柔嫩艾美耳球虫真核起始因子3d基因的酵母表达分析

在毕赤酵母真核表达系统中表达柔嫩艾美耳球虫真核起始因子3d(Emeria tenella eukaryotic initiation factor 3d,EteIF3d)基因,并获得具有天然构象的活性蛋白。将EteIF3d基因克隆到毕赤酵母表达载体pPIC9k上,将构建正确的重组质粒通过电激转化入酵母细胞GS115,用组氨酸缺陷培养基和G418分别进行筛选,获得含重组质粒的酵母表达细胞。在含1%甲醇的BMMY培养基中诱导培养,收集1~3 d的表达上清。表达产物进行SDS-PAGE检测,并用Western blot进行免疫学分析。结果表明,本研究成功构建了重组质粒pPIC9k-EteIF3d,并成功表达该蛋白,且该蛋白能与兔抗EteIF3d血清特异性结合,具有良好的抗原性。EteIF3d基因可在真核表达系统毕赤酵母中表达,且获得蛋白具有抗原性,从而为进一步研究该蛋白的结构和功能奠定了基础。     

枯草芽孢杆菌木聚糖酶Xyn B在毕赤酵母中的表达

编码枯草芽孢杆菌(Bacillus subtilis strain GD3b)木聚糖酶(Xylanse B,Xyn B)基因从重组载体pUC-XynB中切割,并克隆到pPICZa A载体中,经酶切和测序鉴定,成功构建了分泌型毕赤酵母表达载体pPICZa-Xyn B。载体经线性化后转入毕赤酵母X-33,并筛选获得可通过甲醇诱导分泌表达Xyn B重组蛋白的毕赤酵母工程菌X-33/Xyn B。     

兔防御素NP2基因克隆及其毕赤酵母表达的研究

根据兔防御素NP2基因序列和Pichia酵母密码偏爱性，设计兔防御素NP2基因序列片段，构建pGAPZαA酵母表达重组子，转化E．coli JM109，扩增重组子，经限制性内切酶BlnI线性化，转Pichia酵母。PCR扩增挑选阳性转化子，SDS-PAGE电泳分析兔防御素NP2酵母表达、Bradford法蛋白质定量和抗菌试验。结果表明：兔防御素NP2可在Pichia酵母中表达，且对大肠杆菌具有显著的抗菌作用，为兔防御素NP2深入研究和开发利用奠定了一定的物质和理论基础。     

类蜘蛛丝丝素蛋白SPF198在毕赤酵母中的分泌表达

将人工合成的长度为594 bp的类蜘蛛丝丝素蛋白spf198基因克隆到表达载体pP iczαA并导入毕赤酵母(Pichia pastoris)GS115细胞内,利用筛选到的Mut+(m ethanol utilization p lus)重组酵母株进行了表达。初步的研究结果显示,spf198基因在GS115酵母里可以正确表达。     

毕赤酵母Mut+和Muts重组子表达美洲商陆抗病毒蛋白基因的比较

 将N末端信号肽和C末端毒性区域缺失突变的美洲商陆抗病毒蛋白(pokeweed antiviral protein,PAP)基因表达载体pPIC9KP酶切线性化后,通过电击转化整合到巴氏毕赤酵母(Pichia pastoris) GS115菌株细胞中,PCR筛选出表型为利用甲醇快速型(Mut⁺)的重组子。在相同培养条件下比较Mut⁺重组子和利用甲醇缓慢型(Mut^s)重组子在表达缺失突变型PAP方面的异同。结果表明,诱导培养48 h后,Mut⁺重组子表达产物在SDS-PAGE胶上可见清晰目的带,而Mut^s重组子培养72 h才能见到微弱的目的带。抗病毒活性实验表明Mut⁺重组子和Mut^s重组子的表达产物均对TMV病毒侵染有明显的抑制作用,它们的抗病毒活性没有显著的差异。     

Effects of calcium on biocontrol activity of yeast antagonists against the postharvest fungal pathogen Rhizopus stolonifer

Calcium chloride (2% w/v) significantly inhibited the growth of the pathogen Rhizopus stolonifer , but did not affect the colony-forming units (CFU) of yeasts Candida guilliermondii and Pichia membranefaciens in potato dextrose broth. The concentration of yeast suspension influenced spore germination and germ tube growth of R. stolonifer in vitro, as well as disease incidence and lesion development in fruits. There were significant negative relationships between the suspension concentrations of the yeasts and the growth as well as infectivity of the pathogen. The addition of calcium resulted in lower spore germination rates and slower growth of germ tubes in vitro , as well as in lower disease incidences and smaller lesion diameters compared with treatments with yeast antagonists alone. When yeast cell suspensions reached a concentration of 5 × 10⁸ CFU mL⁻¹, growth of the pathogen was completely limited in vitro , and no infection was found in peach and nectarine fruits treated with or without calcium.     

Expression,Purification and Biological Activity Identification of Chicken Growth Hormone in Pichia pastoris

The growth hormone（GH）regulated diverse functions of the target cells through binding its receptor and played important roles in the process of metabolism of the body's growth and development.In order to obtain high purity chicken growth hormone（cGH）with biological activity,the gene segment of the mature cGH with six histidine at the C terminus was cloned into the expression vector pPIC9K.The recombinant plasmid（pPIC9K-cGH）was transformed to Pichia pastoris（GS115）by electroporation to construct the recombinant Pichia pastoris（GS115-pPIC9K-cGH).The GS115-pPIC9K-cGH secreted the recombinant protein（cGH）whose molecular weight was about 25 ku induced by 1% methanol induction for 96 h.The purity of cGH attained at least 95% through using Ni affinity chromatography and polyacrylamide glucan gel chromatography to purify the recombinant protein.The results of Real-time quantitative-PCR showed that the purified cGH recombinant proteins could up-regulate its target gene insulin-like growth factor Ⅰ（IGF-Ⅰ）mRNA in chicken liver cancer cell（LMH).It certified that the recombinant cGH protein had biological activity.This study laid a foundation for research of the structure,function and application of cGH.     

萝卜过氧化物酶基因RsPrx1在毕赤酵母中的表达及其抗性

从成熟心里美萝卜肉质根的皮中提取总RNA,采用RT-PCR方法获得过氧化物酶基因RsPrx1的cDNA,将其克隆到pGEM-T载体中,并构建毕赤酵母重组表达载体pPIC9KH-RsPrx1。用L iC l法转化野生型毕赤酵母GS115获得重组转化子。用不同浓度的NaC l和H2O2处理重组毕赤酵母转化子,结果表明该转化子具有一定的抗盐和抗氧化能力。建立了RsPrx1重组毕赤酵母表达体系,能够有效表达具有生物活性的外源过氧化物酶。     

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。     

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。