显性效应的影响。在选择育种中，田间穗上发芽率以单粒选为好。而穗萌指数同时受制于种子直接遗传效应、母体效应和细胞质效应，但母体植株中只测到显著的母体加性效应。