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。     

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。     

荣昌猪IFN-ω基因毕赤酵母表达载体的构建与表达

将荣昌猪IFN-ω基因亚克隆到酵母表达载体pPICZαB后,氯化锂法转化毕赤酵母菌株X-33,通过酶切、PCR鉴定表明成功构建了表达荣昌猪IFN-ω基因的酵母基因工程菌。经甲醇诱导后,通过SDS-PAGE和Western blotting检测,表明荣昌猪IFN-ω基因在毕赤酵母中实现了高效表达。表达产物在MDBK细胞上抗VSV的比活性为1.21×106U/mg,表明具有抗病毒活性。     

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.     

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。     

Exendin-4基因的克隆及其在毕赤酵母中的表达

根据GenBank中已公布的编号为AAB2200的Exendin-4氨基酸片断,及酵母密码子偏好性,设计了Exendin-4在酵母中的表达序列。利用基因工程技术,将编码Exendin-4的蛋白基因亚克隆到含有分泌信号肽序列的毕赤酵母表达载体pPIC9K中,构建成分泌型重组表达栽体pPIC9K-E4。用电转化法将线性化的pPIC9K-E4转化入毕赤酵母菌株GSll5,转化子经MD平板筛选,得到的阳性菌株再用浓度梯度递增的G418筛选多拷贝重组子。将MM和MD平板筛选得到Muts菌株用甲醇诱导表达6d,每隔24h取样。Tricine-SDS-PAGE检测结果显示,诱导后第6天表达量达到最高。     

Neosartorya fischeri P1来源的外切聚半乳糖醛酸酶异源表达及性质研究

聚半乳糖醛酸酶在食品和饲料行业具有很大的应用价值,发现新的聚半乳糖醛酸酶不仅能够补充该酶的相关信息,还能为工业应用提供更多选择。从一株嗜热费希新萨托菌(Neosartorya fischeri P1)的基因组中克隆得到一个外切聚半乳糖醛酸酶基因(Nf-pg),将Nf-pg在毕赤酵母GS115中进行异源表达,重组酶Nf-PG纯化后进行性质测定。Nf-PG在p H 4.5和55℃时活性最高,在p H 2.0~9.0具有较好的稳定性,50℃热处理60 min仍能保持80%的剩余酶活;去除N-糖基化的酶蛋白Nf-PG-D最适温度为50℃,热稳定性降低,最适p H和酸碱稳定性无明显改变。产物分析证明Nf-PG是严格的外切聚半乳糖醛酸酶,优异的性质使其成为食品加工行业的良好选择。     

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.     

外源基因在甲醇酵母Pichia pastoris中的表达策略

甲醇酵母 Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ表达系统是近几年发展起来的一个真核表达系统,在其乙醇氧化酶 ＡＯＸ１基因启动子的控制下,某些外源基因（如 ＴＮＦ、ＥＧＦ等）的表达量可达克每升以上。虽然该系统是一种优良的表达系统,但有许多因素影响外源基因在其中的表达,包括外源基因的拷贝数及整合到酵母染色体的位置和方式、ｍＲＮＡ的５’和３’非翻译区（ＵＴＲ）、转录起始密码子的上下文、外源基因Ａ＋Ｔ的组成与转录和翻译的阻断、密码子的偏爱性、信号肽的特性、产物的稳定性、宿主菌的生理特性、培养基成分和培养条件等。在应用该表达系统过程中,这些因素都需加以考虑。本文将就这些因素作一概述和讨论,以有利于提高外原基因在该系统中的表达水平。     

杂合抗菌肽Mel-MytB在毕赤酵母中的表达及其抗菌活性测定

为了获得抗菌活性较强的抗菌肽,将蜂毒肽（Melittin）与贻贝素B （Mytilin-B）的核心功能序列杂合,以Melittin （3-14）和Mytilin-B （13-27）的成熟肽段作为模板序列,根据巴斯德毕赤酵母（Pichia pastoris）偏好密码子,采用SOE方法合成杂合抗菌肽Mel-MytB（MEM）基因。改造后的基因克隆到pPICZα-A质粒,构建分泌型重组酵母表达载体pPICZα-A-MEM,而后经Sac Ⅰ酶切线性化后电转入毕赤酵母受体菌X-33。结果表明,在醇氧化酶（AOX）启动子调控下,分子质量约3.0 ku的Mel-MytB杂合抗菌肽获得表达,具有热稳定性和酸稳定性,煮沸40 min、pH 2.0~10.0范围内抑菌活性基本不变。抗菌特性研究结果表明,该表达产物具有广谱抗菌活性,其对大肠杆菌ATCC25922、金黄色葡萄球菌ATCC25923/ATCC6538、肠道沙门氏菌ATCC13076、鼠伤寒沙门氏菌ATCC14028、副溶血弧菌ATCC17802、创伤弧菌ATCC27562、枯草芽孢杆菌ATCC6633的MIC值分别为5.1、2.2、2.0、4.6、7.6、9.4、13.2和59.4 μg/mL。因此,重组抗菌肽Mel-MytB在疾病防治和动物饲料添加剂等方面具备较好的应用前景。