土曲霉植酸酶基因在巴斯德毕赤酵母中的高效表达

参考已报道的植酸酶基因phyA全序列设计一对引物,用PCR方法从土曲霉穴Aspergillusterreus雪总DNA中扩增到去除信号肽和内含子后约1.4kb的phyA基因编码区片段,对该片段进行了克隆与序列测定。进一步用该片段构建了pPIC9K-phyA分泌型表达载体,并转化毕赤酵母穴Pichiapastoris雪GS115。通过表达产物的酶活性检测和SDS-PAGE电泳分析,植酸酶在重组转化子中得到了有效分泌和高效表达。摇瓶诱导发酵132h后发酵液中植酸酶的活性可达167u·mL-1;表达产物在pH2.0～8.0均有活性,最适pH为5.5,最适温度为55℃。     

人源明胶基因在毕赤酵母中的表达

[目的]利用毕赤酵母重组获得明胶蛋白。[方法]构建基因工程菌,利用甲醇诱导表达。[结果]LC-MS/MS检测到了明胶蛋白。[结论]微生物重组表达特定分子量大小的明胶是可行的。     

罗非鱼热休克蛋白70在毕赤酵母中的表达与纯化

以尼罗罗非鱼血液mRNA反转录获得编码罗非鱼Hsp70完整cDNA,重组构建罗非鱼真核表达载体pPIC9K/rtHsp70;在毕赤酵母Pichia pastoris KM71中表达重组罗非鱼的热休克蛋白70（rtHsp70）,对表达上清液进行SDS-PAGE及Western blot分析;采用亲和层析、HiTrap Desalting预装柱脱盐纯化目的蛋白。结果表明：克隆至pMD载体上的基因与目的基因完整编码区序列完全一致;诱导表达上清液经SDS-PAGE及Western blot分析,上清液中蛋白条带大小与目的蛋白相对分子质量大小一致;每升诱导上清液可以获得约2 mg纯化蛋白。本研究中获得的rtHsp70毕赤酵母工程菌及纯化的rtHsp70为后期rtHsp70-肽疫苗研究奠定了基础。     

马属抗菌肽hepcidin在酵母系统中的表达与鉴定

根据GenBank上马属抗菌肽hepcidin的氨基酸序列,设计并合成全长279 bp的hepcidin基因,将该基因插入pPICZαA中构建真核表达载体,PCR鉴定的阳性质粒电转化毕赤酵母菌X-33,用不同浓度Zeocin筛选高拷贝阳性重组菌,甲醇诱导表达,表达上清液经超滤膜纯化后,利用SDS-PAGE电泳和抑菌试验...     

茶树PPO融合蛋白真核表达载体的构建与表达

以迎霜品种茶树基因组为模板,利用高保真聚合酶pfu,通过PCR方法扩增得到茶树PPO基因的编码区序列。通过设计酶切引物将其成功融合至真核表达载体pPICZa中,构建出茶多酚氧化酶的融合蛋白真核表达载体pPICZa-PPO,并将其转化进入巴斯德毕赤酵母（Pichia pastoris）GS115中,成功筛选出多个阳性转化子。对阳性转化子进行甲醇诱导,采用Western-Blotting方法在培养液中成功检测到诱导表达的目的蛋白。酶活检测结果也显示诱导产物具有较高的相对酶活性,能够正确发挥酶蛋白的生物功能。     

猪圆环病毒Ⅱ型ORF2基因在酵母中的表达

为探索猪圆环病毒Ⅱ型(PCVⅡ) ORF2基因的高效表达,将ORF2基因整合到酵母表达载体pPICZαC中,构建重组质粒pPICZαC-ORF2,通过SacⅠ酶切线性化,经电穿孔法转到毕赤酵母菌SMD1168。经Zeocin^TM抗性筛选得到转化子,通过SDS-PAGE分析和Western blotting鉴定,结果表明衣壳蛋白在酵母菌中成功分泌表达。     

Management of the root-knot nematode Meloidogyne incognita on tomato with combinations of different biocontrol organisms

The nematicidal effect of Pseudomonas fluorescens, Paecilomyces lilacinus, Pichia guilliermondii and Calothrix parietina singly or in combination was tested against root-knot nematode, Meloidogyne incognita. Treatments with P. fluorescens and P. lilacinus caused mortality of M. incognita as 45% and 30% of juveniles after 48 h of exposures, respectively compared to water control in vitro. Under greenhouse conditions, all treatments reduced the disease severity and enhanced plant growth compared to untreated control. Application of P. fluorescens, P. lilacinus and P. guilliermondii Moh 10 was more effective compared to C. parietina. There was a negative interaction between C. parietina and either P. lilacinus or P. guilliermondii. Fresh and dry weight of shoots and roots of plants were significantly reduced as a result of infection with M. incognita, however application of biocontrol agents singly or in mix recovered this reduction. Moreover, they enhanced the growth parameters compared with the control. Our results proved that application of different biocontrol agents (P. fluorescens, P. lilacinus and P. guilliermondii) not only has a lethal effect on nematode, but also enhances the plant growth, supplying many nutritional elements and induction the systemic resistance in plants. Presence of C. parietina as a soil inhabitant cyanobacterium could antagonize biocontrol agents leading to the reduction of their practical efficiency in soil.     

Ethanol production from alkali- and ozone-treated cotton stalks using thermotolerant Pichia kudriavzevii HOP-1

In the present study, milled cotton stalks were subjected to alkali pretreatment with NaOH at 1-4% (w/v) concentrations at 121 °C for time ranging from 30 to 90 min. Ozone pretreatment was performed by passing 45 mg/L of ozone gas over 2 mm cotton stalks for 150 min at a flow rate of 0.37 L/min. The residual biomass from 4% alkali pretreatment for 60 min showed 46.6% lignin degradation accompanied by 83.2% increase in glucan content, compared with the untreated biomass. Hydrolysis of 4% alkali-treated and ozone-treated cotton stalks was conducted using enzyme combination of 20 filter paper cellulase units/gram dried substrate (FPU/g-ds), 45 IU/g-ds β-glucosidase and 15 IU/g-ds pectinase. Enzymatic hydrolysis of alkali-treated and ozone-treated biomass after 48 h resulted in 42.29 g/L glucose, 6.82 g/L xylose and 24.13 g/L glucose, 8.3 g/L xylose, respectively. About 99% of glucose was consumed in 24 h by Pichia kudriavzevii HOP-1 cells resulting in 19.82 g/L of ethanol from alkali-treated cotton stalks and 10.96 g/L of ethanol from ozone-treated cotton stalks. Simultaneous saccharification and fermentation of the alkali-treated cotton stalks after 12-h pre-hydrolysis resulted in ethanol concentration, ethanol yield on dry biomass basis and ethanol productivity of 19.48 g/L, 0.21 g/g and 0.41 g/L/h, respectively which holds promise for further scale-up studies. To the best of our knowledge, this is the first study employing SSF for ethanol production from cotton stalks.     

抗菌肽Cecropin AD在毕赤酵母中的组成型表达的研究

为了利用组成型表达方法在Pichia pastoris SMD1168中表达抗菌肽Cecropin AD,利用化学方法合成编码抗菌肽Cecropin AD的基因片段SH,将基因片段SH插入到载体pGAPZa-A中命名为pGAPZa-ASH,将pGAPZa-ASH电转至Pichia pastoris SMD1168中,Zecion抗性筛选阳性重组子。用Tricine-SDS-PAGE分析表达状况,琼脂扩散法检测蛋白的抑菌活性。结果表明:在3.0ku处有目的条带,并且表达产物抗菌肽Cecropin AD有生物活性。研究利用组成型表达方法在毕赤酵母菌株成功表达了抗菌肽Cecropin AD。组成型表达方法在本研究中成功的应用,为以后的实验研究奠定了理论基础。     

牛β-防御素BNBD5基因在毕赤酵母中的表达

本研究在本实验室成功构建的奶牛乳房炎抗性相关cDNA文库的基础上,旨在通过对牛β-防御素基因的分析,进一步揭示乳房炎抗性的分子机理.以牛β-防御素BNBD5基因作为乳房炎抗性的候选基因,通过克隆该基因的编码区,成功构建了毕赤酵母表达载体pPICZαA/BNBD5,并采用毕赤酵母表达系统表达了BNBD5蛋白.该结果为今后BNBD5抗体的制备奠定了基础.