人精液凝固蛋白SgI-52的表达与抗菌活性研究

以人精囊腺cDNA为模板,用嵌套式PCR技术扩增出编码成熟人精液凝固蛋白I(semenogelin I,SgI)第85–136位氨基酸(SgI-52)的核苷酸序列并将其插入载体pMAL-p2X中,成功构建的表达载体pMAL-p2X/SgI-52转化大肠杆菌DH5α后,重组融合蛋白诱导表达于大肠杆菌周质中。经第X因子剪切及超滤处理,得到表达的重组SgI-52。质谱分析结果证明重组SgI-52为目的肽。重组人SgI-52对大肠杆菌ATCC25922标准株以及耐氨苄标准株ML-35P的最低抑制浓度MIC分别为32.45以及46.30?μg/mL。我们的结果及相关研究提示在人精液液化过程中人精液凝固蛋白I的不同降解产物可能行使不同的生物学功能,值得进行深入研究。     

黄瓜砧木根的解剖结构与抗逆性关系研究

利用光学显微技术,对黄瓜(Cucumis sativus)和3种砧木：黑籽南瓜(Cucurbita ficifolia)、南瓜(C. moschata)、丝瓜(Luffa cylindrica) 根的解剖结构进行了比较解剖学研究,探讨了黄瓜及其不同砧木抗病性和抗涝性的植物体结构机制,结果表明：导管的管孔类型、管孔大小、管壁厚度、导管周围木纤维的数量及侵填体的有无等与抗病性相关。木栓厚度,通气组织,管孔直径和导管数量与抗涝性相关。     

玄参的化学成分研究

利用正、反相硅胶以及各种色谱技术进行分离,经理化性质和光谱分析鉴定中药玄参(Scrophularia ningpoensis)有效成分。从玄参(S. ningpoensis)根的乙醇提取物中分离了8个化合物,分别鉴定为14-去氧-12(R)-磺酸基穿心莲内酯（14-deoxy-12(R)-sulfo andrographolide, Ⅷ）、肉桂酸（cinnamic acid,Ⅰ）、对羟基肉桂酸（p-hydroxycinnamic acid,Ⅱ）、丁二酸（succinic acid,Ⅲ）、β-谷甾醇（β-sitosterol, Ⅳ）、胡萝卜甙（daucosteol,Ⅴ）、葡萄糖（gluose,Ⅵ）、熊果酸(ursolic acid,Ⅶ)。Ⅷ为在玄参中首次发现的天然化合物。     

不同寄主来源寄生疫霉菌株的遗传变异分析

从300条RAPD随机引物中筛选出扩增多态性丰富的13条引物,对寄主来源不同的29个寄生疫霉（Phytophthora parasitica）菌株进行基因组DNA遗传变异分析。用筛选出的13条引物对供试菌株进行RAPD-PCR扩增,共产生139条RAPD条带,其中133条为多态性条带,多态检测率为95.7%。利用PopGene Version 1.31软件对供试菌株间的遗传距离进行聚类分析并构建系统树状图,供试29个菌株被划分为5个遗传聚类组,不同菌株间具有丰富的遗传变异。其中寄主为烟草(Nicotiana spp.)、腊梅(Chimonanthus praecox)、凤尾兰(Yucca gloriosa)和西番莲(Passiflora edulis)的菌株的遗传结构与其寄主来源具有明显的相关性,而寄主为刺槐(Sophora chinensis)和番茄(Lycopersicum esculentum)的菌株与其寄主来源的相关性较小,来源于不同寄主植物的菌株之间遗传距离较远。结果提示在寄生疫霉与其不同寄主植物的长期协同进化过程中,寄主对病原菌的遗传结构具有一定的影响。     

绵羊生殖道抗菌肽

以屠宰场收集的新鲜、健康、雌性绵羊生殖器官为原材料．采用乙酸浸提、透析、Sephadex G-50凝胶过滤层析和反相高效液相色谱(RP-HPLC)等方法分离纯化绵羊生殖道抗菌肽．以G+、G-和真菌为抗菌活性检测指示菌株,利用薄层琼脂糖孔穴扩散法、微量肉汤稀释法进行抗菌活性检测．对分离纯化所得纯品进行分子质量质谱测定、纯度鉴定、N端测序,并对其性质进行研究．结果表明：分离纯化所得两个绵羊生殖道抗菌肽分子质量分别为4820.47 u和4012.5 u,N端部分氨基 酸序列分别为AYVLDEPKP和YDSGA．对G+细菌(S. aureus ATCC2592、Streptococcu ATCC55121)、G-细菌(E． coli ATCC25922)、真菌(C． albicans ATCC2002)均具有良好的抑菌活性．对家兔红细胞无溶血活性,对人血液凝固无影响．目前未见有从绵羊生殖道分离纯化得到抗菌肽的报道,并且这一研究结果进一步证实抗菌肽在多种动物生殖道天然免疫防御方面起着重要作用．     

Cr6+对菹草叶绿素荧光参数、抗氧化系统和超微结构的胁迫影响

研究了不同浓度的Cr⁶⁺(0、1、10、30、50 mg·L^-1)对菹草叶片叶绿素、叶绿素荧光参数、抗氧化系统、可溶性蛋白含量、膜脂过氧化产物、可溶性糖含量、超氧阴离子(O₂^—·)产生以及细胞超微结构的胁迫影响。结果表明,随着Cr⁶⁺浓度的增加,总叶绿素、叶绿素a、叶绿素b和叶绿素a/b均呈下降趋势,F_o先升后降,F_v/F_m、F_m及F_v/F_o均逐渐降低;可溶性蛋白和谷胱甘肽(GSH)含量先升后降,抗坏血酸(AsA)含量逐渐下降;超氧化物歧化酶(SOD)活性逐渐下降,而过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性均呈先升后降趋势; O₂^—·产生速率、丙二醛(MDA)和可溶性糖含量均表现先降后升趋势;电镜观察发现：随着Cr⁶⁺浓度的增加,对细胞超微结构的损伤也加剧,表现为叶绿体膨胀,被膜破裂,类囊体片层解体;线粒体嵴数目减少,呈空泡状。可见,Cr⁶⁺破坏了菹草正常生理活动的结构基础,造成菹草生理功能紊乱。     

家蝇抗真菌肽衍生物MAF-1C抗白色念珠菌活性研究

[目的]探究家蝇抗真菌肽衍生物MAF-1C结构特点及抗白色念珠菌活性。[方法]采用氨基酸替代法设计MAF-1A衍生肽MAF-1C,生物信息学分析其理化特性,微量液体稀释法、菌落计数法检测体外抗白色念珠菌活性,扫描电镜观察对菌细胞结构的影响。[结果]MAF-1C为非跨膜阳离子线状多肽,带8个正电荷,二级结构以α螺旋为主。对白色念珠菌具有抗菌活性,MIC、MFC值分别为0.2 mg/m L和0.5 mg/m L,2×MIC浓度的MAF-1C从作用一开始就发挥抗菌作用,其作用后白色念珠菌细胞表面出现明显的凹陷、裂痕,之后细胞皱缩、裂解死亡,并且菌细胞损伤程度与肽浓度及作用时间呈正相关。[结论]MAF-1C结构参数及对白色念珠菌的抗菌活性均显著优于模板肽,对比模板肽改造后的肽阳离子电荷增加了6个,总平均疏水值(GRAVY)由-1.081升高到0.588,不稳定系数由42.27降低到5.83,MIC由0.6 mg/m L降低到0.2 mg/m L,MFC由0.7 mg/m L降低到0.5 mg/m L,其可通过破坏白色念珠菌细胞结构发挥抗菌作用。     

竞争性RT-PCR法及对大肠杆菌acrA基因mRNA表达水平的定量研究

建立了用于检测大肠杆菌(Escherichia coli)ATCC25922的acrA基因mRNA表达水平的定量竞争性RT-PCR(QC-RT-PCR)体系。PCR合成目标片段的突变型片段(321bp)作为内标准模板(Internal standard,IS),与目标片段一致的片段(389bp)作为目标模板(Target standard,TS),优化两种模板共扩增体系;梯度稀释IS与等量大肠杆菌cDNA样本共扩增,扫描电泳条带,软件分析数据。结果表明,引物设计合适,以IS和TS为模板实现共扩增,产物(321bp和389bp)通过1.5%琼脂糖凝胶电泳有效分离;梯度稀释IS与cDNA共扩增产物出现亮度梯度电泳条带;获得一元回归曲线y=-0.345+0.097x(相关系数r=0.959,标准差s=0.05997)。该研究成功构建内标准模板,优化的共扩增PCR体系实现了对大肠杆菌ATCC25922中acrA基因mRNA表达水平的检测,具有简便、高效、敏感度高等优点。     

中国板栗自然居群微卫星（SSR）遗传多样性

采用8对微卫星分子标记对中国板栗(Castanea mollissima)的28个自然居群进行了遗传多样性与遗传结构分析。在849个个体上扩增得到128个等位基因, 每位点平均等位基因数(A)为16。中国板栗居群的平均预期杂合度(H_E)为0.678, 平均观察杂合度(H_O)为0.590。华中地区的中国板栗居群遗传多样性最高(A = 8.112, H_E = 0.705, H_O = 0.618), 其次为西北地区和华东地区, 而西南地区遗传多样性最低(A = 6.611, H_E = 0.640, H_O = 0.559)。基于无限等位基因模型(IAM)和基于逐步突变模型(SMM)的遗传分化系数分别为F_ST = 0.120和R_ST = 0.208。分子方差分析(AMOVA)结果表明中国板栗野生居群的遗传变异主要存在于居群内(87.16%)。Mantel检测揭示遗传距离与地理距离之间无显著相关性, 表明基因流不是主导中国板栗居群遗传结构的关键因素。华中地区(尤其是神农架及其周边地区)是中国板栗遗传多样性的现代分布中心, 因而应该得到优先保护, 同时该区域的野生板栗居群可优先作为栽培板栗遗传育种的材料和基因库。     

乙烯利处理对葡萄花色苷合成相关基因表达的影响

利用荧光定量PCR技术分析‘京优’葡萄果实成熟过程中,花色苷生物合成途径相关酶基因mRNA转录水平的变化以及乙烯利处理对果皮中花色苷含量和关键酶基因转录水平的影响。结果显示,葡萄果实发育进入着色期,花色苷合成过程中主要相关基因(CHSs、CHIs、F3Hs、F3′H、F3′5′H、DFR、LDOX、UFGT、OMT和GST)和转录因子(MybA1和MybA1-2)转录水平都显著提高,其中UFGT、GST、MybA1和CHSs、CHIs、F3Hs基因家族中的CHS3、CHI2、F3H2随着花色苷合成而大量转录;乙烯利处理能够增强花色苷合成相关基因的转录,使其转录时期前移和转录水平提高,其中对GST、UFGT和MybA1转录的促进作用最明显。相关性分析表明,花色苷合成与一些花色苷合成相关基因(CHS3、CHI2、F3H2、F3′5′H、UFGT、GST)和转录因子(MybA1)的转录水平呈显著或极显著正相关;与CHS1、CHS2、CHI1、F3H1、DFR、F3′H、LDOX和OMT转录水平的相关性均不显著。本研究结果为进一步阐明花色苷生物合成机理和花色苷类色素的生产应用提供一定的理论依据。     

Identification of novel semenogelin I-derived antimicrobial peptide from liquefied human seminal plasma.

Semenogelin I (SgI) is one of the most abundant proteins in human seminal plasma. SgI plays a key role in sperm coagulation and spermatozoon immobilization. In addition, SgI and/or its proteolytic fragments are involved in regulating spermatozoon motility, capacitation and inhibin-like activity. However, little is known about the antibacterial activity of SgI-derived peptides. By a combination of ion-exchange, gel filtration and high-performance liquid chromatography, peptides from liquefied human seminal plasma from 40 healthy donors were isolated and characterized. N-terminal amino-acid sequencing and fast atom bombardment mass spectrometry revealed that four isolated peptides were SgI-derived, namely SgI-29 (85-113), SgI-46 (85-130), SgI-47 (85-131) and SgI-52 (85-136). Interestingly, SgI-29, SgI-46 and SgI-47 are newly identified SgI-derived peptides. Antimicrobial activity assay results indicated that synthesized SgI-29 had strong antibacterial activity toward various bacterial strains. Our results indicate that SgI can be digested into small fragments like newly identified SgI-29, SgI-46 and SgI-47 and may have diversified functions.     

The effects of preservation procedures on antibacterial property of amniotic membrane

Amniotic membrane (AM), the innermost layer of the fetal membranes, has been widely employed in the surgical reconstruction and tissue engineering. Expression of the antimicrobial peptides such as defensins, elafin and SLPI which are essential elements of the innate immune system results in antibacterial properties of the AM. Preservation is necessary to reach a ready-to-use source of the AM. However, these methods might change the properties of the AM. The aim of this study was to evaluate antibacterial properties of the AM after preservation. Antibacterial property of the fresh AM was compared with cryopreserved and freeze-dried AM by modified disk diffusion method. Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and two clinical isolated strains of E. coli were cultured in Mueller Hinton agar and a piece of the AM was placed on agar surface. After 24 h incubation, the inhibition zone was measured. In addition, one of the most important antibacterial peptides, elafin, was measured by ELISA assay before and after preservations procedures. Antibacterial properties of the AM were maintained after cryopreservation and freeze-drying. However, the inhibition zone was depending on the bacterial strains. The cryopreservation and freeze-drying procedures significantly decreased elafin which shows that antibacterial property is not limited to the effects of amniotic cells and the other components such as extracellular matrix may contribute in antibacterial effects. The promising results of this study show that the preserved AM is a proper substitute of the fresh AM to be employed in clinical situations.     

抗菌肽及抗菌肽转基因植物研究进展

抗菌肽是一类小分子多肽,在生物体内分布广泛,具有广谱的抗菌性,是生物体内天然防御系统的一部分。主要介绍了抗菌肽的性质,类型,作用机制及抗菌肽转基因植物的研究进展。     

家蝇抗菌肽的抑菌动力学研究及其机理初探

利用鼠伤寒沙门氏菌针刺诱导家蝇幼虫表达抗菌肽,对抗菌肽的抑菌动力学进行研究,并通过抗菌肽样品对不同细菌动力学特性的研究出发对抗菌肽抑菌作用机制进行探讨。研究发现抗菌肽样品的活性与作用时间有关,24h内出现一到两个活性峰,同一抗菌肽样品对不同细菌的抑菌动力学有差异,抗菌肽的抑菌动力学机制应该与它的的抑菌作用机制有关。通过电镜观测、细胞磷代谢、紫外吸收物测定以及抗菌肽与细菌DNA相互作用结果可知,微生物诱导家蝇表达的抗菌肽样品不仅能够造成细菌细胞的快速坍塌破裂而且能够破坏细胞核心,与DNA结合作用。抗菌肽抑菌动力学的解释：微生物诱导产物中含有两类抗菌肽,一类抗菌肽能造成细胞膜的快速坍塌破裂形成第一个活性峰;另一类抗菌肽可进入细胞,破坏细胞核心,造成紫外吸收物大量外泄形成第二个活性峰。     

杂合抗菌肽在毕赤酵母中的表达及其活性测定

为获得溶血活性低、抗菌活性高的杂合抗菌肽,以家蝇抗菌肽Cec Md和中国林蛙抗菌肽Chensirin为母体肽,并结合毕赤酵母偏爱密码子的原则,设计出6条具有抗菌潜力的新型杂合抗菌肽,将其命名为CC22、CC28、CC29、CC30和CC34(1),CC34,利用SOE-PCR技术合成所需的目的基因,并将其克隆至毕赤酵母表达载体pGAPZαA,通过电击转化技术,将其转化至毕赤酵母SMD1168中,经含有Zeocin的抗性平板筛选阳性转化子,YPD液体培养72h后,经Tricine-SDS-PAGE检测出目的蛋白,然后采用高效液相色谱法对其进行纯化。检测结果显示,表达产物CC29对大肠杆菌、鸡沙门氏菌的最小抑菌浓度(MIC)均为25μg/ml;CC34(1)对大肠杆菌表现相对较弱的抑制作用,最小抑菌浓度为100μg/ml;CC34对鸡沙门氏菌和金黄色葡萄球菌的最小抑菌浓度为50μg/ml;且杂合抗菌肽对有益菌均没有表现出抑制作用。6条杂合肽的溶血活性均呈现较低水平,其中表现出抗菌活性的3条抗菌肽中,以CC29的溶血活性最低,CC34(1)和CC34相对次之。结合抑菌活性,CC29和CC34的抑菌效果较为明显,从而确定溶血活性低且抗菌活性较高的CC29和CC34为新型杂合抗菌肽。     

Bovine hemoglobin: an attractive source of antibacterial peptides

A peptic hemoglobin hydrolysate was fractioned by a semi-preparative reversed-phase HPLC and some fractions have an antibacterial activity against four bacteria strains: Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis. These fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize the peptides in these fractions. Each fraction contains at least three peptides and some fractions contain five peptides. All these fractions were purified several times by HPLC to obtain pure peptides. Thirty antibacterial peptides were identified. From the isolated antibacterial peptides, 24 peptides were derived from the chains of hemoglobin and 6 peptides were derived from the β chains of hemoglobin. The lowest concentration of these peptides (minimum inhibitory concentration (MIC)) necessary to completely inhibit the growth of four bacteria strain was determined. The cell population of all of the tested bacteria species decreased by at least 97% after a 24-h incubation with any of the peptides at the minimum inhibitory concentration.     

家蝇幼虫抗菌肽的抗菌谱及其与抗生素的协同作用研究

研究3种家蝇幼虫抗菌肽的抗菌谱以及每种抗菌肽的最小抑菌浓度(MIC),初步探讨3种抗菌肽分别与青霉素、链霉素相结合后对抗菌活性的影响,并采用分级抑制浓度指数(Fractionalinhibitoryconcentrationindex,FIC)来定量检测抗菌肽与抗生素之间的抗菌作用关系。结果表明3种抗菌肽的抗菌谱不同,不同的抗菌肽对不同病原菌的抗菌活性不同。3种抗菌肽与链霉素、青霉素之间的抗菌协同关系因细菌种类不同。抗菌肽与抗生素之间并不是都存在协同关系,有些不相关,甚至表现为对抗关系,表明抗菌肽、抗生素与细菌三者的相互作用关系非常复杂。     

Design,synthesis and antibacterial evaluation of honokiol derivatives

Staphylococcus aureus is a major and dangerous human pathogen that causes a range of clinical manifestations of varying severity, and is the most commonly isolated pathogen in the setting of skin and soft tissue infections, pneumonia, suppurative arthritis, endovascular infections, foreign-body associated infections, septicemia, osteomyelitis, and toxic shocksyndrome. Honokiol, a pharmacologically active natural compound derived from the bark of Magnolia officinalis, has antibacterial activity against Staphylococcus aureus which provides a great inspiration for the discovery of potential antibacterial agents. Herein, honokiol derivatives were designed, synthesized and evaluated for their antibacterial activity by determining the minimum inhibitory concentration (MIC) against S. aureus ATCC25923 and Escherichia coli ATCC25922 in vitro. 7c exhibited better antibacterial activity than other derivatives and honokiol. The structure-activity relationships indicated piperidine ring with amino group is helpful to improve antibacterial activity. Further more, 7c showed broad spectrum antibacterial efficiency against various bacterial strains including eleven gram-positive and seven gram-negative species. Time-kill kinetics against S. aureus ATCC25923 in vitro revealed that 7c displayed a concentration-dependent effect and more rapid bactericidal kinetics better than linezolid and vancomycin with the same concentration. Gram staining assays of S. aureus ATCC25923 suggested that 7c could destroy the cell walls of bacteria at 1 × MIC and 4 × MIC.     

弱酸性家蝇蛆抗菌肽MD7095的分离纯化及性质研究

家蝇抗菌肽多是碱性蛋白,目前尚无弱酸性家蝇抗菌肽的报道。通过稀醋酸低温浸提,海藻酸吸附,稀盐酸低温洗脱、盐析、Sephadex G25凝胶过滤和CMC23弱阳离子交换柱层析等方法,利用灵敏的杀菌活性检测手段,从家蝇蛆(Musca domesticalarvae)中分离纯化出一组弱酸性抗菌肽,对苏云金芽孢杆菌(Bacillus thuringiensis)等革兰氏阳性菌和几种革兰氏阴性菌有强烈的杀灭作用,有极强的耐热、耐冻融的特性。通过电洗脱方法进一步纯化出抗菌肽MD7095,质谱测定其分子量7095Da,IEF电泳测得其等电点5.59,经肽质量指纹谱(PMF)鉴定为一新肽。扫描电镜超微结构观察表明,弱酸性家蝇蛆抗菌肽对苏云金芽孢杆菌的杀菌机制主要是使细胞膜穿孔,内容物外泄,最终使细菌完全解体死亡。     

ROCK-I Derived Bi-functional Peptides with Antibacterial Infection Activity

Rho-associated protein kinase I (ROCK-I) dimerization is an important biological event in activation of ROCK-I signaling pathway, which is involved in a variety of physiological and pathological processes. Targeting and disrupting the dimerization have been recognized as an attractive approach to molecular therapeutics. Here, we reported successful application of structural bioinformatics analysis to dissect the intermolecular interaction in ROCK-I dimerization based on a high-resolution complex crystal structure of ROCK-I dimer. Three helical peptides that can independently bind to ROCK-I monomer were derived from the dimerization domain; they have high or moderate affinity towards the ROCK-I monomer. These peptides are amphipathic helices which use their hydrophobic side to contact ROCK-I, while expose their hydrophilic side to solvent. Antimicrobial susceptibility test demonstrated that these amphipathic peptides also exhibited antibacterial activity against three clinical strains, including Escherichia coli ATCC 25922, Streptococcus pneumoniae ATCC 49619 and Staphylococcus aureus ATCC 25923. Thus, the three helical peptides can be considered as bi-functional molecular entities that possess both dimerization-disrupting potency and antibacterial activity.