天蚕抗菌肽Ａ与蜂毒素杂合肽基因的合成及在大肠杆菌中的克隆与表达

根据天蚕抗菌肽Ａ（cecropin A,CA）Ｎ端第１～７个 氨基酸残基、 蜂毒素（melittin,M）Ｎ端第５～１２个氨基酸残基,以大肠杆菌偏爱的密码子设计合成了杂合肽ＣＡ（１～７）-Ｍ（５～1２）基因,同载体pＧＥＭＥＸ-１连接后转化大肠杆菌ＪＭ１０９,经打点杂交筛选和序列测定,获得一个与设计一致的克隆。将此克隆中的质粒亚克隆至JM103(DE3) ,经IPTG诱导、BrCN切割和抑菌圈试验表明,克隆的杂合肽基因在JM109(DE3)中获得了表达。 Abstract:According to the studies of David Andreu and other scientists,we designed and synthesized a gene encoding shortened cecropin A-melittin hybrid.This hybrid consists of CA(1-7) and M(5-12).The gene was inserted into pGEMEX-1 in the site of EcoRI and BamHI.A positive clone was obtained by hybridization and DNA sequencing and was expressed in JM109(DE3).     

杂合抗菌肽CecA-Mag的人工合成及其在Pichia pastoris中的分泌表达

根据抗菌肽天蚕素A(cecropinA,CA)N端第1~7个氨基酸残基,马盖宁(magainin,M)N端第2~12个氨基酸残基,以毕赤酵母偏爱的密码子设计合成了杂合肽CA(1~7)-M(2~12)基因,同载体pPICZα-A连接后转化Pichia pastoris受体菌SMD1168,在醇氧化酶(AOX)启动子调控下,分子量约1.9kDa的CecA-Mag杂合抗菌肽获得表达,抗菌特性研究表明,该表达产物具有广谱抗菌活性,对多数G-菌及G 菌均有较好的抑菌活性。初步抑菌活性测定,显示该杂合肽对金黄色葡萄球菌、耐氨苄青霉素的大肠杆菌及枯草芽孢杆菌有良好的抑杀活性。酸稳定实验显示pH为3.2时仍具有相当高的活性。热稳定性实验显示该杂合肽100℃加热5min后仍具有抑菌活性。这些特点使得重组抗菌肽CecA-mag在疾病防治和动物饲料添加剂等方面显露出很好的应用前景。     

家蚕天蚕素cDNA原核表达及抗菌活性检测

采用RT-PCR方法从家蚕Bombyx mori新疆品种新蚕三号组织中扩增天蚕素cDNA片段,回收并克隆至Pmd18-T载体,进行序列分析。基因序列分析结果与已发表的天蚕素B的序列同源性为98%,表明所克隆的新疆家蚕天蚕素cDNA为独特的cDNA片段。将天蚕素基因与Pgex-4T-1融合表达载体中的谷胱甘肽转移酶基因融合,在大肠杆菌中表达, 结果表明经IPTG 诱导30 min后,pGEX-4T-1/天蚕素转化后的大肠杆菌生长明显受到抑制;当诱导210 min 后,大肠杆菌数量又开始增加,逐渐恢复至正常水平。说明天蚕素与谷胱甘肽转移酶基因融合表达后,在IPTG存在的短时间内仍然对原核细胞有较强的抗菌抑杀作用。     

杂合抗菌肽牛乳铁蛋白素-天蚕素在大肠杆菌中的高效表达及其活性鉴定

【目的】菌株耐药性问题日益突出,研制新型安全高效的抗菌药物成为目前的研究热点之一。抗菌肽具有多种优良特性,高活性抗菌肽的开发及其重组表达对解决菌株耐药性问题具有重要意义。【方法】根据牛乳铁蛋白素与天蚕素的结构,设计一种新型的杂合抗菌肽牛乳铁蛋白素-天蚕素(LfcinB-Cecropin),根据Escherichia coli密码子偏爱性合成其编码基因,利用同尾酶法构建含有不同LfcinB-Cecropin基因片段拷贝数的重组表达载体,转化到E.coli BL21(DE3)进行重组表达。【结果】经IPTG诱导,LfcinB-Cecropin融合蛋白成功获得表达。经超声破碎、包涵体纯化、甲酸裂解后,获得具有明显抑菌活性的杂合肽LfcinB-Cecropin。【结论】获得一种高活性的新型抗菌肽LfcinB-Cecropin,并实现了在E.coli中的高效重组表达。     

融合基因Mdc-hly的构建及原核表达

构建家蝇天蚕素-人溶菌酶(Mdc-hly)融合基因,实现Mdc-hly基因在大肠杆菌中的表达。通过RT-PCR分别扩增出家蝇天蚕素和人溶菌酶的成熟肽基因序列,再利用Gene-SOEing技术构建融合基因,将融合基因克隆至pET32a表达载体,转化E.coli BL21(DE3),经IPTG诱导得到高效表达,融合蛋白分子量约为38kD。Western blotting杂交证实了表达蛋白的抗原活性。成功构建了融合其因并进行了原核表达,为进一步的生物活性研究打下基础。     

鸽β-防御素5基因克隆及其抗菌活性

从鸽的骨髓和肝组织中扩增到2个禽β-防御素(AvBD)基因,在大肠杆菌中高效表达,检测其重组蛋白的抗菌活性。应用RT-PCR方法从鸽的骨髓和肝脏组织中扩增AvBD5基因,根据已发现的禽β-防御素和部分哺乳类动物β-防御素-5的氨基酸序列构建系统进化树,将该基因克隆到大肠杆菌原核表达载体pGEX-6p-1上进行原核表达,对该重组蛋白进行纯化,测定体外抗菌活性与理化特性。测序得到这2个基因的cDNA大小均为201 bp,编码66个氨基酸残基,内含6个位置保守的半胱氨酸残基。经遗传进化分析发现,该基因推导的两组氨基酸序列与鸭AvBD5的同源性最高,分别为87.9%和78.8%,这两组氨基酸序列的同源性为83.3%。因此,将其分别命名为鸽AvBD5α(骨髓)和鸽AvBD5β(肝脏)。进一步将这两个基因分别亚克隆到大肠杆菌pGEX-6p-1载体中进行原核表达。两个重组融合蛋白经纯化后,通过菌落计数法测定其体外抗菌活性,盐离子浓度对其抗菌活性的影响,及其融合蛋白的溶血活性。结果表明,重组鸽AvBD 5α、AvBD 5β融合蛋白的分子量约为32 kDa。具有明显的抗菌活性,对12种细菌均有不同程度的抑制作用,高盐浓度对其抗菌活性显著影响。此外,这两个重组蛋白对红细胞无显著溶血活性。     

虾夷扇贝过敏原tropomyosin的克隆表达、纯化及免疫学鉴定

从虾夷扇贝(Patinopecten yessoensis)肌肉中提取总RNA,RT-PCR克隆虾夷扇贝中变应原原肌球蛋白的全长基因,根据序列设计带有酶切位点的特异性引物,扩增扇贝tropomyosin的完整开放阅读框,与pET-28a载体连接并转化大肠杆菌Escherichia.coli BL21(DE3),诱导表达后,Ni2+亲和层析柱纯化重组蛋白,Western-blot检测其免疫学活性。经序列测定,该基因含有长度为855bp的开放阅读框,编码284个氨基酸,其在GenBank数据库中的登录号为EU839640。SDS-PAGE检测该重组变应原在大肠杆菌中高效表达36kD的目的蛋白,且重组变应原具有良好的IgE结合活性。研究获得了具有变应原活性的重组虾夷扇贝tropomyosin,为扇贝过敏性疾病的诊断和治疗奠定了基础。       

鹅β-防御素3基因的分离、鉴定及其表达产物的生物学特性

为了克隆鹅β-防御素(AvBD)3基因,并在原核表达重组鹅AvBD3蛋白,进一步研究鹅AvBD3蛋白的生物学特性,利用RT-PCR方法从鹅脾脏和法氏囊组织中扩增到鹅AvBD3基因片段,其cDNA片段大小为182 bp,编码60个氨基酸残基.经同源性分析发现鹅AvBD3氨基酸序列与鸡AvBD3氨基酸序列同源性最高,为100％.将该基因亚克隆到原核表达载体pGEX-6p-1的BamH Ⅰ和SalⅠ双酶切位点上,构建重组表达质粒pGEX-goose AvBD3.将重组质粒转化大肠杆菌BL21,于37℃用IPTG诱导表达,SDS-PAGE电泳表明,重组鹅AvBD3蛋白在原核高效表达(分子量约31 kDa).该重组蛋白经纯化后测定其体外抗菌活性与理化特性,结果显示,重组鹅AvBD3蛋白具有广谱的抗菌活性,对12种细菌,包括革兰氏阳性菌和革兰氏阴性菌均具有抑菌作用.高盐离子浓度显著降低重组鹅AvBD3蛋白的抗菌活性.此外,该重组蛋白的溶血活性极低,并对酸碱度具有较高的稳定性.     

大肠杆菌可溶性表达人碱性成纤维细胞生长因子的研究

包涵体的形成与外源基因在大肠杆菌中的表达量高度相关,在适当的范围内,降低hbFGF在表达宿主BL21(DE3)plysS中的表达,成为实现高可溶性表达的关键。在不改变氨基酸序列的条件下,对hbFGF高表达菌株突变重组子起始密码ATG下游前3个密码子的摇摆碱基进行随机回复突变,共有7种组合,合成引物PCR扩增后,克隆至表达载体pET3c, 将重组子转导BL21(DE3)plysS后,IPTG诱导表达,发现其中1株有较高可溶性和活性的菌株。可见部分降低外源蛋白的表达量可以避免与减少包涵体的形成。     

抗菌肽ABP3基因的克隆及其在Pichia pastoris中的表达

用化学合成法合成以植物偏爱密码子编码的新抗菌肽ＡＢＰ３基因片段,合成片段拼接后,与ｐＵＣ１９重组,经限制酶片段分析与核苷酸序列分析,获得抗菌肽ＡＢＰ３基因。ＡＢＰ３基因与表达载体ｐＢＩＣ９重组,构建受乙醇氧化酶１基因（ＡＯＸ１）的启动子与转录终止区控制的酵母表达质粒,转化ＧＳ１１５宿主菌,经表型筛选,阳性克隆用甲醇诱导表达,重组ＡＢＰ３以分泌型表达,具抗菌活性,且符合ＡＢＰ３的抗菌特性。     

A necessary and sufficient determinant for protein-selective glycosylation in vivo

A limited number of glycoproteins including luteinizing hormone and carbonic anhydrase-VI (CA6) bear N-linked oligosaccharides that are modified with beta1,4-linked N-acetylgalactosamine (GalNAc). The selective addition of GalNAc to these glycoproteins requires that the beta1,4-N-acetylgalactosaminyltransferase (betaGT) recognize both the oligosaccharide acceptor and a peptide recognition determinant on the substrate glycoprotein. We report here that two recently cloned betaGTs, betaGT3 and betaGT4, that are able to transfer GalNAc to GlcNAc in beta1,4-linkage display the necessary glycoprotein specificity in vivo. Both betaGTs transfer GalNAc to N-linked oligosaccharides on the luteinizing hormone alpha subunit and CA6 but not to those on transferrin (Trf). A single peptide recognition determinant encoded in the carboxyl-terminal 19-amino acid sequence of bovine CA6 mediates transfer of GalNAc to each of its two N-linked oligosaccharides. The addition of this 19-amino acid sequence to the carboxyl terminus of Trf confers full acceptor activity onto Trf for both betaGT3 and betaGT4 in vivo. The complete 19-amino acid sequence is required for optimal GalNAc addition in vivo, indicating that the peptide sequence is both necessary and sufficient for recognition by betaGT3 and betaGT4.     

The amino acid sequence of a flavodoxin from the eukaryotic red alga Chondrus crispus.

The amino acid sequence of the constitutive flavodoxin from the red alga Chondrus crispus was determined from the analyses of peptide fragments derived by enzymic digestions of the carboxymethylated protein. This is the first sequence reported for a flavodoxin from a eukaryote. The protein is composed of 173 amino acid residues and is a member of the longer-chain group of flavodoxins. The extent of sequence homology to the three other flavodoxins in the group for which sequences are available is in the range 36-39%, with the most strongly conserved regions being those implicated in binding of the FMN, the redox-active prosthetic group. Nevertheless, Chondrus crispus flavodoxin stands apart in a number of respects, in particular the possession of an unusually high content of proline, with these residues distributed more or less regularly along the peptide chain.     

Evidence for production of a new lantibiotic (butyrivibriocin OR79A) by the ruminal anaerobe Butyrivibrio fibrisolvens OR79: characterization of the structural gene encoding butyrivibriocin OR79A

The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of bvi79A was a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5' region of bvi79A or with degenerate oligonucleotides based on the predicted sequence of the second peptide.     

Determination of the amino acid residues required for the activity of the anti-rhizobial peptide antibiotic trifolitoxin

AIMS: The first aim was to determine those amino acid residues required for the biological activity of the potent peptide antibiotic, trifolitoxin (TFX). The second aim was to determine the concentrations of TFX1 and TFX2 that cause 50% inhibition of bacterial growth (Ki), the two predominant isomeric forms of TFX made by Rhizobium. METHODS AND RESULTS: Site-directed mutagenesis of tfxA was used to produce strains that made mutant TFX peptides. The mutant tfxA genes were placed on a vector and inserted in Rhizobium leguminosarum b. trifolii Tn54A112, a tfxA mutant of strain T24 that lacks trifolitoxin activity. Our standard bioassay was used to assess the activity of these mutants. TFX1 and TFX2 were purified by reverse phase chromatography. Several concentrations of each peptide were assayed for biological activity to determine Ki. The unmodified TFX peptide (DIGGSRQGCVA) was synthesized and was found to lack any biological activity. Four of the 11 amino acid residues in ribosomally synthesized, post-translationally modified peptide were required for TFX activity. These required amino acids include arginine (R37), glutamine (Q38), glycine (G39) and cysteine (C40). S36T and S36Y mutants showed reduced TFX activity. The numbering system is based on the 42-amino acid TfxA peptide that is post-translationally modified to form the active TFX peptide. The Ki of TFX2 was determined to be 10-fold lower than TFX1. CONCLUSIONS: The post-translational modifications of the TfxA peptide are required for biological activity. TFX2 is far more active than TFX1. SIGNIFICANCE AND IMPACT OF THE STUDY: The sequence of the TfxA peptide appears to have been optimized for maximum activity through the course of evolution. Even conservative changes to any of the amino acid residues required for activity results in a complete loss of activity. The understanding of the action of this peptide is critical for its proposed action as a control agent for crown gall disease.     

In Vitro Selection of a Peptide Inhibitor of Human IL-6 Using mRNA Display

Interleukin-6 (IL-6) plays a crucial role in malignant diseases, such as rheumatoid arthritis, Castleman disease, and multiple myeloma, and as such, is an attractive therapeutic target. Here, the authors isolated a novel IL-6 inhibitor peptide by in vitro selection using mRNA display. The authors first used a random-primed human cDNA library to isolate IL-6-binding peptides. After four rounds of selection, a 19-amino acid peptide named CA11 was selected and confirmed to specifically interact with IL-6. The authors then performed an alanine scan analysis of CA11 and determined the amino acid residues necessary to interact with IL-6. Next, the authors constructed a CA11-based partially randomized library and after ten more rounds of selection, isolated several groups of peptides. The most frequently occurring sequence, RA07, bound to IL-6 with 3 to 4-fold higher affinity than CA11. Furthermore, RA07 inhibited IL-6-dependent KT-3 cell proliferation in a dose-dependent manner. ELISAs revealed that RA07 could not inhibit IL-6 from binding to the IL-6 receptor (IL-6R), but could inhibit the IL-6/IL-6 complex binding to gp130.