解淀粉芽孢杆菌TF28抗菌蛋白AP1的表达、纯化与抑菌活性

本研究采用人工合成方法合成抗菌蛋白基因AP1,连接到p ET32a(+)表达载体,导入E.coli BL21菌株进行原核表达,表达产物经HIS柱纯化和肠激酶切割后测定抑菌活性。人工合成了分子量为321 bp的抗菌蛋白基因AP1,获得了p ET32a(+)-AP1-BL21基因工程菌株,该菌株在0.1 mmol/L IPTG,25℃诱导2 h,蛋白表达率为43.2%,HIS柱纯化后获得SDS-PAGE电泳一条带分子量为27 k D的融合蛋白,其含量为124.82 mg/L,收率为92%,肠激酶切割后的蛋白能抑制大豆根腐病菌和水稻恶苗病菌的生长。获得高效表达抗菌蛋白AP1的工程菌株,该菌株表达的抗菌蛋白纯化后具有抑菌活性,在植病生防方面具有应用潜力。     

猪防御素PD基因表达载体的构建

目的：构建猪防御素PD基因表达载体。方法：化学合成经过适当改造的带有双酶切位点的PD基因,将该基因定向插入带有相同酶切位点的融合表达质粒PinPoin^TMXa-3的多克隆位点构建表达质粒。结果：构建的表达载体经双酶切电泳分析及插入基因片段序列分析,表明PD基因表达载体构建成功。结论：PD基因表达载体的构建,为进一步获得PD基因的表达产物,研究其抗菌活性、抗菌机理打下基础。     

抗菌肽ABP3基因的克隆及其在Pichia pastoris中的表达

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pPIC9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。     

解淀粉芽孢杆菌TF28抗菌蛋白基因TasA克隆与表达

[目的]克隆解淀粉芽孢杆菌TF28抗菌蛋白基因Tas A并进行原核表达和抑菌活性研究。[方法]采用PCR方法扩增抗菌蛋白基因Tas A,连接p ET22b载体,导入E.coli BL21(DE3)菌株,进行IPTG低温诱导表达,用His柱纯化表达产物,采用纸片方法测定其抑菌活性。[结果]从解淀粉芽孢杆菌TF28中克隆了抗菌蛋白基因Tas A,以p ET22b为表达载体构建高效表达抗菌蛋白Tas A的基因工程菌株,该菌株在0.05 mmol/L IPTG 15℃诱导4 h,Tas A蛋白表达率为34.2%,经His柱纯化后获得SDS-PAGE电泳一条带的纯化Tas A蛋白,其含量为67.8 mg/L,收率为90.8%。该蛋白抑制番茄灰霉病和叶霉病、玉米茎基腐病和水稻稻曲病菌生长。[结论]实现抗菌蛋白基因Tas A的原核表达,表达率34.2%,表达蛋白具有广谱抑菌活性,在植病生防方面具有应用潜力。     

颗粒裂解肽抗菌结构域在大肠杆菌中表达水平的分析

为实现颗粒裂解肽抗菌结构域的高效表达并避免其对宿主菌的毒性,研究设计了共表达阴离子配体重组生产阳离子抗菌肽的方法。结果证明该方法可有效提高阳离子抗菌肽的表达产量。同时研究分析了颗粒裂解肽不同抗菌结构域在同一原核表达载体pACYCDuet-1中表达水平的实验数据。通过计算机程序对mRNA二级结构的模拟分析,计算并比较出其二级结构生成自由能之间的差异,提出了预测颗粒裂解肽抗菌结构域在E.coli中表达水平的几点依据,参考这些依据可以对不同抗菌肽基因进行改造以获得高效表达。     

蜂毒素分子的改造及其基因在毕赤酵母中的表达

为获得保留有抗菌活性而降低溶血作用的蜂毒素,对蜂毒素的分子结构进行了改造。将第5位的Val变为Arg,第15位Ala变为Arg,删除了第16位的Leu。用PCR技术获得了改造后的蜂毒素基因,将其克隆入酵母表达载体pPICZa-A,获得重组表达质粒pPICZa-A-MEA。该质粒转化毕赤酵母菌GS115,甲醇诱导下表达,发酵上清液经抑菌活性、溶血活性测定及亲和层析纯化,结果表明,蜂毒素基因成功地在毕赤酵母中表达,经改造后表达的蜂毒素保留了抗菌活性且溶血活性显著降低,经纯化后用Bradford法测定表达蜂毒素的含量约为0.29mg／ml。     

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。     

猪PR-39抗菌肽基因在毕赤酵母中的表达

[目的]在毕赤酵母中表达抗菌肽PR-39基因,获得有抗菌活性的PR-39。[方法]根据酵母和猪密码子偏好性,对其密码子进行优化改造。将经SOE-PCR获得的PR-39基因与毕赤酵母表达载体pPIC9K连接,构建重组载体pPIC9K-PR-39。经SacⅠ线性化电击转化毕赤酵母GS115,取阳性克隆进行髙拷贝转化子筛选和诱导表达。[结果]pPIC9K-PR-39重组质粒构建成功,pPIC9K-PR-39菌株发酵产物检测结果对DH5α大肠杆菌和金黄色葡萄球菌都有抑菌效果。[结论]获得了PR-39基因的重组酵母,并用毕赤酵母系统成功地分泌表达了具有明显抗菌活性的抗菌肽PR-39。     

利用水稻抗菌蛋白开发抗稻瘟病水稻

《日经生物技术》１９９９年４月１２日第１２页报道：岩手县生物工程研究中心和岩手县农业研究中心、叶大学小组在高表达水稻抗菌蛋白的基础上,成功地获得抗水稻重要病害之一稻瘟病的抗性水稻。导入的是从水稻悬浮培养细胞中克隆的タウマチン样抗菌蛋白—米胶蛋白（ォリゼマチン）基因。是该小组独自克隆的基因。用导入基因的水稻当代的叶片,进行稻瘟病菌孢子的接种试验,获得抗性病斑,几乎没有患病性的个体。详细情况于４月４日在宇都宫市召开的日本育种学会上发表。目前该小组把米胶蛋白基因导入烟草获得了灰色霉病抗性。即使用水稻过…     

蜂毒素分子的改造及其基因在毕赤酵母中的表达

为获得保留有抗菌活性而降低溶血作用的蜂毒素,对蜂毒素的分子结构进行了改造.将第5位的Val变为Arg,第15位Ala变为Arg,删除了第16位的Leu.用PCR技术获得了改造后的蜂毒素基因,将其克隆入酵母表达载体pPICZa-A,获得重组表达质粒pPICZa-A-MEA.该质粒转化毕赤酵母菌GS115,甲醇诱导下表达,发酵上清液经抑菌活性、溶血活性测定及亲和层析纯化,结果表明,蜂毒素基因成功地在毕赤酵母中表达,经改造后表达的蜂毒素保留了抗菌活性且溶血活性显著降低,经纯化后用Bradford法测定表达蜂毒素的含量约为0.29mg/ml.     

Inhibitory effect of a defensin gene from the Andean crop maca (Lepidium meyenii) against Phytophthora infestans

In this study, we report the isolation of a defensin gene, lm-def, isolated from the Andean crop 'maca' (Lepidium meyenii) with activity against the pathogen Phytophthora infestans responsible of late blight disease of the potato and tomato crops. The lm-def gene has been isolated by polymerase chain reaction (PCR) using degenerate primers corresponding to conserved regions of 13 plant defensin genes of the Brassicaceae family assuming that defensin genes are highly conserved among cruciferous species. The lm-def gene belongs to a small multigene family of at least 10 members possibly including pseudogenes as assessed by genomic hybridization and nucleotide sequence analyses. The deduced mature Lm-Def peptide is 51 amino acids in length and has 74-94% sequence identity with other plant defensins of the Brassicaceae family. The Lm-Def peptide was produced as a fusion protein using the pET-44a expression vector and purified using an immobilized metal ion affinity chromatography. The recombinant protein (NusA:Lm-Def) exhibited in vitro activity against P. infestans. The NusA:Lm-Def protein caused growth inhibition and hyphal damage at concentration not greater than 0.4 microM. In contrast, the NusA protein alone expressed and purified similarly did not show any activity against P. infestans. Therefore, these results indicate that the lm-def gene isolated from maca belong to the plant defensin family with activity against P. infestans. Its expression in potato, as a transgene, might help to control the late blight disease caused by P. infestans with the advantage of being of plant origin.     

Tandem combination of Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) generates a more potent antifungal protein

Plant defensins are small (45 to 54 amino acids) positively charged antimicrobial peptides produced by the plant species, which can inhibit the growth of a broad range of fungi at micro-molar concentrations. These basic peptides share a common characteristic three-dimensional folding pattern with one α-helix and three β-sheets that are stabilized by eight disulfide-linked cysteine residues. Instead of using two single-gene constructs, it is beneficial when two effective genes are made into a single fusion gene with one promoter and terminator. In this approach, we have linked two plant defensins namely Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) genes by a linker peptide sequence (occurring in the seeds of Impatiens balsamina) and made into a single-fusion gene construct. We used pET-32a+ vector system to express Tfgd2-RsAFP2 fusion gene with hexahistidine tag in Escherichia coli BL21 (DE3) pLysS cells. Induction of these cells with 1 mM IPTG achieved expression of the fusion protein. The solubilized His₆-tagged recombinant fusion protein was purified by immobilized-metal (Ni²⁺) affinity column chromatography. The final yield of the fusion protein was 500 ng/μL. This method produced biologically active recombinant His₆-tagged fusion protein, which exhibited potent antifungal action towards the plant pathogenic fungi (Botrytis cinerea, Fusarium moniliforme, Fusarium oxysporum, Phaeoisariopsis personata and Rhizoctonia solani along with an oomycete pathogen Phytophthora parasitica var nicotianae) at lower concentrations under in vitro conditions. This strategy of combining activity of two defensin genes into a single-fusion gene will definitely be a promising utility for biotechnological applications.     

Constitutive expression of a fusion gene comprising Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) confers enhanced disease and insect resistance in transgenic tobacco

Plant defensins are small, basic cysteine-rich peptides that can inhibit the growth of a broad range of fungi or bacteria at micro-molar concentrations. They have been introduced as transgenes into different species to enhance host resistance to pathogens. In this study, a fusion gene of two defensins, Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) fused by a linker peptide of a polyprotein precursor from Impatiens balsamina was introduced into tobacco (Nicotiana tabacum var. Xanthi) via Agrobacterium-mediated leaf section transformation. Putative transgenic plants were confirmed by PCR analysis and integration of the fusion gene was confirmed by Southern blotting. RT-PCR analysis showed that the fusion gene was expressed in several confirmed transgenic plants. Western blotting analysis of crude protein extracts from leaves of the transgenic plants with anti-Tfgd2 and anti-RsAFP2 antibodies exhibited an 8 and 9 kDa bands corresponding to size of the fusion gene and confirmed the expression of fusion protein. When the leaves of transgenic plants were challenged with Rhizoctonia solani and Phytophthora parasitica var. nicotianae pathogens, they showed enhanced levels of disease resistance along with resistance to the generalist herbivore, Spodoptera litura larvae compared to control. Our results demonstrate that Tfgd2–RsAFP2 fusion protein is effective in protecting the transgenic plants against fungal and insect pathogens.     

Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae)

We previously purified and determined the partial amino acid sequence of a 4 kDa peptide having high homology with scorpion defensin from the hemolymph of adult fed female soft ticks, Ornithodoros moubata. In this study, the full length sequences of two defensin isoforms were obtained. Deduced amino acid sequences reveal a precursor protein of 73 amino acid residues with a mature portion consisting of 37 amino acid residues. This mature peptide contains six cysteine residues conserved in the same location as other invertebrate defensins. Phylogenetic analysis reveals that Ornithodoros defensin is most closely related to scorpion defensin and other more ancient arthropods. Ornithodoros defensin mRNA is constitutively expressed and up-regulated by blood-feeding and bacterial injection. Ornithodoros defensin gene expression occurs mainly in the midgut. This is the first report of the cloning and gene expression of an antibacterial peptide from the Acari.     

Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway.

A 5-kD plant defensin was purified from Arabidopsis leaves challenged with the fungus Alternaria brassicicola and shown to possess antifungal properties in vitro. The corresponding plant defensin gene was induced after treatment of leaves with methyl jasmonate or ethylene but not with salicylic acid or 2,6-dichloroisonicotinic acid. When challenged with A. brassicicola, the levels of the plant defensin protein and mRNA rose both in inoculated leaves and in nontreated leaves of inoculated plants (systemic leaves). These events coincided with an increase in the endogenous jasmonic acid content of both types of leaves. Systemic pathogen-induced expression of the plant defensin gene was unaffected in Arabidopsis transformants (nahG) or mutants (npr1 and cpr1) affected in the salicylic acid response but was strongly reduced in the Arabidopsis mutants eln2 and col1 that are blocked in their response to ethylene and methyl jasmonate, respectively. Our results indicate that systemic pathogen-induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response.     

δ-睡眠肽与GFP融合蛋白的表达和纯化

构建δ-睡眠肽(DSIP)蛋白与GFP的融合基因表达载体,高效表达和纯化GFP-DSIP融合蛋白。通过SOE-PCR拼接DSIP全长编码基因,并使得DSIP上游具有肠激酶识别位点,经双酶切定向克隆至表达载体pET-28a,构建重组载体pET-28a-DSIP,通过PCR扩增GFP全长编码基因,经双酶切定向克隆至pET-28a-DSIP,构建原核重组表达载体pET-28a-GFP-DSIP,通过双酶切和测序鉴定后,导入E.coli BL21宿主菌中,IPTG诱导表达融合蛋白,采用镍亲和层析和分子筛凝胶层析获得高纯度蛋白,SDS-PAGE分析鉴定。经测序鉴定成功构建了原核重组表达载体pET-28a-GFP-DSIP,在IPTG诱导下获得可溶性的绿色荧光蛋白与睡眠肽的融合蛋白,经Ni-NTA亲和层析纯化成功获得高纯度的融合蛋白。成功构建了DSIP与GFP融合基因的重组表达载体,确定了GFP-DSIP融合蛋白诱导表达的最佳条件,获得了较高纯度的融合蛋白,为进一步研究DSIP蛋白的生物学功能奠定了基础。     

人β防御素3在大肠杆菌中可溶性表达及其生物活性的鉴定

根据大肠杆菌对精氨酸密码子使用的偏好,设计引物并通过酶促法合成了人β防御素3(hBD-3)全基因序列,克隆进pGEX-4T-2中构建pGEX-4T-2-hBD-3融合表达载体．将表达载体转化Ecoli宿主菌DH5α,进行IPTG诱导表达．控制诱导条件,提高可溶性蛋白的表达量．将菌体进行反复冻溶使细胞膜穿孔,释放可溶性蛋白．融合蛋白GST-hBD-3经凝血酶切割得到重组人防御素蛋白．用琼脂孔穴扩散抑菌法检测表明,重组人β防御素3对金黄色葡萄球菌有抑菌活性．     

人胰岛素原基因在大肠杆菌中高效外分泌表达

将胰岛素原基因融合到金色葡萄球菌蛋白Ａ的基因上,构建成大肠杆菌中基因融合的外分泌表达载体。它能高效表达且有效地分泌表达产物。利用亲和层析能方便地从培养液中分离出融合蛋白。融合蛋白经ＣＮＢｒ裂解后,经反相ＨＰＬＣ分析,分离得到具有天然结构的胰岛素原并进行了鉴定。