家蚕抗菌肽-死亡素杂合肽基因在大肠杆菌中的克隆与表达

近年来的研究发现 ,抗菌蛋白在生物体非专一性防御系统有着重要的作用 ,已有数十种具有抗菌活性的多肽被分离 ,这些多肽可大致分为 3类 ,即含分子内二硫桥的抗菌肽 ;具有双亲α 螺旋结构的抗菌肽 ;以及富含某种氨基酸残基的抗菌肽[1 ] ,一般来说 ,这些抗菌肽具有分子量小 ,稳定性好 ,无细胞毒性 ,抗菌谱广等特点。多种抗菌肽的一级结构和二级结构已经确定[2 ] ,但作用机理仍不明了。一般认为可能存在两种作用模式 ,即 1)通过肽 脂膜相关作用杀菌 ;2 )通过受体介导的识别过程起作用[1 ] 。ＣｅｃｒｏｐｉｎＢ是一种较早从家蚕中分离得到 ,由 …     

抗菌肽Cec4的结构改造及抗菌活性研究

[目的]对抗菌肽Cec4进行结构改造,并评估优化小肽的稳定性及溶血性。[方法]采用序列截短、氨基酸替换的方式来改造Cec4。采用微量稀释法,检测改造后的Cec4对各类鲍曼不动杆菌、肺炎克雷伯菌的最小抑菌浓度(MIC)。绘制时间-杀菌曲线,并评估不同因素对抗菌肽抑菌活性的影响,最后通过溶血实验检测其安全性。[结果]①截短后的Cec4失去抑菌效果。②Cec4氨基酸替换后的Cec4-1、Cec4-2未能提升抑菌能力。Cec4-4的MIC值为Cec4的50%。③在24h,时间-杀菌曲线显示,Cec4-4的抑菌活性较Cec4高30%。④血清会影响抗菌肽活性,并且高浓度胰蛋白酶(1. 0 mg/m L)会抑制Cec4-4抗菌活性。⑤溶血实验表明Cec4-4在1 mg/m L浓度下无溶血反应。[结论]截短使Cec4失去抑菌活性,氨基酸替换后的Cec4-4对各测试菌的抑菌活性较母肽提升了1倍,且抗菌肽在1mg/m L浓度下对人体无溶血反应。     

昆虫抗菌肽基因工程研究进展

昆虫抗菌肽对细菌、真菌、病毒和原虫都具有杀灭作用 ,甚至对肿瘤细胞也具有杀伤作用。昆虫抗菌肽并且有独特的作用机制 ,成为众多表达系统外源导入基因的侯选对象之一 ,综述了昆虫抗菌肽的种类及其在微生物转基因工程和植物转基因工程中的进展。     

昆虫抗菌肽的功能、作用机理与分子生物学研究最新进展

昆虫虽然没有完善的免疫防御体系,但却具有高效的无细胞免疫系统。抗菌肽是昆虫免疫后血淋巴中的一类抗菌多肽,它具有分子量小,热稳定,水溶性好,无免疫原性,抗菌谱广等特点。现在,它被认为是从细菌到高等哺乳动物普遍存在的一类防御性多肽,称之为“第二防御体系”。抗菌肽不仅抗菌谱广,而且可以抑杀某些真菌、病毒及原虫,并对多种癌细胞及动物实体瘤有明显的杀伤作用,而不破坏正常细胞。近年来,对昆虫抗菌物质的研究,特别是对昆虫抗菌肽的研究已成为一个迅速发展的新领域,越来越引起人们的关注和重视。抗菌肽可望成为新一代的抗菌、抗病毒、抗癌药物。但天然抗菌肽的来源少,成本高,无法满足临床试用和基础研究的需要。因此通过 Ｄ Ｎ Ａ 重组技术来获得大量抗菌肽,成为人们普遍关注的焦点。同时,对抗菌肽抗菌、抗肿瘤机理的深入研究也越来越具有重大的理论意义和实际应用价值,前景十分广阔     

昆虫抗菌肽对肿瘤细胞的抑制作用研究进展

昆虫抗菌肽(antimicrobial peptide,AMPs)是一类昆虫先天免疫系统中十分重要的效应因子,它分子量小、热稳定,并且具有广谱抗菌性,能够抑制、杀死多种细菌、真菌.近几年来,由于昆虫抗菌肽具有抗肿瘤的活性且其不具有抗原性而受到研究者的广泛关注.昆虫种类多、分布广,因此昆虫抗菌肽具有很高的开发潜力和实际应用价值.但是,目前对于昆虫抗菌肽抗肿瘤能力的研究尚不够深入,对其作用机制还没有一套准确并且系统的理论.现在普遍认为昆虫抗菌肽的抗肿瘤机制与其抗菌机制类似,可以分为破坏细胞膜机制以及非破坏细胞膜机制,并且同一种昆虫抗菌肽可以通过多种方式来抑制甚至杀死肿瘤细胞,但是对正常真核细胞无明显的毒副作用.相较于传统化疗药物的无差别杀伤,昆虫抗菌肽在肿瘤治疗领域有着巨大潜力.本文简要综述了昆虫抗菌肽对肿瘤细胞的抑制作用及其作用机制,并对其开发潜力和实际应用价值进行了展望,以期为今后的研究提供理论支持.     

昆虫抗菌肽对肿瘤细胞的抑制作用研究进展

昆虫抗菌肽(antimicrobial peptide,AMPs)是一类昆虫先天免疫系统中十分重要的效应因子,它分子量小、热稳定,并且具有广谱抗菌性,能够抑制、杀死多种细菌、真菌.近几年来,由于昆虫抗菌肽具有抗肿瘤的活性且其不具有抗原性而受到研究者的广泛关注.昆虫种类多、分布广,因此昆虫抗菌肽具有很高的开发潜力和实际应用价值.但是,目前对于昆虫抗菌肽抗肿瘤能力的研究尚不够深入,对其作用机制还没有一套准确并且系统的理论.现在普遍认为昆虫抗菌肽的抗肿瘤机制与其抗菌机制类似,可以分为破坏细胞膜机制以及非破坏细胞膜机制,并且同一种昆虫抗菌肽可以通过多种方式来抑制甚至杀死肿瘤细胞,但是对正常真核细胞无明显的毒副作用.相较于传统化疗药物的无差别杀伤,昆虫抗菌肽在肿瘤治疗领域有着巨大潜力.本文简要综述了昆虫抗菌肽对肿瘤细胞的抑制作用及其作用机制,并对其开发潜力和实际应用价值进行了展望,以期为今后的研究提供理论支持.     

昆虫抗菌肽的研究进展

昆虫抗菌肽是昆虫免疫后存在于血淋巴中的一类肽类活性物质.具有相对分子质量小、热稳定性强、无免疫原性、抗菌谱广等特点.结合昆虫抗菌肽的研究现状和发展前景,对昆虫抗菌肽的分类、作用机理、基因工程等方面进行了综述.     

昆虫抗菌肽的结构和功能研究现状

昆虫抗菌肽是昆虫血淋巴中的一类活性肽。在昆虫感染病菌或体壁受到损伤的情况下其脂肪体能迅速合成一系列抗菌活性物质并释放到血淋巴中发挥作用,抑杀多种致病菌、真菌、病毒等,但不破坏生物体的正常细胞。对昆虫抗菌肽的结构特征、生物活性及抗菌肽分子的作用模式的最新研究进展作了综述。     

哺乳动物抗菌肽研究进展

自昆虫中发现抗菌肽以来,迄今已有约2000多种抗菌肽在生物体中被分离,仅在哺乳动物中就发现了上百种抗菌肽,哺乳动物抗菌肽是哺乳动物抵抗外界病原物质侵染而产生的一系列非专一性免疫应答物,它具有分子量低,热稳定,广播谱抗菌等特点,本文就近年来发现的哺乳动物的抗菌从分类,结构、生物学特性,表达控制,作用机制及应用前景方面作一介绍。     

昆虫抗菌肽的生物学特点及药物研发进展

昆虫抗菌肽是由昆虫细胞特定基因编码、由细胞核糖体合成的,具有体液免疫功能的一类碱性多肽,对细菌、真菌、病毒和原虫,甚至癌细胞都具有杀伤作用,有望开发成为新一代的抗菌药物。随着抗菌肽家族的不断扩大,其各方面的研究也日益深入。简要综述了昆虫抗菌肽的种类及结构特点、作用机制、生物活性、构效关系、药物开发情况。     

Defective Escherichia coli signal peptides function in yeast

To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.     

A lipopeptide-encoding sequence upstream from the IysA gene of Pseudomonas aeruginosa

An open reading frame (ORF) of 141 bp was observed upstream from the Pseudomonas aeruginosa lysA gene. The translation product of this ORF contains a signal peptide with a lipoprotein box, Ile-Ala-Ala-Cys, at the predicted signal peptidase cleavage site. The Escherichia coli phoA gene without its signal sequence was fused in frame to this ORF in a broad host-range plasmid. The resulting construct expressed a hybrid protein exhibiting alkaline phosphatase activity in phoA mutants of both E. coli and P. aeruginosa. This indicates that the ORF encodes a peptide, part of which acts as an export signal. The hybrid peptide was identified by immunoblotting with alkaline phosphatase antiserum. The accumulation of a precursor form was observed when P. aeruginosa cells carrying this gene fusion on a plasmid were treated with globomycin. Moreover, the mature form could be labelled with 2-[3H]-glycerol, indicating that lipidic residues may be linked to the hybrid protein. Taken together, these results strongly suggest that the ORF encodes a lipopeptide. We propose that the gene is called IppL.     

The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli

The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.     

A colicin A fragment containing the receptor binding domain can be directed to the periplasmic space in E. coli through gene fusion

The central region of the colicin A polypeptide chain has been fused to the N-terminal part of beta-lactamase through genetic recombination. This region comprising amino acid residues 70-335 confers on the hybrid protein the ability to protect sensitive cells from the lethal action of colicin A. Although colicin A belongs to the cytoplasmic compartment of E. coli, export of the hybrid protein to the periplasmic space was promoted by the signal peptide of beta-lactamase.     

牛乳铁蛋白素(1-15)-蜂毒素(5-12)杂合肽基因的合成及在大肠杆菌中的表达

为了构建新型的杂合肽,设计了一种新型杂合抗菌肽牛乳铁蛋白素(1-15)-蜂毒素(5-12),由牛乳铁蛋白素(LfcinB)N端第1~15个氨基酸残基和蜂毒素(Melittin)N端第5~12个氨基酸残基组成。根据大肠杆菌密码子的偏爱性,设计合成了杂合肽LfcinB(1-15)-Melittin(5-12)的基因片段,插入到表达载体pET-32a的NcoI和SalI的酶切位点之间,构建重组表达质粒。重组表达质粒转化到Escherichia coli BL21(DE3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,融合蛋白以可溶形式在Escherichia coli BL21(DE3)中获得成功表达,表达量占菌体总蛋白的35%以上,每升培养物可获得35mg融合蛋白。带His标签的融合蛋白经His-Bind纯化试剂盒纯化、肠激酶切割和抑菌实验表明,杂合肽具有明显的抑菌效果。这为利用基因工程方法生产抗菌肽奠定了理论基础。     

Illicit secretion of a cytoplasmic protein into the periplasm of Escherichia coli requires a signal peptide plus a portion of the cognate secreted protein. Demarcation of the critical region of the mature protein

The beta-lactamase signal peptide alone is not sufficient to direct secretion of chicken muscle triosephosphate isomerase, a normally cytoplasmic protein, into the periplasm of Escherichia coli. The signal peptide and at least the first 3 residues of the mature beta-lactamase are required before any secretion of the isomerase can be observed. At this point the level of secretion is very low, but the addition of further residues of the mature beta-lactamase enhances the secretion of the hybrid protein. The maximum level of secretion is achieved when 12 or more residues of the mature beta-lactamase intervene between the signal peptide and the isomerase. It is the proximity of an arginine residue at position 3 of the isomerase that is responsible for the blockade to secretion of these hybrid proteins (see Summers, R.G., Harris, C.R., and Knowles, J.R. (1989) J. Biol. Chem. 264, 20082-20088). With 12 residues of the mature beta-lactamase between the signal peptide and the isomerase, the offending arginine now lies at position 15 of the hybrid. The 14 residues that immediately follow the signal peptide therefore define a region of constrained properties that is critical to the secretability of proteins from E. coli.     

Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence

A DNA fragment coding for the E. coli phoA signal peptide was synthesized and inserted into the expression vector pKK223-3. A single HindIII restriction site is located just at the end of the signal sequence. A gene coding for the proteinase inhibitor hirudin, which has previously been synthesized, was inserted into this HindIII site. The hybrid protein was expressed under control of the tac-promoter and secreted into the periplasm of E. coli. From the periplasmic fraction two processed proteins were isolated. One of these was identical with desulfatohirudin and also had similar biological properties.     

东亚钳蝎K+通道阻断肽cDNA的克隆与表达

目的：克隆东亚钳蝎毒素基因,以进一步研究其生物学和药理学功能。方法：利用已知蝎神经毒素基因序列,设计引物,用RT-PCR方法克隆从蝎毒腺组织蝎毒素cDNA。结果：成功地克隆了一个新的东亚钳蝎毒素基因,该基因开放阅读框架编码59个氨基酸残基,其中前22个为信号肽,成熟肽为37个氨基酸残基,经PCR扩增除去信号肽序列,克隆到pTreHisA质粒中,在E．coli中表达了分子质量为7ku左右融合蛋白,表达产物占菌体总蛋白的21％左右。结论：其结构中含有三对二硫链,6个Cys残基组成蝎K^ 通道毒素共同特征序列-CXXXC-、-GXC-、-CXC-,推断其为K^ 通道阻断肽,命名为KChTX1。已被Gene-bank收录,收录号为AY129234。     

The molecular cloning of the gene encoding the Escherichia coli 75-kDa helicase and the determination of its nucleotide sequence and gentic map position

A previously unreported DNA unwinding enzyme, referred to as the 75-kDa helicase, was recently purified from Escherichia coli cell extracts and biochemically characterized (Wood, E. R., and Matson, S. W. (1987) J. Biol. Chem. 262, 15269-15276). In order to initiate the genetic analysis of the 75-kDa helicase, the gene encoding this enzyme was cloned. DNA sequencing confirmed the identity of the gene since the predicted amino acid sequence of the encoded polypeptide precisely matched the sequence of the first 27 NH2-terminal amino acid residues of the 75-kDa helicase as determined by peptide sequencing. The predicted amino acid sequence of the 75-kDa helicase is similar in several regions to the amino acid sequences of two other E. coli helicases, Rep protein and helicase II. The gene encoding the 75-kDa helicase was mapped to 22 min on the E. coli chromosome. We propose that this newly defined locus be referred to as helD, and, to avoid confusion with other E. coli helicases with a similar molecular size, we propose that the 75-kDa helicase be referred to as helicase IV.