凯氏定氮法测定啤酒酵母水溶性多糖中蛋白质含量

从啤酒酵母中提取出水溶性粗多糖SA,进一步纯化得SA1,采用凯氏定氮法,经消化、蒸馏、滴定后计算其蛋白质含量,分别为7.36%和2.45%.     

Cloning， expression and characterization of a nucleoside diphosphate kinase (NDPK) gene from tobacco

Nucleoside diphosphate kinase (NDPK) is a housekeeping enzyme that maintains the intracellular levels of all (d)NTPs used in biosynthesis except ATP.Here we report that a full-length cDNA encoding nucleoside diphosphate kinase (NDPK) was cloned us- ing yeast two-hybrid approach.A tobacco NDPK gene was obtained and designated as NtNDPK1.NtNDPK1 is 704 bp in length and en- codes a putative 16.2 kD protein of 148 amino acids.Phylogenic analysis showed that NtNDPK1 is highly homologous to other plant NDPK genes and identified as type I (NDPK1).RNA-gel blot analysis showed that there was no significant difference of NtNDPK1 ex- pression in roots,stems,leaves and buds.And expression of NtNDPK1 was induced by ABA and PEG and repressed by NaCl,but not significantly affected by Paraquat,wounding and low temperature (4℃) treatments,indicating that NtNDPK1 may play a certain role in response to abiotic stress.In vitro phosphorylation assay demonstrated that NtNDPK1 had autophosphoryLation activity.     

Cloning and characterization of a new actin gene from Oryza sativa L.

Using Rho family member osRACD as bait, a new member of actin gene family -Act was isolated from Oryza sativa by yeast two-hybrid system. The full-length cDNA was cloned with 5' RACE technology, which contains an open reading frame of 1134 bp with a predicted protein of 377 amino acids. Sequence alignment revealed 96% to 81.8% identities with some known actin proteins in plants. The method of bioinformatics was used to analyze the protein modification sites, structure and evolution of the gene. Southern blot analysis showed that Act is a single-copy gene in the genome. The result of RT-PCR showed it is ubiquitously expressed in root, shoot, callus and panicle in a temporal fashion. The relationship between Rho family and actin family in evolution and function was also studied.     

细胞不对称分裂机制--芽殖酵母接合型不对称转换

芽列酵母的母细胞与子细胞呈不对称接合型转换，其原因是只有母细胞可表达编码核酸内切酶的基因HO，使相反接合型的缄默基因转位到活动位点取代了原来的接合型基因。HO的不对称表达是因在细胞分裂的末期至G1早期，子细胞核中存在有Ashlp转录抑制因子。Ashlp的不对称分布是由其mRNA的定向转运而实现的：ASH1 mRNA在有丝分裂期被转录出之后，通过接头蛋白She2p和She3p与肌球蛋白Myo4p结合成核糖核蛋白颗粒，经肌动蛋白纤维转运到子细胞远端皮层而锚定并翻译。     

酒曲根霉糖化性质的研究

研究了Rhizopus34产生糖化酶、液化酶、蛋白酶的最佳培养条件，利用Rhizopus34制曲的最佳培养时间为3～4d；在制曲过程中添加5％～10％的玉米粉能显著提高3种酶的活性；制曲和糖化过程中产生3种酶的最佳pH为5．8；3种酶的最佳作用pH值范围为4．6—5．2。     

hTERT主要HLA-A2.1相关基因的表达

为了研究hTERT的表达特性及意义。根据hTERT基因中与HLA-A2.1分子相关性较强的几个9mer肽段合成引物，通过RT-PCR技术分别对14例RCC组织和2例肿瘤细胞株进行了扩增，扩增片段大小为306bp(小片段）和1002bp(大片段），结果有13例癌肿组织表达hTERT基因小片段，1例组织不表达hTERT基因；Hela细胞和786-0肾腺癌细胞株均表达hTERT；对大片段进行克隆，酵母表达，构建了pPICZαA-hTERT重组表达载体和Pichia pastorisX33/hTERT酵母工程菌；SDS-PAGE电泳显示毕赤酵母能分泌表达39kD的目标蛋白。得出，大部分RCC肿瘤组织和细胞表达hTERTmRNA，而正常组织则不表达，提示hTERT可作为通用的抗肿瘤治疗尤其是免疫治疗的靶点。     

酵母甘露聚糖乙酰解产物的制备和分析

从啤酒酵母(Saccnaromyces Cerevisiae)中提取甘露聚糖Sp_1。将Sp_1进行选择性(2.5hr,5hr)乙酰解反应,经冷冻干燥得乙酰解产物Sp_1 2.5和Sp_1 5。通过Sephadex G-50柱层析、醋酸纤维薄膜电泳、红外光谱法、比旋光度测定等方法分析其理化性质。Sp_1 2.5和Sp_1 5在3400cm~(-1)、2900cm~(-1)、2300cm~(-1)、1600cm~(-1)、1000cm~(-1)左右有多糖的特征吸收峰。     

Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system on the human tumor cell growth

Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the sensitivity of chemotherapy and radiotherapy. E1A have the ability to integrate into the host genome, resulting in long-time expression that induces Rb gene inactivation and animal cells immortalization. This prompted us to select the E1A protein for treatment of cancer in order to overcome the limitations of E1A gene therapy. Thus, we firstly constructed E1A eucaryotic expression vector (pPIC9/E1A), transformated the pichia pastoris yeast cells (GS115) and screened the high-expressing recombinant strains. The positive yeast strains were cultured in the shake flask, and induced for 3 d. The crude E1A protein was purified using two steps of column chromatography on HiTrap Q and HiTrap SP. The purified E1A protein was identified by SDS-PAGE and Western blot. E1A protein was mostly located at cellular nuclear when Chariot delivered E1A protein into cells. The analysis in vitro indicated that the E1A protein arrested LN686 cell cycle at G2/M phase, and significantly inhibited the growth of LN686 tumor cells. The current studies firstly provided an experimental basis to further develop E1A protein for tumor treatment.     

蛋白体外结合实验联合质谱分析鉴定 Num1 的互作蛋白

目的: 为了解析芽殖酵母 Num1 蛋白在纺锤体定位和线粒体中的调节机制．方法: 利用大肠杆菌原核表达 并纯化了在 Num1 功能中起核心作用的补丁组装(PA) 结构域的重组蛋白, 通过蛋白体外结合实验(Pull-down) 分 离了酵母细胞中与 PA 结构域相结合的蛋白复合物,并对其进行了质谱分析．结果: 鉴定了一系列新的 Num1 互作蛋 白,包括核糖体蛋白,参与蛋白折叠、分配及转运的内质网和高尔基复合体蛋白, 参与基因转录和翻译的核酸酶和 蛋白酶,及其他功能的蛋白．结论: Num1 的互作蛋白的鉴定为进一步研究 Num1 的作用机制奠定了基础.     

酵母胞外槐糖脂产生条件优化及其抑菌作用

通过单因子试验和正交试验对筛选得到的1株未见报道的产胞外槐糖脂的酵母菌Y2A菌株的培养基组成及培养条件进行了优化,结果表明,优化的最适产槐糖脂培养基配方为：质量分数6．0％葡萄糖、体积分数8．0％菜子油、质量分数0．2％酵母粉;优化后发酵条件为：初始pH值6．0、接种量体积分数2．5％、发酵时间120h,在此条件下,槐糖脂产量达到39．4g/L,然后发酵液经分离纯化并制备,得到纯品槐糖脂,将槐糖脂进行抑菌试验的研究结果表明,它对某些试验菌株的生长有抑制作用。