家蝇防御素在大肠杆菌中的表达、纯化与抗体制备

家蝇防御素是从家蝇中克隆得到的1种抗菌肽。为了进一步研究家蝇防御素的功能和制备特异性抗体,采用大肠杆菌表达外源蛋白的方法, 进行了家蝇防御素原核表达的研究。根据克隆到的家蝇防御素基因(Mdde) 的cDNA序列, 设计特异性引物, PCR 扩增成熟肽的cDNA片段, 将成熟肽序列重组到表达载体pGEX 4T 1中, 构建m Mdde/pGEX 4T 1重组表达载体, 在大肠杆菌BL21 中诱导表达, 重组表达的融合蛋白GST Mdde占菌体总蛋白的33 4%。纯化得到GST Mdde后, 再用凝血酶将其从特定位点切开, 得到表达的m Mdde。液体抑菌实验结果初步表明, 表达的融合蛋白GST Mdde对细菌生长有一定的抑制作用。利用纯化的GST Mdde融合蛋白, 制备了抗血清。     

家蚕抗菌肽CD高效表达及其抗BmNPV感染作用

通过大肠杆菌JM109诱导家蚕,提取其脂肪体总mRNA后,通过RT-PCR 得到 cDNA,根据GenBank上家蚕抗菌肽 Cecropin D 的cDNA序列,设计并合成引物,然后PCR扩增得到 Cecropin D肽基因并克隆到pGEM-T载体中,经过EcoRΙ和 Xho I 酶切,连接并将Cecropin D肽基因插入pET32a表达载体中.用重组质粒pET32a- ecropin D 转化大肠杆菌BL21(DE3),在IPTG诱导下,融合蛋白Trx-Cecropin D 以可溶形式得到高效表达,经SDS-PAGE检测显示分子量为23kDa与预期大小相符,表达量约为总蛋白的30%.融合蛋白经Ni2+ 柱纯化后通过肠激酶切割后释放为 Trx(18kDa)和 Cecropin D (5kDa),最后通过超滤管分离得到重组抗菌肽.通过抑菌实验测得重组CecropinD对于革兰氏阴性及阳性菌均有抑菌活性.并将重组Cecropin D 与家蚕病毒BmNPV作用混合4h后,一起投喂家蚕,发现病毒感染力有明显降低,说明其有抗病毒感染作用.     

重组抗癫痫肽二串体的表达、纯化与鉴定

目的：利用基因工程方法表达抗癫痫肽（AEP）串联体蛋白,并进行纯化、鉴定。方法：PCR扩增AEP及AEP—His基因片段,并分别克隆至测序载体中;测序正确后,利用基因重组技术获得二串体基因,构建毕赤酵母表达载体pPIC9K-AEP2-His,甲醇诱导AEP2-His在毕赤酵母中表达,用Ni—chelating SepharoseFF柱纯化表达的融合蛋白,并用抗His单克隆抗体进行Westernblot鉴定。结果：构建了AEP二串体酵母表达载体,获得了纯化的AEP2-His蛋白,其相对分子质量约20000。结论：用基因串联思路表达小分子多肽是一种可行的方法;AEP2-His蛋白的成功表达为其功能研究奠定了基础。     

抗菌肽Cecropin B-人溶菌酶融合蛋白表达载体的构建

目的是构建抗菌肽B(Cecropin B)和人溶菌酶(hLyso)的融合蛋白表达载体。从pUC118～hLyso上卸下人溶菌酶基因后,通过重叠区扩增法人工合成抗菌肽B基因,并将其融合到人溶菌酶基因的5’端。将抗菌肽B基因和人溶菌酶基因按正确的阅读框架定向克隆至大肠杆菌高效表达载体pET32a,终止子位于人溶菌酶基因的3’端。PCR鉴定及序列分析表明,所转化的BL21(DE3)菌落中含有插入Cecropin B-hLyso基因的重组质粒pET32a-CB-hLyso。     

家蝇幼虫消减文库的构建及差异表达基因的鉴定

为了鉴定家蝇Musca domestica免疫相关基因,应用抑制性消减杂交技术,构建刺激家蝇幼虫差异表达cDNA消减文库。以大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus诱导12 h的家蝇幼虫与未诱导的家蝇幼虫为消减杂交对象,获得了差异表达基因的cDNA片段,将其与T/A载体连接并转化大肠杆菌DH5α,构建了刺激家蝇幼虫cDNA消减文库。PCR检测发现,文库的阳性克隆中插入的cDNA片段大小在200~1 000 bp之间,随机挑选了161个含大小不等差异片段的克隆进行测序和同源性分析,鉴定了36种蛋白的基因片段,包括抗菌肽、酶、核糖体蛋白、其他功能蛋白以及功能不明的蛋白。用半定量RT-PCR分析了其中6种蛋白基因的表达,结果显示：防御素和攻击素基因在细菌刺激后24 h内明显上调表达,而溶菌酶、酚氧化酶原活化因子、糜蛋白酶和蛋白质合成起始因子基因在细菌刺激后0-4 h内表达受抑制,12 h后上调表达。该研究结果为家蝇免疫相关基因的克隆和家蝇免疫防御机制的探讨奠定了良好的基础。     

家蝇抗菌肽Defensin基因在COS-7细胞中的瞬时表达

目的:研究家蝇抗菌肽Defensin cDNA在非洲绿猴肾细胞株COS-7中的表达情况,并对表达产物的抗菌作用进行初步检测。方法:以Defensin基因为模板设计特异性引物,扩增在C端含6×His标签的Defensin开放阅读框序列,将此序列与真核表达载体pcDNA3.1( )进行重组,构建重组质粒pcDNA3.1( )/Defensin-His 6。以阳离子脂质体LipofectamineTM2000为载体,对宿主细胞COS-7进行重组质粒pcDNA3.1( )/Defensin-His 6和空载体pcDNA3.1( )的转染,72h后收集细胞培养上清液,表达产物经His-Trap HP亲合层析柱分离纯化和Western blotting鉴定后,进行杀菌活性的初步检测。结果:重组质粒pcDNA3.1( )/Defensin-His 6组细胞培养上清液的纯化物,行Western blotting得到了分子量大小约为10.0kD的单一目的条带,与预期相符;杀菌活性试验中发现:该纯化物对大肠杆菌E.coliK12D31具有一定的杀菌活性。结论:家蝇抗菌肽Defensin基因在宿主细胞COS-7中得到了正确表达。     

一种新型抗菌肽Cecropin B真核表达载体的构建

通过重叠区扩增法人工设计和合成一种新型抗菌肽Cecropin B基因,按正确的阅读框架定向克隆至巴斯德毕赤酵母的高效表达载体pPIC9K上。经PCR鉴定及序列分析,所转化的大肠杆菌DH5ct菌落中含有插入Cecropin B基因的重组质粒pPIC9K—CB,表明成功构建了新型抗菌肽Cecropin B表达载体pPIC9K—CB。     

中华蜜蜂蜂毒镇静肽基因的cDNA克隆和表达

从中华蜜蜂 (Apisceranacerana)工蜂毒腺中快速抽提总RNA ,用RT PCR扩增得到大小约为2 5 0bp的cDNA片段 ,测序得到的片段长度为 2 34bp ,为蜂毒前镇静肽原 (preprosecapin)基因编码区的cDNA .以 3′RACE方法 ,扩增和测定了 3′端非编码区 2 19bp序列 .中蜂前镇静肽原cDNA序列与已报道的欧洲意蜂该基因cDNA序列具有 92 %同源性 ,氨基酸序列具有 87%同源性 .代表成熟肽镇静肽的最后 2 5个氨基酸序列 ,中蜂与意蜂同源性为 88% .3′端非编码区cDNA序列与欧洲意蜂序列有 73 1%同源性 .将中华蜜蜂蜂毒镇静肽成熟肽编码区与 3′非编码区部分克隆 ,构建了镇静肽与谷胱甘肽转移酶融合表达的载体pGEX AcSecapin .将载体转化大肠杆菌BL2 1(DE3)进行融合表达 .表达产物与抗GST抗体在 2 9kD处有很强的交叉反应 .大肠杆菌超声破碎后的上清液用SDS PAGE检测到表达的蛋白多为可溶性融合蛋白 ,通过亲和层析柱纯化和凝血酶的切割得到了镇静肽蛋白     

家蝇抗菌肽Defensin基因同向串联表达载体的构建和鉴定

目的:构建家蝇抗菌肽Defensin基因多拷贝串联体,并克隆到甲醇酵母分泌表达载体pPIC9K上。方法:PCR法扩增家蝇抗菌肽Defensin基因成熟肽片断,目的片断的上游5′端带有EcoRⅠ和NheⅠ位点,下游5′端带有NotⅠ和XbaⅠ位点,目的片断首先克隆入pMD18-T载体,利用pMD18-T载体的NdeⅠ位点和目的片断上的一对同尾酶(NheⅠ和XbaⅠ),多次酶切连接,串联成多拷贝的Defensin成熟肽基因,再用EcoRⅠ和NotⅠ双酶切,最后克隆入甲醇酵母分泌表达载体pPIC9K。结果:PCR鉴定、酶切鉴定和DNA测序证明多拷贝基因重组质粒构建成功。结论:该方法能方便高效地获得所需的多拷贝基因,为进一步进行高效表达打下基础。     

果蝇唾液腺特异基因CG8419多克隆抗体的制备及检测

果蝇CG8419基因与脊椎动物中TRIM45基因为同源基因.以果蝇cDNA为模板通过PCR扩增出亲水性和特异性好的果蝇CG8419基因片段,将其克隆入表达载体PET-28a,构建出重组表达质粒PET-28a-CG8419.将重组质粒转化大肠杆茵Rosetta,经IPTG诱导出带His标签的重组融合蛋白.通过尿素洗涤包涵体并切胶回收纯化融合蛋白,然后再免疫新西兰大白兔制备多克隆抗体.Western blot实验分别验证抗体的效价和特异性.果蝇胚胎抗体染色显示该基因在果蝇唾液腺中表达.     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Effect of soil salinity and water stresses on growth and content of nitrogen,chloride and phosphate of wheat and triticale

Summary Three wheats and one triticale were grown, up to flowering stage, in pots on calcareous soil adjusted to a range of salinities (S₁=3.5, S₂=6, S₃=8.5, and S₄=11 mmhos/cm, 20°C, soilpaste extract) by adding solution consisting of 3∶2∶1 of Na-, Ca- and Mg chlorides in chemical equivalent amounts. Moisture in the pots was kept at 100% (W₁), 40% (W₂) and 20% (W₃) of the available water. The vegetative growth, nitrogen and phosphate were affected by S and W treatments, chloride was affected only by S. The interaction S×W affected only dry weight. Varietal effect was observed between wheat as a group and triticale. Multiple quadratic regression equations of these properties on salinity and water revealed that the higher the available water the wider the range of tolerable salinity. Triticale was relatively more tolerant to water stress. Salinity increases Cl and decreases N, whereas water stress enhances N accumulation to a certain extent. However, in triticale at S₃ and S₄ the effect of water stress on N was overshadowed by the excessive salinity. This did not occur for the wheat (Florence). P trends were described. R² for P was low (0.7435–0.3603) which made interpretations rather difficult.     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭示草地放牧演替的生理机制有重要意义.大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段.放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势.牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关.放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关.