大腹园蛛丝腺及其细胞形态的初步观察

运用显微解剖和石蜡切片技术,我们首次对大腹园蛛Araneus ventricosus腹部的5种丝腺的形态进行了分离、拍照和切片观察.研究发现壶状腺腹腔较大,有多根纺管;梨状腺为多细胞腺体、成簇;集合腺有不规则的分支,纺管外附着小结节;葡萄状腺成簇.各类型丝腺腺体细胞明显可见,但未能明显见到不同丝腺腺体细胞的形态区别.同时也发现了几个存疑形态：壶状腺上附着的腺体、壶状腺下导管和壶状腺腺体细胞,它们可能与蜘蛛腺体的退化或发育等相关.     

大腹园蛛主壶腹腺蛛丝蛋白末端结构域的克隆表达

牵引丝(dragline silk)由主壶腹腺蛛丝蛋白(major ampullate spidroin, MaSp)组成,是蜘蛛丝中强度最好的丝,同时具有极佳的生物相容性和可降解性,因此引起研究者的研究热潮。目前关于大腹园蛛MaSp结构和成丝机理方面的研究甚少,限制了其仿生应用。本文以大腹园蛛牵引丝的组成蛋白质之一MaSp1为研究对象,通过锚定PCR的方法首次获取了大腹园蛛MaSp1 NT的完整编码基因,并对其进行了克隆、表达、纯化,产量可达60 mg/L;同时对该MaSp1的CT进行表达纯化,产量可达80 mg/L。另外,通过CD色谱分析了MaSp1 NT和CT的二级结构,结果表明二者的二级结构均以α-螺旋为主。上述结果为大腹园蛛MaSp1的结构和成丝机理研究奠定了基础。     

大腹园蛛大壶状腺丝蛋白-1基因部分cDNA的克隆和序列分析

大腹园蛛大壶状腺表达拖丝蛋白新基因的克隆, 为进一步研究蛛丝蛋白基因以及人工表达蛛丝蛋白提供参考依据。文章利用“通用方法”即反转录—置换法构建大腹园蛛(Araneus ventricosus)大壶状腺(Major ampullate gland) cDNA文库, 并筛选出具有典型重复结构的大腹园蛛大壶状腺丝蛋白-1部分cDNA序列AvMaSp1 (GenBank登录号: AY177203)。该部分序列大小为1 408 bp, 编码区为1 288 bp, 编码氨基酸429个, 预测分子量为34.07 kDa, 典型的重复结构为 (GA)nAm(GA)N, 与十字园蛛(Araneus diadematus)丝蛋白基因ADF-1 (GenBank登录号: ADU47853)同源关系最近, 一致性为75.0%。     

大腹圆蛛主壶腹腺cDNA文库构建和丝蛋白基因筛选

首次通过反转录-置换法和使用pUC18质粒成功构建大腹圆蛛（Araneus ventricosus)主壶腹腺（major ampullate gland)cDNA基因文库,并以鸟枪法从中筛选出具有典型重复结构的大腹圆蛛主壶腹丝蛋白cDNA基因AvF1,大小为1744bp,编码区为1572bp,编码氨基酸524个,分子量为42489.55Da,典型的重复结构为（GGP）nGGX。与现有已知的蛛丝蛋白基因中三带金蛛（Argiope trifas-ciata)鞭毛样丝基因（AtfF)有最高的同源性69.3%。大腹圆蛛主壶腹腺cDNA文库的构建和蛛丝蛋白新基因的克隆,为提供大腹圆蛛蛛丝蛋白基因背景和进一步研究蛛丝蛋白奠定了基础。     

大腹园蛛鞭毛样丝蛋白cDNA克隆

应用RT-PCR技术,从大腹圆蛛（Araneus ventricosus)壶腹腺中扩增出鞭毛样丝蛋白基因（flagelldid-form silk protein gene),经1.5%琼脂糖凝胶电泳分离,WizardPCR Preps DNA Purification System回收后,将其克隆在pGEM-T载体中,经限制性核酸内切酶鉴定和核苷酸序列分析证实,构建的重组擀粒pSF1中含有蜂蛛鞭毛样丝蛋白基因,且含有3个重复序列。     

大腹园蛛次壶腹腺丝的表达

基于大腹园蛛次壶腹腺丝Minor Ampullate Spidroin全长编码基因最新报道,研究了该基因的表达。利用PCR扩增该基因重复区一段长1 348 bp的片段P1,融合his-tag标签,构建酵母表达载体,在毕赤酵母菌GS115进行表达。同时构建大肠杆菌表达载体,在大肠杆菌BL21(DE3)中进行表达。SDS-PAGE和Western blotting检测结果表明,P1在两种表达系统中均可实现表达。研究结果显示:P1在GS115中的表达经优化后产量、产率有较大提高,且远高于BL21(DE3)中的表达,相应的纯化效率GS115也远高于对照BL21(DE3)的表达。研究表明酵母表达系统更适合重复度高、且富含Gly/Ala的天然蛛丝蛋白基因的表达,为表达全长天然MiSp编码序列提供前期实验基础,也为大规模蛛丝蛋白的重组表达建立了平台。     

大腹园蛛鞭状腺蛛丝蛋白N端结构域的克隆表达及圆二色谱分析

鞭状腺蛛丝是6种蛛丝中延展性最好的丝,由鞭状腺蛛丝蛋白(FlSp)构成,具有极大的潜在应用价值。目前,有关FlSp的末端功能模块(NT和CT)编码基因的报道极少,且其结构和功能均尚未明确,这在一定程度上限制了鞭状腺蛛丝的仿生。现利用5′-RACE的方法,以大腹园蛛总RNA和基因组DNA为模板,首次获取了大腹园蛛FlSp NT模块(FlSp_A.v-NT)的完整编码基因,并对其成功进行了克隆、表达及纯化,产量可达60 mg/L;同时,利用圆二色谱(circular dichroism,CD)对FlSp_A.v-NT的二级结构进行了分析,结果显示其主要以α螺旋构象存在,这是首次对FlSp NT的二级结构进行探索,为后续FlSp NT结构功能的研究提供了材料,奠定了研究基础。     

大腹园蛛(Araneus ventricosus)粗毒双向电泳及质谱分析

以大腹园蛛粗毒为材料,用固相pH梯度等电聚焦IPG(immobilized pH gradient)和SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)获得蛋白质组双向电泳图谱,经Bio-Rad公司的PDQUEST软件进行图像分析,检测到500个左右的蛋白质点．对其图谱的部分蛋白质点酶解后使用Micromass公司的ESI-Q-TOF进行了鉴定．得到了质量较好的MS／MS数据．然后将其在MS-Fit中的genepeptide数据库和Mascot的Swissprot中进行搜索从而对蛋白质点进行鉴定．目前初步获得5个组分的鉴定结果．     

大腹园蛛(Araneus ventricosus)12SrRNA基因片段序列分析

对大腹园蛛(Araneus ventricosus)的12SrRNA基因部分序列进行测定,得到276bp的碱基序列,其中A、T、G、C的含量分别为104bp(37.68％)、96bp(34.78％)、33bp(11.95％)、43bp(15.57％)。并尝试为我国蜘蛛目的分子系统学研究提供一些资料。     

不同体重肩斑银鳞蛛和大腹园蛛圆网的结构特征

对在自然条件下不同体重肩斑银鳞蛛Leucauge blanda和大腹园蛛Araneus ventricosus所织圆网的结构特征进行测量研究.结果表明:体重小于35.1 mg的肩斑银鳞蛛所织圆网的捕丝长度、捕食面面积、捕丝间距和半径丝根数均与个体体重呈显著正相关,而体重大于35.1 mg的个体中,这种关系并不显著,但其圆网的上、下部捕丝长度比与体重呈显著负相关,即圆网随个体的体重增加而表现出更强的不对称性;体重小于144.9 mg的大腹园蛛所织圆网的捕丝长度和捕食面面积均与个体体重呈显著正相关,体重小于103.8 mg的大腹园蛛所织圆网的半径丝根数与个体体重呈显著正相关,而大于这一体重分界值的个体中,这种关系同样不显著.大腹园蛛圆网的平均捕丝间距与体重未呈现出相关关系,体重大于85.4 mg的个体中,其网的上、下部捕丝长度比与体重呈显著负相关.两种蜘蛛圆网结构特征的变化及圆网结构特征与个体体重关系变化的不同体重分界值,可能反映了它们在不同生境下不同生长阶段的捕食投入与捕食策略.     

Gel electrophoresis in studies of protein conformation and folding

Electrophoresis through polyacrylamide gels is a useful method for distinguishing conformational states of proteins and analyzing the thermodynamic and kinetic properties of transitions between conformations. Although the relationship between protein conformation and electrophoretic mobility is quite complex, relative mobilities provide qualitative estimates of compactness. Conformational states which interconvert slowly on the time scale of the electrophoretic separation can often be resolved, and the rates of interconversion can be estimated. If the transitions are more rapid, then the electrophoretic mobility represents the equilibrium distribution of conformations. Protein unfolding transitions induced by urea are readily studied using slab gels containing a gradient of urea concentration perpendicular to the direction of electrophoresis. Protein applied across the top of such a gel migrates in the presence of continuously varying urea concentrations, and a profile of the unfolding transition is generated directly. Transitions induced by other agents could be studied using analogous gradient gels. Electrophoretic methods are especially suited for studying small quantities of protein, and complex mixtures, since the different components can be separated during the electrophoresis.     

Apparatus and method for preparative gel electrophoresis

A new apparatus for preparative gel electrophoresis with continuous elution which includes a miniaturized electrode and elution chamber system is described. The design provides high resolution, high yield, applicability for small and large amounts of peptide material, and easy operation. Furthermore, the apparatus enables a very accurate gel column or gel gradient to be formed. A method for preparative gel electrophoresis in sodium dodecyl sulfate which allows the purification of peptides and proteins without concurrently modifying tryptophane residues or blocking N-terminal α-amino groups is also described.     

Prolein polymorphism in a randombred chicken population

Polymorphism in plasma amylase, plasma alkaline phosphatase, non-specific esterase and red cell esterase-D of the Athens-Canadian randombred (ACRB) population of chickens was determined by polyacrylamide and starch gel electrophoresis. Amylase alleles Amy-1^A and Amy-1^B were segregating in the ACRB population with frequencies of 0.45 and 0.55 respectively. For the plasma alkaline phosphatase the F and S bands, the B band and a new isozyme migrating at a faster rate than the previously reported F band were detected. A genetic nomenclature for plasma alkaline phosphatase is suggested which considers the difference between the F and S bands as the presence or absence of sialic acid attached to a primary protein.
Plasma esterase activity was observed in all four of the regions previously reported, but there was no polymorphism found in any of the loci. All birds in this population showed the same red-cell esterase-D phenotype which consisted of a main band with sub-bands on each side.     

Protein polymorphism in quail populations selected for large body size

The polymorphism of five enzyme loci (amylase, alkaline phosphatase, albumin, for 4-week body weight was compared to that of the unselected control line (C). for 4 week body weight was compared to that of the unselected control line (C). Three loci in the C line and two in the P line demonstrated polymorphism. Plasma amylase was separated into six bands and zymograms were classified on the basis of these bands into nine phenotypes. Three of the nine types were of relatively high activity and six were of relatively low activity. All nine types were found in the C line, whereas, all birds of the P line had only the most active type. Two alkaline phosphatase alleles (Akp-2^B and Akp-2^C) were segregating in the C line. Gene frequencies of alkaline phosphatase for the Akp-2^B allele were 0.92 in the C line and 1.00 in the P line. Two albumin alleles (Alb^Q1 and Alb^Q2) were segregating in both populations. Gene frequencies for the Alb^Q1 allele were 0.74 in the C line and 0.81 in the P line. Two red cell esterase-D alleles (Es-D^F and Es-D^s) were segregating in both populations. The gene frequency for the Es-D^s allele (0.61) was higher than that of the Es-D^F allele in the C line. In the P line the frequency of the Es-D^F allele was higher than that of the Es-D^s allele. Heterozygosities of the C and P lines were estimated as 0.2258 and 0.1560 respectively. The relative inbreeding coefficient of the P line, calculated from heterozygosities was 0.31.     

2DE中IPG胶条转移到SDS-PAGE中的技术改进

2-维凝胶电泳(Two—dimensional gel electrophoresis,2DE)因其高通量、高分辨率等特点,被广泛用于蛋白质组的分离．然而,在蛋白质从第一向一固相(Immobilized pH gradients,IPG)胶条转移到第二向一十二烷基磺酸钠聚丙烯酰胺凝胶(Sodium dodecyl sulfate,SDS;Polyacrylamide gel electrophoresis．PAGE)时,常会引起蛋白质的损失．尤其是当将IPG胶条放入浓缩胶上时,在胶面不平、技术不熟练等情况下,常会在IPG胶条下引入气泡,导致蛋白质更多的损失．现将IPG胶条转移过程进行改进：先在第二向的十二烷基磺酸钠聚丙烯酰胺凝胶上加上薄薄的一层(约1～2mm厚)琼脂糖溶液,然后将IPG胶条转移上去,最后再封住胶条的上面．这样的转移不会引起气泡,可以大大提高实验的成功率及重复性,有利于蛋白质的转移(尤其是相对分子质量大于40kDa的蛋白质)．提高质谱鉴定的准确性．此法操作简便,可以减少因操作不熟练而带来的麻烦．     

Electrophoretical studies of proteins of the hypopharyngeal glands and of the larval food ofMelipona quadrifasciata anthidioides Lep. (Hymenoptera,Meliponinae)

Summary The electrophoretical protein patterns of hypopharyngeal glands, larval food ofMelipona, and royal jelly ofApis were compared.Since protein patterns of hypopharyngeal glands from newly emerged workers, brood cell provisioners and foragers are similar to freshly deposited larval food, the identical protein bands probably represent actual gland secretion. This suggests that, as inApis, the glands secrete proteins to the larval food, and maintain this ability throughout life, although at slightly different intensities, according to the activity of the bees.The similarity on the electrophoretic profiles of the major larval food protein inApis andMelipona is an interesting finding because of its probable evolutionary significance.     

G-electrode-loading method for isoelectric focusing, enabling separation of low-abundance and high-molecular-mass proteins

The G-electrode-loading method (GELM) is a technique enabling a large number of proteins from rat liver to enter an immobilized pH gradient (IPG) gel strip for isoelectric focusing (IEF). In this method, three slips containing the sample solution are placed on the cathodic edge of an IPG gel strip and a slip containing Chaps solution, a filtration membrane, and an electrode slip are placed on top. Finally, a G-electrode is placed on these slips. The Chaps solution (an amphoteric compound) is supplied gently to the sample solution during IEF and helps the proteins in the sample solution to enter the IPG gel strips with a high solubilization capacity. This method was compared with traditional slip-loading and in-gel rehydration, and it showed the best results for protein separation, including high-molecular-mass proteins.