基于rDNA序列的酵母整合载体的构建及应用

目的构建一个基于rDNA序列的酵母整合载体。方法通过PCR扩增得到酿酒酵母rDNA序列;通过常规分子生物学技术构建以rDNA序列为整合位点的酵母整合载体。为了确证该载体的效能,将青蒿紫穗槐-4,11-二烯合酶基因克隆到该载体上,构建了含紫穗槐-4,11-二烯合酶基因的整合型酿酒酵母工程菌。提取该工程酵母的基因组,通过紫穗槐-4,11-二烯合酶基因的特异引物进行扩增,确认紫穗槐-4,11-二烯合酶基因是否已整合到酵母基因组上。发酵酵母工程菌,对发酵产物进行GC—MS检测,确认是否产生紫穗槐-4,11-二烯。结果构建了1个酵母整合载体,具有如下特点：①能将外源基因表达盒序列整合到酿酒酵母rDNA序列上,增加目的基因的拷贝数;②选择标记能反复应用,方便获得多拷贝的整合形式;③整合片段不需要酶切线性化,节省了内切酶,而且简化了操作程序。酵母基因组PCR结果表明,紫穗槐-4,11-二烯合酶基因已经整合到了酵母基因组上。以朱栾倍半萜为标准品,对该酿酒酵母工程菌的代谢产物进行GC—MS检测,发现其能产生紫穗槐-4,11-二烯,产量达到（91．33±7．57）mg／L朱栾倍半萜当量。结论初步成功构建了一个基于rDNA序列的酵母整合载体。     

化学发光法测定盐藻β-胡萝卜素清除自由基的能力

β-胡萝卜素是一类重要的天然抗氧化剂,以往多以脂溶性β-胡萝卜素(以反式结构为主)为研究对象,本研究以从杜氏盐藻中提取的纳米水溶性β-胡萝卜素(以顺式结构为主)为研究对象,利用化学发光法测定其清除自由基的能力,结果表明,纳米水溶性盐藻β-胡萝卜素具有较强的清除自由基的能力,其中对单线态氧的清除能力最强。     

小鼠Dectin-1胞外区的融合表达及其识别白念珠菌β-葡聚糖的研究

目的 克隆、表达并纯化小鼠腹腔巨噬细胞Dectin-1基因胞外区,探讨其识别并结合真菌β-葡聚糖的能力.方法 应用RT-PCR方法从小鼠腹腔巨噬细胞RNA中扩增Dectin-1基因胞外区,构建原核重组表达载体pET28-CRD,进行融合表达、纯化和复性.将融合蛋白与白念珠菌酵母相共同孵育,检测其识别、结合白念珠菌细胞壁β-葡聚糖的功能.结果 成功克隆并构建原核重组表达质粒pET28-CRD,表达并纯化了融合蛋白,该蛋白能识别、结合白念珠菌酵母细胞壁β-葡聚糖.结论 构建的原核重组表达载体能够在原核细胞内表达,表达产物有识别、结合真菌胞壁β-葡聚糖的功能,为进一步研究相应的真菌检测方法奠定了基础.     

卡介苗吸入对LPS致肺炎大鼠肺β-防御素mRNA的影响

目的探讨卡介苗（BCG）雾化吸入是否能增强肺炎大鼠活体内肺组织β-防御素-1、β-防御素-2的mRNA表达。方法采用RT-PCR方法研究肺炎时大鼠活体内β-防御素-1（RBD-1）、β-防御素-2（RBD-2）mRNA的表达，卡介苗雾化吸入时脂多糖所致肺炎大鼠活体内的肺组织RBD-1、RBD-2mRNA的表达。结果脂多糖（LPS）、卡介苗不能增强下呼吸道RBD-1mRNA的表达，但可增强RBD-2mRNA表达。结论通过卡介苗雾化吸入能增强机体表达内源性β-防御素-2，协助治疗呼吸道感染，其在诱导β-防御素方面的作用是一个值得进一步探索的研究领域。     

可溶性HLA-A2-肽组装复合物的研制及初步鉴定

目的:诱导表达HLA-A2的两个亚基-重链(HC)和轻链β2m,并将其纯化产物和特异性肽段结合为HLA-A2-肽复合体。方法:将HC工程菌和β2m工程菌经0.5 mmol/L IPTG诱导5 h。经超声破菌及专利技术处理获得精制包涵体,再复性、 超滤浓缩后,用DEAE Sepharose Fast Flow阴离子交换层析进行纯化。然后,将HC、β2m与两条特异性肽段分别结合,用Superdex 75凝胶过滤纯化,并用其天然结构的单克隆抗体(mAb)W6/32进行初步鉴定。结果:HC工程菌和β2m工程菌诱导表达的水平分别为43％和47％,HC和β2m纯化后的纯度均达到95％。两条特异性肽段与HC和β2m可组装成HLA-A2-肽复合体,纯化的HLA-A2-肽复合物在4℃可保存2个月左右,并可与mAb W6/32特异性结合。结论:HLA-A2的HC工程菌和轻链β2m工程菌均具有较高的表达水平,与相应的特异性肽段可形成稳定的可溶性HLA-A2-肽复合物,为进一步研究细胞毒性T淋巴细胞(CTL)的识别和应答奠定了基础。     

柔性肽串联四价Aβ15重组腺病毒的构建及纯化

目的构建并纯化含有柔性肽串联四价β-淀粉样蛋白（Aβ15）的重组腺病毒疫苗,用于老年性痴呆的预防和治疗。方法酶切获取带有IgGк轻链信号肽序列的柔性肽串联四价Aβ15基因,定向克隆于穿梭质粒pAdTrack-CMV中。通过与腺病毒骨架载体pAdeasy-1在细菌内同源重组形成重组腺病毒质粒pAd-4×Aβ15,转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒（Ad-4×Aβ15）。Western检测4×Aβ15在培养液中的分泌表达。氯化铯密度梯度离心纯化重组腺病毒Ad-4×Aβ15。结果 pAdTrack-4×Aβ15测序结果和预期一致,同源重组并经293细胞包装后形成重组腺病毒Ad-4×Aβ15,western检测4×Aβ15在培养上清中表达。经氯化铯密度梯度离心纯化后Ad-4×Aβ15病毒颗粒数约为4.6×1012 VP/mL。结论成功制备了重组腺病毒Ad-4×Aβ15,感染细胞证实其呈分泌表达,经氯化铯密度梯度离心纯化后得到高滴度的重组腺病毒。为老年性痴呆（Alzheimer’s disease,AD）的实验治疗打下基础。     

红色荧光蛋白在酿酒酵母中的表达特性研究

目的研究红色荧光蛋白编码基因Dsred在酿酒酵母菌中的快速克隆与表达。方法根据已发表基因序列设计引物,采用连续重叠PCR方法快速克隆获得全长Dsred基因,将其与pMD-18T载体连接后进行测序鉴定。通过In-fusion方法将鉴定正确的pMD-Dsred重组载体与酿酒酵母表达载体pYeDP60进行连接,测序后利用LiAc方法将鉴定正确的pYeDP60-Dsred重组表达载体转化至酿酒酵母菌W303-1B中,PCR扩增筛选阳性克隆,获得的W303B[pYeDP60-Dsred]工程菌经诱导培养后进行SDS-PAGE电泳分析和绿光激发荧光成像检测。将工程菌分别接种至YPD、YPG、SCG和SCD培养基,培养48、72、96、120和144h后分别测定吸光度(A600)值。取诱导表达后的工程菌(菌液组)进行离心(菌体组)、离心后加甘油(高渗组)处理,分别置于–70、–20、4、28和37℃条件培养,荧光显微镜观察其表达特性。结果连续重叠PCR扩增和测序鉴定结果表明,扩增获得的Dsred基因长为678bp,其序列与已发表的基因序列完全一致;Dsred基因已成功插入pYeDP60-Dsred重组表达载体,且受酵母诱导型启动子GAL10-CYC1调控表达。PCR扩增筛选和SDS-PAGE分析显示,pYeDP60-Dsred重组表达载体已成功导入酿酒酵母菌中,诱导表达产物相对分子质量与预期相符,且诱导后工程菌可在绿光激发下发出红色荧光。4种培养基内工程菌的菌体生长情况无明显差别,重组Dsred红色荧光蛋白表达不会抑制酿酒酵母菌体生长。荧光显微镜观察显示,工程菌经不同处理后,高渗组红色荧光蛋白成熟时间最短,菌液组时间最长;红色荧光蛋白在37℃条件下培养时成熟最早,但降解速度较快。结论成功构建了pYeDP60-Dsred重组酿酒酵母表达载体,实现了Dsred基因在酿酒酵母菌体内的异源表达。红色荧光蛋白表达对酿酒酵母菌体生长无明显影响,且离心保留菌体和加入甘油等缺水高渗处理都有利于红色荧光蛋白的成熟。     

β-1,4半乳糖基转移酶-Ⅰ在钳夹伤后大鼠坐骨神经中表达的变化

为了观察β-1，4-半乳糖基转移酶-Ⅰ（β-1，4-galactosyltransferase-Ⅰ，β-1，4-GalT-Ⅰ）在正常和钳夹伤后大鼠坐骨神经中表达的变化，本研究采用RT—PCR方法，从小鼠坐骨神经中特异性地扩增β-1，4-GalT-ⅠcDNA片段并将其克隆到pGEM-T载体。采用体外转录的方法合成地高辛标记的正、反义β-1，4-GalT-ⅠRNA探针。通过原位杂交和图像分析的方法，观察β-1，4-GalT-ⅠmRNA在正常和夹伤大鼠坐骨神经中的表达及其变化。结果表明：β-1，4-GalT-Ⅰ mRNA在大鼠坐骨神经的髓鞘中表达，在夹伤坐骨神经后1-2d，β-1，4-GalTⅠmRNA在坐骨神经的表达最高，于夹伤后1周开始下降，在夹伤后1个月恢复至正常水平。上述结果提示坐骨神经夹伤后β-1，4-GalTⅠmRNA的表达发生变化，并且主要表达在坐骨神经的Schwann细胞。本研究结果为进一步分析β-1，4-GalT-Ⅰ在周围神经再生中的调控机制奠定了基础。     

Ad-BMP-2和Ad-TGF-β1共转染人退变椎间盘髓核细胞对细胞外基质代谢的影响

目的探讨骨形态发生蛋白腺病毒表达载体（Ad—BMP-2）和转化生长因子-β1腺病毒表达载体（Ad—TGF—β1）共转染人退变椎间盘髓核细胞后对aggrecan和Ⅱ型胶原合成的影响。方法人退变椎间盘髓核细胞体外分离培养至第2代。为检测转入目的基因后蛋白表达情况,将其分为空白对照组、目的基因组和共转染组,细胞分别转染Ad—BMP-2（50MOI）、Ad-TGF-β1（50 MOI）及共转染（各50 MOI）,再用Western blot方法检测转染后细胞目的蛋白的表达;为探讨目的基因对细胞外基质代谢的影响,将研究对象分为空白对照组（A组）、Ad—BMP-2100 MOI（B组）、Ad-TGF—β1100 MOI（C组）、Ad-BMP-2和Ad-TGF—β1各50 MOI（D组）,用ELISA法分别检测转染第3天和第6天细胞上清液中aggrecan和Ⅱ型胶原的蛋白表达量。RT-PCR检测第6天髓核细胞内Ⅱ型胶原和aggrecan的mRNA的表达水平。应用方差分析比较组间差异,以P〈0．05表示差异有统计学意义。结果在转染目的基因载体后,目的蛋白表达增加。在转染后第3天和第6天各转染组细胞培养基中aggrecan和Ⅱ型胶原蛋白表达量高于对照组（P〈0．01）,与转染单个基因组相比,共转染组的aggrecan和Ⅱ型胶原蛋白表达量明显增加（P〈0．01）;并且在转染后第6天共转染组髓核细胞内aggrecan和Ⅱ型胶原mRNA表达也明显增加（P〈0．01）。结论转染Ad-BMP-2、Ad-TGF-β1能促进aggrecan和Ⅱ型胶原合成,同时上调aggrecan和Ⅱ型胶原mRNA的表达;共转染Ad-BMP-2和Ad-TGF-β1对细胞外基质的mRNA和蛋白合成有协同作用。     

17β-雌二醇对培养的乳鼠心肌细胞肥大的影响

目的观测17β-雌二醇（17β-estradiol，E2）对肾上腺素（PE）诱导的乳鼠心肌细胞肥大的影响并探讨其机制。方法以培养的乳鼠心肌细胞为模型并分组给药，用计算机图像分析软件测量心肌细胞表面积，[^3H]标记亮氨酸掺入法测定心肌细胞蛋白质合成速率，免疫细胞化学方法检测心肌细胞原癌基因c-fos蛋白表达，半定量RT-PCR检测心肌细胞肥大特征性胚胎型基因β-肌球蛋白重链（pMHC）、α-骨骼肌肌动蛋白（α-skA）和心房肽（ANP）的mRNA表达。结果17β-雌二醇明显抑制肾上腺素诱导的心肌细胞表面积和蛋白质合成速率的增加；17β-雌二醇减弱肥大心肌细胞c-fos蛋白表达；17β-雌二醇降低pMHC、α-skA的mRNA表达，但增加ANPmRNA表达。结论17β-雌二醇可抑制心肌细胞肥大，其作用机制可能与抑制原癌基因c-fos的蛋白表达，逆转收缩蛋白基因（β-MHC和α-skA）向胚胎型转化及促进ANPmRNA表达有关。     

Characterization of yeasts with high L[+]-lactic acid production: lactic acid specific soft-agar overlay (LASSO) and TAFE-patterns

Only few yeast strains are known for the high level production of L[+]-lactate. We report indications for the conspecifity of Kluyveromyces thermotolerans (formerly Saccharomyces veronae) strain CBS 4728 with Stamm 42 (formerly Saccharomyces pretoriensis, RADLER 1984). We suggest that Stamm 42 has little, if any relationship to Saccharomyces cerevisiae. Furthermore, we have optimized the method of Subden et al. (1982) for the detection of lactate producing microorganisms. Using this method in a screening with 100 yeast strains of our institute collection, we could not find additional strains with high L[+]-lactate production. This method may provide a useful tool for the molecular cloning of the unique yeast L[+]-LDH1) gene (s).     

鼠疫耶尔森氏菌F1抗原在酿酒酵母中的表达及鉴定

目的：构建含鼠疫杆菌F1抗原结构基因caf1的重组载体，并在酿酒酵母中表达。方法：将鼠疫杆菌的F1抗原结构基因caf1插入到pHSS6-mTn-3xHA／locz质粒中，构建重组载体pHSS6-mTn-3xHA／locz—caf1。将重组载体经NotⅠ酶切后，以醋酸锂法转化酿酒酵母，将转化的酿酒酵母接种于SCUra平板上筛选阳性克隆，表达产物用SDS—PAGE和Western blot进行鉴定。结果：经SDS—PAGE鉴定和Western blot分析表明，转化的酿酒酵母可表达鼠疫杆菌F1表面抗原蛋白。结论：成功地构建了重组载体pHSS6-mTn-3xHA／lacZ—caf1，并在酿酒酵母中稳定表达鼠疫杆菌F1表面抗原，为制备可经消化道途径免疫的鼠疫杆菌基因疫苗奠定了基础。     

Towards diacetyl-less brewers' yeast. Influence of ilv2 and ilv5 mutations

During alcoholic fermentations, the off-flavour compound diacetyl is formed non-enzymatically from acetolactate leaking out from the cells. Acetolactate is an intermediate in the biosynthesis of valine. In beer fermentation, the amount of diacetyl is reduced to acceptable levels during maturation. A reduction of the time needed for maturation may be achieved by the use of a brewing yeast that produces less diacetyl. Saccharomyces cerevisiae laboratory strains with an inactive ilv2 gene can not form acetolactate, while ilv5 strains, blocked in the subsequent step, leak acetolactate in high amounts. Induction of recessive mutations in production strains of Saccharomyces carlsbergensis has not yet been achieved, as the yeast is polyploid and possibly a hybrid between S. cerevisiae and another Saccharomyces species. Thus, all chromosomes investigated so far are present in at least two genetically different versions. Genetic and molecular analysis has shown that the brewing yeast is structurally heterozygous for ILV2 and ILV5. Genetic modification of brewers' yeast to reduce diacetyl formation is being carried out by mutation of ILV2. Deletion mutations in both ILV2 alleles have been constructed in vitro to be used for gene replacement in the brewing strain. In addition, partial inactivation of the ILV2 function is carried out by selecting spontaneous dominant mutations resistant to the herbicide sulfometuron methyl. Among these mutants some produce only half the amount of diacetyl compared to the parental strain. An alternative way to reduce diacetyl production might be to increase the activity of the ILV5 gene product. Model experiments in S. cerevisiae show that the presence of the ILV5 gene on a 2-micron based multi-copy vector can reduce the diacetyl production by half.     

Comparative genomic hybridization provides new insights into the molecular taxonomy of the Saccharomyces sensu stricto complex

The science of taxonomy is constantly improving as new techniques are developed. Current practice is to construct phylogenetic trees based on the analysis of the DNA sequence of single genes, or parts of single genes. However, this approach has recently been brought into question as several tree topologies may be produced for the same clade when the sequences for various different genes are used. The availability of complete genome sequences for several organisms has seen the adoption of microarray technology to construct molecular phylogenies of bacteria, based on all of the genes. Similar techniques have been used to reveal the relationships between different strains of the yeast Saccharomyces cerevisiae. We have exploited microarray technology to construct a molecular phylogeny for the Saccharomyces sensu stricto complex of yeast species, which is based on all of the protein-encoding genes revealed by the complete genome sequence of the paradigmatic species, S. cerevisiae. We also analyze different strains of S. cerevisiae itself, as well as the putative species S. boulardii. We show that in addition to the phylogeny produced, we can identify and analyze individual ORF traits and interpret the results to give a detailed explanation of evolutionary events underlying the phylogeny.     

Genetic effects of PR toxin in eukaryotic microorganisms.

The genetic activity of PR toxin, a mycotoxin from Penicillium roqueforti, was studied in Saccharomyces cerevisiae and Neurospora crassa. The results show that PR toxin, without enzymic activation, causes gene conversion in S. cerevisiae strains D4 and D7, reverse mutation in S. cerevisiae strain D7 and N. crassa strain N24, and mitotic crossing-over in S. cerevisiae strain D7, In the log phase cells of S. cerevisiae the effects are more pronounced at alkaline than at acid pH. The active site responsible for the genetic activity and toxicity is known. The carbonyl groups appear to play an important role in the biologic activity of this molecule.     

Genetic effects of pr toxin in eukaryotic microorganisms

The genetic activity of PR toxin, a mycotoxin from Penicillium roqueforti, was studied in Saccharomyces cerevisiae and Neurospora crassa. The results show that PR toxin, without enzymic activation, causes gene conversion in S. cerevisiae strains D4 and D7, reverse mutation in S. cerevisiae strain D7 and N. crassa strain N24, and mitotic crossing-over in S. cerevisiae strain D7, In the log phase cells of S. cerevisiae the effects are more pronounced at alkaline than at acid pH. The active site responsible for the genetic activity and toxicity is known. The carbonyl groups appear to play an important role in the biologic activity of this molecule.     

Promoter strength of folic acid synthesis genes affects sulfa drug resistance in Saccharomyces cerevisiae

The enzyme dihydropteroate synthase (DHPS) is an important target for sulfa drugs in both prokaryotic and eukaryotic microbes. However, the understanding of DHPS function and the action of antifolates in eukaryotes has been limited due to technical difficulties and the complexity of DHPS being a part of a bifunctional or trifunctional protein that comprises the upstream enzymes involved in folic acid synthesis (FAS). Here, yeast strains have been constructed to study the effects of FOL1 expression on growth and sulfa drug resistance. A DHPS knockout yeast strain was complemented by yeast vectors expressing the FOL1 gene under the control of promoters of different strengths. An inverse relationship was observed between the growth rate of the strains and FOL1 expression levels. The use of stronger promoters to drive FOL1 expression led to increased sulfamethoxazole resistance when para-aminobenzoic acid (pABA) levels were elevated. However, high FOL1 expression levels resulted in increased susceptibility to sulfamethoxazole in pABA free media. These data suggest that up-regulation of FOL1 expression can lead to sulfa drug resistance in Saccharomyces cerevisiae.     

Analysis of the Saccharomyces cerevisiae pan-genome reveals a pool of copy number variants distributed in diverse yeast strains from differing industrial environments

Although the budding yeast Saccharomyces cerevisiae is arguably one of the most well-studied organisms on earth, the genome-wide variation within this species--i.e., its "pan-genome"--has been less explored. We created a multispecies microarray platform containing probes covering the genomes of several Saccharomyces species: S. cerevisiae, including regions not found in the standard laboratory S288c strain, as well as the mitochondrial and 2-μm circle genomes-plus S. paradoxus, S. mikatae, S. kudriavzevii, S. uvarum, S. kluyveri, and S. castellii. We performed array-Comparative Genomic Hybridization (aCGH) on 83 different S. cerevisiae strains collected across a wide range of habitats; of these, 69 were commercial wine strains, while the remaining 14 were from a diverse set of other industrial and natural environments. We observed interspecific hybridization events, introgression events, and pervasive copy number variation (CNV) in all but a few of the strains. These CNVs were distributed throughout the strains such that they did not produce any clear phylogeny, suggesting extensive mating in both industrial and wild strains. To validate our results and to determine whether apparently similar introgressions and CNVs were identical by descent or recurrent, we also performed whole-genome sequencing on nine of these strains. These data may help pinpoint genomic regions involved in adaptation to different industrial milieus, as well as shed light on the course of domestication of S. cerevisiae.     

Protein extracts for nutritional purposes from fragile strains of Saccharomyces cerevisiae: construction of strains and conditions for lysis

A superfragile strain, Saccharomyces cerevisiae 211, carrying three nonallelic nuclear determinants of cell lysis by osmotic shock was isolated from the haploid progeny of a cross between two laboratory fragile strains VY 1160 and SY 15. The strain Sacch. cerevisiae 211 is prototrophic, grows well in both laboratory and industrial media, only when supplemented with osmotic stabilizers. The average protein content of its biomass after growth in a molasses-based nutritional medium is 48.7%. In laboratory conditions this strain releases about 60% of its cellular proteins by spontaneous lysis in water. However, on a pilot scale the protein yield decreases to about 25%. After a single disintegration step at 650 atm, practically all of the cellular protein--91.5%, is extracted without any additional chemical treatment. This result cannot be reached in any of the wild type strains tested even after triple treatment at 650 atm.