Hydrophilic and hydrophobic copolymers of a polyaspartylhydrazide bearing positive charges as vector for gene therapy

BACKGROUND: The design of polymeric vectors for gene delivery provided with specific properties is one of the most critical aspects for a successful gene therapy. These polymers should be biocompatible as well as able to carry efficiently DNA to target tissues and to transfect it into cells. RESULTS: The formation of complexes of poly[(α,β‐asparthylhydrazide)–poly(ethylene glycol)] and poly[(α,β‐asparthylhydrazide)–hexadecylamine] copolymers functionalised with glycidyltrimethylammonium chloride (PAHy–PEG‐GTA and PAHy–C₁₆‐GTA, respectively) with DNA was studied. The effects of the introduction of hydrophilic (PEG) or hydrophobic (C₁₆) moieties on the chains of PAHy–GTA copolymers, such as the stabilising effect on the DNA structure, were evaluated. In particular, we observed a high DNA protection by PAHy–PEG‐GTA copolymers. Degradation studies led us to suppose a particular aqueous conformation of the polyionic complex of PAHy–PEG₂₀₀₀‐GTA in which DNA should be internalised into an inner core surrounded by a PEG hydrophilic shell; while no significant protection was detected with PAHy–C₁₆‐GTA in which DNA should be disposed on the surface of the complex, freely exposed to DNase II action. CONCLUSION: The insertion of PEG or C₁₆ chains into the polymeric structure of PAHy–GTA copolymers changes significantly the DNA complexing and protecting ability of the PAHy–GTA copolymers, showing that hydrophilic and hydrophobic side chains can play a crucial role in supramolecular arrangements of interpolyelectrolyte complexes between DNA and PAHy copolymers. Copyright © 2008 Society of Chemical Industry     

Functional properties of soy proteins

Soy protein ingredients must possess appropriate functional properties for food applications and consumer acceptability. these are the intrinsic physicochemical characteristics which affect the behavior of protein in food systems during processing, manufacturing, storage and preparation, e.g., sorption, solubility, gelation, surfactancy, ligand-binding, and film formation. These properties reflect the composition and conformation of the proteins, their interactions with other food components, and they are affected by processing treatments and the environment. Because functional properties are influenced by the composition, structure and conformation of ingredient proteins, systematic elucidation of the physical properties of component protein is expedient for understanding the mechanism of particular functional traints. The composition and properties of the major components of soy proteins are summarized, and the functional properties of soy proteins of importance in current applications (e.g., hydration, gelation, emulsifying, foaming and flavorbinding characteristics) are briefly reviewed.     

N-Glycosidase-carbohydrate-binding module fusion proteins as immobilized enzymes for protein deglycosylation

A carbohydrate-binding module (CBM) was fused to the N-termini of mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (EndoF1) and peptide N-glycosidase F (PNGaseF), two glycosidases from Chryseobacterium meningosepticum that are used to remove N-linked glycans from glycoproteins. The fusion proteins CBM-EndoF1 and CBM-PNGaseF also carry a hexahistidine tag for purification by immobilized metal affinity chromatography after production by Escherichia coli. CBM-EndoF1 is as effective as native EndoF1 at deglycosylating RNaseB; the glycans released by both enzymes are identical. Like native PNGaseF, CBM-PNGaseF is active on denatured but not on native RNaseB. Both fusion proteins are as active on RNaseB when immobilized on cellulose as they are in solution. They retain activity in the immobilized state for at least 1 month at 4 degrees C. The hexahistidine tag can be removed with thrombin, leaving the CBM as the only affinity tag. The CBM can be removed with factor Xa if required.     

Functional properties of oilseed proteins

The functional and physical properties, rather than the nutritional value, of protein in protein-containing products will largely determine their acceptability as ingredients in prepared foods. The nature of the protein per se, the presence of other constituents naturally present in protein-containing products, the degree to which the protein product is refined, the presence of other ingredients in a food system to which protein products are added, manufacturing conditions and a number of other factors, working singly or together, will influence the way proteins (as well as other ingredients) exert their functional characteristics. Differences in these characteristics are presented with examples of the manner in which various factors can influence functionality. Nonfunctional properties may be of interest for certain applications. Presented at the AOCS-AACC Joint Protein Short Course, French Lick, Indiana, July 13–16, 1969.     

DNA-polymer complexes for gene therapy

The condensation of DNA induced by vinyl saccharide graft copolymers of N-methacryloy-lamino-D-glucose with N,N-dimethylaminoethyl methacrylate and N-vinylpyrrolidone with N-vinylamine and induced by the linear polycation poly(L-lysine) in solution, as well as the conformational changes of a DNA molecule during its interaction with polycations in solution, is studied by atomic-force microscopy, low-gradient viscometry, dynamic birefringence, spectrophotometry, circular dichroism, and electrophoresis. The differences in the conformational changes of DNA related to the effect of polycations and other agents inducing its condensation are discussed.     

LINKER: a program to generate linker sequences for fusion proteins

The construction of functional fusion proteins often requiresa linker sequence that adopts an extended conformation to allowfor maximal flexibility. Linker sequences are generally selectedbased on intuition. Without a reliable selection criterion,the design of such linkers is often difficult, particularlyin situations where longer linker sequences are required. Herewe describe a program called LINKER which can automaticallygenerate a set of linker sequences that are known to adopt extendedconformations as determined by X-ray crystallography and NMR.The only required input to the program is the desired linkersequence length. The program is specifically designed to assistin fusion protein construction. A number of optional input parametershave been incorporated so that users are able to enhance sequenceselection based on specific applications. The program outputsimply contains a set of sequences with a specified length.This program should be a useful tool in both the biotechnologyindustry and biomedical research. It can be accessed throughthe Web page http://www.fccc.edu/research/labs/feng/linker.html.     

Functional properties of proteins in foods

The measurement of functionality of protein food ingredients has developed somewhat haphazardly, probably due to the wide range of proteins used as ingredients and the diversity of foods. Studies of the physiochemical properties of proteins should enable prediction of a proteins response to process environments and prove more fruitful than many of the empirical measurements of functionality. The effects of pH, salt type and concentration on the phase behaviour of the oilseed globulin and arachin, demonstrates the complexity of protein solubility and the inadequacies of simple tests that have arisen. Studies of the effects of salts and conditioning on meat fibres, coupled with measurement of the location of water in pellets from water holding tests enable the latter to be applied with increased confidence. Comparison of the endothermic transitions observed on heating with the development of storage and loss moduli allow the contributions of domains of skeletal muscle myosin to gel structure to be investigated.     

壳聚糖复合纳米基因载体的制备

目的制备壳聚糖复合纳米基因载体,并探讨其理化性质、细胞毒性、稳定性及体外转染效率。方法采用复凝聚法制备包封聚乙烯亚胺(Polyethylenimine,PEI)/DNA复合物的壳聚糖复合纳米基因载体,用纳米粒度分析仪测定其粒径和Zeta电位;透射电镜观察其形态;MTT法检测其细胞毒性;在PBS溶液(pH 7.4)及含10%小牛血清的RPMI1640培养基中,于37℃条件下放置0、1、3、5 d,1%琼脂糖凝胶电泳检测其稳定性;体外转染CNE细胞,评价其转染活性。结果当N(PEI的氨基)/P(DNA的磷酸根)≥6时,能够形成稳定的壳聚糖复合纳米粒,平均粒径约为300 nm,表面电荷约为30 mV;壳聚糖复合纳米基因载体呈球形,圆整且分散性好;复合纳米基因载体的细胞毒性较低;1%琼脂糖凝胶电泳分析显示,DNA被完全包裹在复合纳米载体中,且5 d内无游离DNA释放;体外转染活性与壳聚糖/DNA复合物相比,提高了约1 000倍,且转染能力不受血清的干扰。结论制备的壳聚糖复合纳米基因载体是一种高效、低毒的非病毒载体,具有作为体内基因治疗载体的应用潜力。     

IgG-Fc融合蛋白的研究进展

IgG-Fc融合蛋白是将生物活性蛋白与IgG的铰链区和Fc片段进行基因重组而表达的融合蛋白。IgG-Fc融合蛋白不仅可发挥所融合蛋白的生物学活性,还赋予其类似抗体的特性,包括延长血浆半衰期以及Fc片段特有的一系列效应功能,并已广泛应用于临床治疗、生物医学研究等领域。合理优化IgG-Fc融合蛋白是未来发展趋势,选择合适的IgG亚类作为融合载体,采用Fc片段单个或多个氨基酸序列修饰、糖基化改造等,提高融合蛋白的血浆半衰期、优化融合蛋白的效应功能,最终达到最佳免疫治疗效果。本文对IgG-Fc融合蛋白的功能、相应优化策略作一综述,并介绍了其临床研究现状。     

The construction and cloning of synthetic genes coding for artificial proteins and expression studies to obtain fusion proteins

Synthetic genes coding for artificial proteins with predeflnedand nutritionally valuable amino acid compositions have beenconstructed and cloned In bacterial plasmid vector pKK233-2.The genes were constructed from three easily interchangeable‘cassettes’ encoding either essential, non-essentialor branched-chain amino acid residues. A potential hairpin loopstructure in the mRNA around the region of the ribosome bindingsite was probably the reason for blockage of translation fromthis vector. Two selected genes, AHB (containing one copy ofeach cassette) and A (consisting of six copies concatemerizedA₆cassette) were cloned into pUR300, a (ß-Gal fusionvector and expressed as fusion proteins (ß-Gal-AHBand (ß-Gal-A₆.     

Functional and nutritional properties of acylated rapeseed proteins

Rapeseed flour was acylated to various degrees with acetic and succinic anhydrides to produce acetylated rapeseed protein concentrates (SRPC) and succinylated rapeseed protein concentrates (SRPC), respectively. With both acylating agents, approximately 5–82% (ARPC-5 to ARPC-82) and 5–56% (SRPC-5 to SRPC-56) of ε-amino groups of lysine were acylated. Changes in functional properties were monitored; nitrogen solubility, emulsifying properties and specific viscosity were improved by acylation. The effects on functional properties were more pronounced with succinylation than acetylation. The nutritive value of succinylated rapeseed protein concentrates was determined and compared with those of unmodified rapessed protein concentrate and rapessed flour. Succinylation reduced the availability of lysine and decreased (p<0.05) the net protein ratio (NPR) and apparent digestibility coefficient (ADC) of nitrogen in rapessed protein concentrates. Supplementation of SRPC with lysine improved the NPR compared to that of SRPC-56.     

Functional properties of chemically modified egg white proteins

Functional properties of egg white proteins can be altered through selected chemical reactions. Acylations with acid anhydrides have received the greatest amount of attention. Oleic acid and sodium dodecyl sulfate (SDS) have also been used to affect function of egg white proteins. The charge characteristics of acylated proteins are altered through modification of the N-terminal and epsilon-amino groups. The acid anhydride used and the extent of modification have a major effect on the ionic properties of the protein. The altered ionic properties have been shown to affect the optical properties of protein sols, heat stability, foaming, performance in angel cakes, initiation of gelation, ultimate strength and freeze-thaw stability of heat-set gels. Although exact explanations of the mechanisms for the interactions of oleic acid and SDS with egg white protein are not available, increases in charge occur and result in gels with physical properties very similar to gels made from succinylated egg protein.     

hepaCAM基因重组腺病毒质粒的构建及鉴定

目的构建表达肝细胞黏附分子(Hepatocyte cell adhesion molecule,hepaCAM)基因的重组腺病毒质粒,并进行鉴定。方法以质粒pEGFP-N2-hepaCAM为模板,PCR扩增hepaCAM基因,克隆至腺病毒穿梭质粒pAdtrack-CMV上,经酶切、PCR及测序鉴定正确后,将重组腺病毒穿梭质粒pAdtrack-CMV-hepaCAM经PmeⅠ线性化,转化至含腺病毒骨架质粒pAdEasy-1的感受态大肠杆菌BJ5183中进行同源重组。重组腺病毒质粒pAdEasy-1-hepaCAM经PmeⅠ线性化后,转染HEK-293细胞,包装重组腺病毒AdI-hepaCAM,经大量扩增后,检测病毒滴度。RT-qPCR及Westernblot检测感染的BIU-87细胞内hepaCAM的表达。结果酶切鉴定证实重组腺病毒质粒pAdEasy-1-hepaCAM构建正确,重组腺病毒AdI-hepaCAM滴度可达1.6×1012pfu/ml,与空载体腺病毒组比较,经重组腺病毒感染的BIU-87细胞中hepaCAM mRNA及蛋白的表达均明显升高(P<0.05)。结论已成功构建携带hepaCAM的重组腺病毒载体AdI-hepaCAM,为研究hepaCAM基因对膀胱癌细胞增殖的调控提供依据。     

瓣状内切核酸酶1基因重组真核表达质粒的构建及鉴定

目的构建瓣状内切核酸酶1(Flap endonuclease 1,FEN1)基因重组真核表达质粒,并进行鉴定。方法提取L02细胞总RNA,逆转录合成cDNA,经巢式PCR扩增FEN1基因,定向克隆至载体pcDNA3.1,构建重组真核表达质粒,经酶切及测序进行鉴定。将鉴定正确的真核表达质粒转染293T细胞,Western blot检测FEN1蛋白的表达。结果 FEN1基因重组真核表达质粒经酶切及测序鉴定证明构建正确;在相对分子质量约47 000处可见目的蛋白条带,转染细胞中FEN1蛋白表达量较空载体转染组及空白细胞对照组提高约3倍。结论已成功构建了FEN1基因重组真核表达质粒,并可在293T细胞中过表达FEN1。     

脑心肌炎病毒VP2基因真核表达质粒的构建

目的构建脑心肌炎病毒(encephalomyocarditis virus,EMCV)VP2基因真核表达质粒,并于CHO细胞中表达。方法 RT-PCR法扩增EMCV VP2目的基因,克隆至载体pcDNA3. 1中,构建重组质粒pcDNA3. 1-VP2,脂质体法转染CHO细胞。转染48 h后,RT-PCR法检测EMCV VP2基因mRNA的转录情况,免疫组化法及Western blot法检测EMCV VP2蛋白的表达情况。结果经双酶切及测序鉴定,重组质粒pcDNA3. 1-VP2构建正确。EMCV VP2基因m RNA及蛋白可在CHO细胞中正常转录及表达,且EMCV VP2蛋白可与兔抗EMCV阳性血清发生特异性结合,主要分布于CHO细胞膜上。结论成功构建了重组质粒pcDNA3. 1-VP2,并有效地在CHO细胞中进行了表达,为EMCV VP2基因体外表达及其功能的进一步研究奠定了基础。