林可链霉菌中的同源重组

为研究链霉菌中的同源整合频率和机制 ,采用不能在链霉菌中复制的大肠杆菌质粒转化链霉菌StreptomyceslincolnensisB48。质粒pYYE0 4a1上携带的被硫链丝菌素抗性基因灭活的林可霉素生物合成基因与染色体DNA上的同源基因发生重组 ,经过低抗筛选 ,得到两个突变子S .lincolnensisYY1和S .lincolnensisYY2。进一步以硫链丝菌素抗性基因为探针杂交染色体DNASmaⅠ片段 ,S .lincolnensisYY1和S .lincolnensisYY2都得到 1 5kb的阳性条带 ;而以缺失的lacZ基因为探针杂交染色体DNAHindⅢ和SmaⅠ联合酶切片段 ,只有S .lincolnensisYY2得到 4 4kb的阳性条带。Southern杂交结果表明S .lincolnensisYY1是由同源交换或二次重组产生的 ,而S .lincolnensisYY2为同源整合的结果。为验证同源整合子上大肠杆菌复制子和氨苄抗性基因的存在 ,用SphⅠ酶切染色体DNA后连接 ,连接液转化E .coliJM83感受态细胞 ,在氨苄抗性板上得到 2个转化子 ,命名为pSLE1。对…     

棒状杆菌2,5-二酮基-D-葡萄糖酸还原酶Ⅰ基因在大肠杆菌中的克隆与表达

从棒状杆菌(Corynebacterium sp.SCB3058)初步纯化得到两个2,5-二酮基-D-葡萄糖酸(2,5 DKG)还原酶,在此基础上利用PCR技术,以基因组DNA为模板,扩增得到含有2,5-DKG还原酶Ⅰ基因的片段,定向连接到PGEM3Zf(+)并转化大肠杆菌DH5α,筛选得到阳性克隆pGEM813.序列分析表明,片段全长1107bp,含有一个834bp的开放阅读框架,编码产生由278个氨基酸组成的分子量为34kD的蛋白.将目的基因的调控序列进行缺失突变后,克隆到原核表达载体pBL上获得表达质粒pBL4.通过温度诱导,经SDS-PAGE分析有明显的表达条带,约占菌体总蛋白的20%,并且表达的蛋白具有较高的酶活力.构建的优良基因工程菌为最终实现从葡萄糖一步发酵产生维生素C前体2-酮基-L-古龙酸打下了基础.     

苏云金芽胞杆菌cryⅡ基因的克隆和表达

分离的苏云金芽胞杆菌(Bacillus-thuringiensis)YBT-791对鳞翅目小菜蛾(Plutellaxylostella)有毒力,将其质粒DNA提纯,经HindⅢ酶切后与杀虫晶体蛋白cryⅡ基因探针杂交,显示出分子量分别为5kb和9．4kb两条DNA阳性片段。把5kb的DNA阳性片段克隆到pUCl8的HindⅢ位点上并转化大肠杆菌TGl,经酶切和杂交检测,证明斑点杂交阳性克隆子中含有5kb的cryI片段。把这个含cryⅡ基因的5kbHindⅢ片段进行亚克隆,将4kb的BamHI－Pstl酶切片段插入穿梭载体pXl61中,并用电脉冲法克隆于苏云金杆菌不产伴胞晶体的突变株中,得到产生单一cry Ⅱ基因编码的杀虫晶体蛋白的克隆菌株M一5。经电镜观察,该克隆菌株能形成出发菌YBT－791多种形态伴胞晶体中的一种方形伴胞晶体;经免疫双扩试验,它只能与Cry Ⅱ晶体蛋白抗血清形成沉淀线;经SDS—PAGE电泳,克隆菌的伴胞晶体只含有一种65kDa的晶体蛋白。生物测定结果表明它既对鳞翅目小菜蛾(Piutella xylostella)有毒性,又对双翅目致倦库蚊(Culex quinquefasciatus)有毒性。     

苏云金杆菌δ—内毒素基因在大肠杆菌中的克隆及表达

分离了苏云金杆菌肯尼亚亚种7404(Bacillus huringiensis subsp．Kenyae 7404)和库斯塔克亚种(Bacillus thuringiensis subsp．Kwrstaki HD-1)的质粒。经凝胶原位杂交证明苏云金杆菌肯尼亚亚种7404的δ—内毒素基因位于约47Md大小的一个质粒上。用蔗糖密度梯度离心法从sau 3 A1部分酶解的上述两种苏云金杆菌质粒DNA酶解片段中分离出大于 4kb的DNA片段,将这些片段克隆到pBR332的Barn HI位点上并转化大肠杆菌HB101。通过菌落原位杂交、菌落原位放射免疫试验及Western blct分析等方法,选到了带有δ—内毒素基因并能在大肠杆菌中表达此毒蛋白的转化体。初步的生物学试验表明,在四个试验过的转化体中带有肯尼亚亚种δ—内毒素基因的转化体TK89及带有库斯塔克亚种δ—内毒素基因的转化体THl2和TH48对烟青虫(Heliothis assulta)有毒杀活性。     

串珠镰孢霉D-泛解酸内酯水解酶基因的克隆及在大肠杆菌中的表达

利用D_泛解酸内酯水解酶N末端序列,并根据NCBI中公布的D_泛解酸内酯水解酶cDNA序列设计了一个特异引物,该引物结合Oligo(dT) 1 5,以串珠镰孢霉(Fusariummoniliforme)CGMCC 0 5 36mRNA反转录得到的总cDNA为模板进行扩增,获得约1 5kb左右的片段,将其克隆到T载体上进行测序,对测得的序列进行分析,重新设计了一对引物,并在引物两端分别加上限制酶EcoRⅠ和SalⅠ的识别位点序列,利用热启动PCR成功地扩增出了D_泛解酸内酯水解酶基因,基因片段长度为114 6bp ,该序列同来源于尖镰孢霉菌(F .oxysporum)AKU 370 2菌株的编码D_泛解酸内酯水解酶cDNA结构基因的同源性为90 0 6 %。将所得片段定向克隆到pTrc99a载体中,转化至JM10 9感受态细胞,筛选出了阳性克隆。经IPTG诱导阳性菌,进行SDS_PAGE电泳,检测出在约4 0kD处有一蛋白表达带。对两株重组基因工程菌的比活力进行测定,结果分别为37U和4 1U。     

琥珀酸弧菌L-天门冬酰胺酶基因的初级克隆和表达

本文以表达型噬菌体λgtll为载体,以及¹²⁵I标记的放射免疫抗体为探针,从EcDR I酶切的琥珀酸弧菌(Vyibrto succinogenes)染色体DNA片段中克隆得到携带天门冬酰胺酶基因的目的片段,在宿主菌E．Coil Y 1090 中得到表达。经酶解和凝胶电泳分析表明该插入DNA片段的分子量为5．8kb．重组DNA感染另一宿主菌E．ColiYl089后所产生的酶蛋白具有L-天门冬酰胺酶活力。用重组DNA(λgt11-AS8)为探针进行southern DNA杂交,琥珀酸弧菌染色体DNA的Ec0R I酶切片段中,出现一条位置在5．8kb处的杂交带,证明我们克隆到的携带L-天门冬酰胺酶基因的目的片段来自琥珀酸弧菌。     

苜蓿尺蠖核型多角体病毒后期Voly(A)+RNA的互补DNA的合成

从苜蓿尺蠖(Autographa californnica)核型多角体病毒感染的草地贪夜蛾(Spodoptera frugi-perda)培养细胞分离病毒的后期poly(A)+RNA,经离成髓细胞白血病病毒(Amv)逆转录酶、E. coli DNA聚合酶I、Sl核酸酶等一系列处理得到了混合的双链cDNA。经限制酶分析在聚丙烯酰胺凝胶上可以检出被-{h 1切割的混合cDNA产生的均一片段,证明合成的cDNA,混合物中含有相对丰富的周源cDNA,它们代表着病毒后期poty(A)+aNA中某些相对富含的种类。     

DNA错配修复基因mutS的高效表达及表达产物活性鉴定

将DNA错配修复基因mutS（2.56kb）克隆于分泌型原核表达载体pET32a（+）上,以N端融合6个组氨酸的形式在E.coliAD494(DE3) 中进行了IPTG诱导表达。SDSPAGE分析证实有一与预期分子量相应的诱导表达条带,其表达量占全菌蛋白质的35％左右,且表达蛋白以可溶形式存在。利用固定化金属离子（Ni²⁺）配体亲和层析柱纯化目的蛋白,其纯度为90％以上。与含有错配碱基DNA双链的结合反应证明该蛋白具有特异性识别、结合含有错配碱基DNA双链的生物活性。     

欧文氏菌2-酮基醛糖还原酶基因敲除的研究

根据已知基因序列,利用PCR技术,从克隆质粒和欧文氏菌(Erwinia sp.)SCB125染色体中重新扩增得到含有2酮基醛糖还原酶A和B(2KRA和B)基因的片段,分别用于基因表达和敲除。用于表达的片段定向连接到表达载体pBL并转化大肠杆菌DH5α后,获得高酶活表达。在证实发生了突变的基因表达产物仍具有酶活的基础上,对其进行基因敲除的研究。在体外将链霉素抗性基因插入到tkrA内部使其突变失活并作为阳性筛选标记,再将此突变基因构建到分配不稳定型质粒pBR322上,导入宿主菌后与染色体上正常基因进行同源重组交换,筛选质粒丢失的阳性菌落进行进一步鉴定。这项工作将为阻断旁路代谢,实现从葡萄糖一步发酵产生维生素C前体2酮基古龙酸(2KLG)打下基础。     

嗜水气单胞菌外膜蛋白基因ompTS的高效表达及其免疫原性

根据嗜水气单胞菌外膜蛋白基因ompTS的核苷酸序列设计引物,运用聚合酶链式反应（PCR）扩增出与预期大小相符的基因片段。将此基因片段克隆至质粒pRSET A的BamHI和EcoRI位点,构建重组质粒,转化大肠杆菌BL21（DE3）,经IPTG诱导获得高效表达,SDS-PAGE蛋白电泳表明在39.9kD处出现超强特异带,占总蛋白的51％。以 Ni-NTA-Conjugate抗体进行Western blot分析证明该399kD的蛋白为所表达的融合蛋白。纯化融合蛋白注射雄性新西兰大白兔可诱导产生特异抗体。ELISA和Western blot检测结果显示,该抗体与表达的融合蛋白和从嗜水气单胞菌中提取的36.9 kD外膜蛋白均呈阳性反应,表明所表达的融合蛋白仍保持原有外膜蛋白的免疫原性,为此融合蛋白作为疫苗的候选成份提供理论基础。     

家蝇幼虫差异表达基因的克隆筛选与分析

目的是利用mRNA差异显示(DDRT-PCR)技术对诱导后家蝇幼虫差异表达基因进行克隆分析。取诱导和未诱导家蝇(Musca domestica vicina)三龄幼虫总RNA进行mRNA差异显示反应,产物经6％非变性聚丙烯酰胺凝胶电泳展开,银染分析后,回收差异显示条带。随机选取4条诱导家蝇三龄幼虫中上调表达的基因条带,进行Northern杂交验证,有2条被验证为真实带,命名为YD1和YD2。对这2条基因片段进行T-A克隆和测序,长度分别为495bp和265bp,登录NCBI运用Blastn程序进行同源性比较,发现两者与任何已知基因同源性都低于70％,提示为两个未报道的新基因,在家蝇免疫反应中可能起着一定的作用。     

Proteins induced in Escherichia coli by benzoic acid.

Proteins induced by benzoic acid in Escherichia coli were observed on two-dimensional electrophoretic gels (2-D gels). Cultures were grown in glucose-rich medium in the presence or absence of 20 mM benzoate at an external pH of 6.5, where the pH gradient (deltapH) is large and benzoate accumulates, and at an external pH of 8.0, where deltapH is inverted and little benzoate is taken up. Radiolabeled proteins were separated on 2-D gels and were identified on the basis of the index of VanBogelen and Neidhardt. In the absence of benzoic acid, little difference was seen between pH 6.5 and pH 8.0; this confirms that the mechanisms of protein homeostasis in this range are constitutive, including the transition between positive and inverted deltapH. Addition of benzoate at pH 6.5 increased the expression of 33 proteins. Twelve of the benzoate-induced proteins were induced at pH 8.0 as well, and nine of these matched proteins induced by the uncoupler dinitrophenol. Eighteen proteins were induced by benzoate only at pH 6.5, not at pH 8.0, and were not induced by dinitrophenol. One may be the iron and pH regulator Fur, which regulates acid tolerance in Salmonella spp. The other 13 proteins had not been identified previously. The proteins induced by benzoate only at a low pH may reflect responses to internal acidification or to accumulation of benzoate.     

在海藻酸钠凝胶上诱导骨髓间充质干细胞分化为成骨细胞

通过在海藻酸钠凝胶上诱导bMSCs向成骨细胞分化,探讨其对骨髓间充质干细胞（bone mesenchymal stem cells, bMSCs）的生物学效应。采用MTT、甲苯胺蓝染色、von Kossa染色和RT-PCR分别检测细胞的增殖、生长形态、诱导后细胞的钙化结节和成骨相关基因的表达。实验组bMSCs生长状况良好、细胞增殖迅速,与对照组的增殖无差异;bMSCs成集落样生长明显,集落中央细胞重叠生长形成钙化结节;培养至12d,实验组和对照组的成骨相关基因,包括碱性磷酸酶、I型胶原和骨钙素,均为阳性表达,但实验组的表达量高于对照组。海藻酸钠凝胶能够促进bMSCs向成骨细胞的分化,是良好的骨组织工程支架材料。     

Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling

Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland.     

Albumin indirectly modulates fibrin and protofibrin ultrastructure

Albumin modulation of fibrin and protofibrin coagulation parameters was studied. Cation-depleted, fatty acid free, human and bovine albumins decrease fibrin clot turbidity in a concentration-dependent manner. Albumin also inhibits the formation of protofibrin gels, induced by addition of 25 microM Zn(II) to protofibrils, though it does not bind to (proto)fibrin. In order to verify that competition for cations underlies the influence of albumin, fibrinogen was dialyzed against cation-depleted albumin. Elemental analysis indicates a redistribution of Zn(II) from the fibrinogen to the albumin compartment, and the resultant fibrin clots are less turbid. Apparently, cation-depleted albumin acts as a competitor for divalent cations. The ability of albumin to compete for available Zn(II) was also expressed in gels formed by pH-jump experiments, in which fibrin monomer, maintained soluble at pH 4.9, is induced to change phase by addition of NaOH to pH 7.4. While turbidimetric evidence indicates that individual fibrin fibers simply become thinner with albumin, scanning electron micrographs (SEM) reveal a more complex effect on ultrastructure. Though albumin does not bind to the gels, fibrin gels produced with albumin show major changes in fiber ultrastructure, particularly evident in gels formed in the presence of Zn(II). These structural modifications are discussed within the context of the "excluded volume" effect, in which "crowding" by albumin alters (proto)fibrin reactivity and ultrastructure.     

Polymerization of acrylamide at acid pH using uranyl nitrate

A new photopolymerizing reagent, uranyl nitrate, is used for the polymerization of acrylamide gels at low pH. The amount of uranyl nitrate (0.2 mg/ml) required for the polymerization of gels at pH 3.0 is considerably less than that of persulfate (7 mg/ml). Use of this reagent obviates the need for the removal of excess of persulfate by preelectrophoresis. The electrophoretic separation of basic proteins in uranium-polymerized gels showed faster movement and better resolution of proteins and proved the gels to be versatile, uniform, and reproducible. Electrophoresis of trypsin in these gels does not affect the enzymatic activity. The catalyst can also be used for the polymerization of gels containing 3 M urea.     

Role of D and E domains in the migration of vascular smooth muscle cells into fibrin gels

The structure of fibrin plays an important role in the organization of thrombi, the development of atherosclerosis, and restenosis after PTCA. In this study, we examined the mechanisms of the migration of vascular smooth muscle cells (SMCs) into fibrin gels, using an in vitro assay system. Cultured SMCs from bovine fetal aortic media migrated into fibrin gels prepared with thrombin, which cleaves both fibrinopeptides A and B from fibrinogen, without other chemotactic stimuli. Both desA fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, and desB fibrin gels prepared with Agkistrodon contortrix thrombin-like enzyme (ACTE), which cleaves only fibrinopeptide B, similarly induced the migration of SMCs compared to fibrin gels prepared with thrombin. These results suggest that the cleavage of fibrinopeptides is not necessary, but rather that the three-dimensional structure of the gel may be important for the migration of SMCs. Furthermore, gels prepared with protamine sulfate, which forms fibrin-like gels non-enzymatically, similarly induced the migration of SMCs compared to the gels prepared with thrombin. Both anti-fibrin(ogen) fragment D and anti-fibrin(ogen) E antibodies inhibited the migration of SMCs into fibrin gels, suggesting that both the D and E domains of fibrin(ogen) are involved in the migration of SMCs into fibrin gels. The addition of GRGDS, a synthetic RGD-containing peptide, but not that of GRGES, a control peptide, partially inhibited the migration of SMCs into fibrin gels, suggesting that the migration of SMCs into fibrin gels is at least in part dependent on the RGD-containing region of the alpha chain. The migration of SMCs into fibrin gels was also inhibited by a monoclonal antibody for integrin alpha v beta 3 and alpha 5 beta 1, indicating that migration is dependent on these integrins. Furthermore, both fibrin(ogen) fragments D and E inhibited the migration of SMCs into fibrin gels, suggesting that these fragments, generated during fibrino(geno)lysis, may be relevant in the regulation of SMC migration into fibrin gels.     

Ruthenium (II) tris-bathophenanthroline disulfonate is well suitable for Tris-Glycine PAGE but not for Bis-Tris gels

Pre-cast bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) gels have proven to be very suitable for pre-fractionation for LC-MS/MS analysis due to high reliability and long stability. To visualize proteins within gels fluorescence dyes proved to be a good tradeoff between sensitivity and MS-compatibility. The custom-made ruthenium dye represents a low-cost alternative regarding fluorescence-based protein visualization with high sensitivity. We demonstrate, that this dye is incompatible with Bis-Tris gels, while using Tris-Glycine gels a competitive sensitivity to commercially available stains can be achieved.     

Double replica electroblotting: a method to produce two replicas from one gel

Electroblotting is a method by which proteins or nucleic acids, separated by electrophoresis, are transferred, also by electrophoresis, from a gel to a so-called transfer medium, e.g a nitrocellulose membrane. In some experiments, it is desirable to be able to obtain more than one replica from each gel and it has now proved possible to produce two replicas, which are almost identical, from one gel. This is achieved by applying one membrane on each side of the gel and change the direction of the current several times in such a way that the efficient transfer time is short in the beginning of the electroblotting and is increased for each cycle. This procedure will be referred to as 'double replica electroblotting'. Proteins were transferred at 100 V and the duration of an experiment with 2 h efficient transfer time in each direction was 7 h. The gel was more efficiently depleted of proteins after double replica electroblotting as compared to ordinary electrotransfer in one direction. Cathodically migrating proteins are also trapped on the membranes with this technique. Double replica electroblotting was used to produce two replicas from ordinary sodium dodecyl sulfate polyacrylamide gels as well as from 2-dimensional gels.     

Microengineering neocartilage scaffolds

Advances in micropatterning methodologies have made it possible to create structures with precise architecture on the surface of cell culture substrata. We applied these techniques to fabricate microfeatures (15-65 microm wide; 40 microm deep) on the surface of a flexible, biocompatible polysaccharide gel. The micropatterned polymer gels were subsequently applied as scaffolds for chondrocyte culture and proved effective in maintaining key aspects of the chondrogenic phenotype. These were rounded cell morphology and a positive and statistically significant (p < 0.0001) immunofluorescence assay for the production of type II collagen throughout the maximum culture time of 10 days after cell seeding. Further, cells housed within individual surface features were observed to proliferate, while serial application of chondrocytes resulted in the formation of cellular aggregates. These methods represent a novel approach to the problem of engineering reparative cartilage in vitro.