表达乙肝病毒融合表面抗原基因SA-28的二倍体酵母工程菌的选育

将乙肝病毒融合表达抗原基因ＳＡ－２８的单倍体酵母工程菌Ｙ１９／ＹＦＤ１５８和与其不同接合型的单倍体酵母菌Ｙ９５接合,筛以二倍体酵母工程菌Ｙ９５ｘＹ１９／ＹＦＤ１５８。对两种工程菌的研究表明：三倍体工程菌发酵密度为单倍休工程菌的３倍;表达质粒在二倍体酵母中的稳定性明显高于单倍体工程菌;二倍体工程菌对融合抗原的表达量为单倍体的３倍以上,表达质粒在二倍体细菌中的平均拷贝数略低于单倍体工程菌。     

基因拷贝数与染色体位置对酵母表达乙肝病毒融合表面抗原SA-28基因的影响

应用ＦＬＰ重组酶介导的染色体定点整合技术,将带有不同拷贝数的乙肝病毒融合表面抗原ＳＡ－２８基因表达单元的质粒整合在酵母不同的染色体位点,并测定了ＳＡ－２８基因的表达情况,初步研究了基因拷贝数与染色体位置对酵母表达外源基因的影响。结果表明ＳＡ－２８基因在ＨＩＳ３位点整 合时的表达水平随基因拷贝数的增加而提高,遵循基因剂量效应;在某些染色体位点整２合时,插入方向对其表达有不同程度的影响,呈现出明显的染     

三角酵母二倍体菌株的选育

由三角酵母(Trigonopsis variabilis)原生质体融合,获得了35株原养型融合子。通过对其中四株进行详绌分析表明,融合子细胞体积和DNA含量为两亲株之和,苏木精染色显示单核,这些结果证明融合子为亲株的二倍体。此外,融合子的生长速度、D-氨基酸氧化酶以及细胞蛋白质含量均明显高于亲株。电镜形态观察：进一步证明融合子细胞大于亲株。     

乙肝病毒表面抗原基因在人参细胞中的表达

为获得表达乙肝病毒表面抗原(HBsAg)的人参细胞系,构建携带HBsAg基因的植物细胞表达载体pBIBSa,采用以农杆菌LBA4404感染的方法,经G418筛选后得到13株具有抗性的人参细胞.提取基因组DNA进行PCR反应,其中8株得到约700bp的HBsAg基因片段;提取mRNA进行RT-PCR反应,其中6株得到约700bp的HBsAg基因片段;用ELISA方法检测HBsAg,6株均为阳性.每克人参细胞中最高含HBsAg184 ng,占细胞可溶性蛋白的0.009%.免疫组化切片染色显示HBsAg主要分布在细胞膜上,少量位于细胞核中.这些结果表明人参细胞整合了HBsAg基因,并能稳定表达HBsAg.     

乙肝病毒表面抗原基因在胡萝卜中的克隆及表达

构建HBV表达抗原（HBsAg)植物表达载体并在胡萝卜植株中表达。采用自行构建adr亚型HBsAg基因克隆T-adr,再次酶切获得860bp含PreS2的HBsAg基因片段,将其插入到植物表达载体pBPC55,新质粒命名为pBPC91adr。将其与含除草剂抗性基因及GUS蛋白基因的筛选质粒pBPC93共同经基因枪（PDS-1000/He)转化胡萝卜悬浮细胞,经含除草剂（Biolaphos)的培养基筛选及植物激素诱导分化,获得除草剂抗性胡萝卜幼苗,结果为转化后8周,自胡萝卜细胞中分化出除草剂抗性胡萝卜幼苗,提取新分化幼苗总DNA,特异性引物PCR扩增后可见860bp扩增带;Southern-Blot证明有HBsAg基因整合,胡萝卜蛋白萃取物的Western-Blot及ELISA检测证实有HBsAg蛋白表达。利用基因枪转化使质粒pBPC91adr中HBsAg基因在胡萝卜幼苗内整合并表达,提示以植物生产疫苗具有可行性。     

乙肝病毒表面抗原preS1与人肿瘤坏死因子α融合基因的表达

用ＰＣＲ法获得了ＨＢｓＡｇｐｒｅＳ１（１－６５）肽段基因,将该基因融合在肿瘤坏死因子（ｈＴＮＦα）之后,插入表达载体ＰＳＢ－９２中,使融合基因的５′端直接置于大肠肝菌ＰＬ启动子下游,采用３０℃培养,４２℃诱导,获得了ＴＮＦ与ｐｒｅＳ１（１－６５）融合蛋白的表达产物。ＳＤＳ－ＰＡＧＥ电泳显示表达产物为２５ｋＤ,约占细菌总蛋白的３５％。表达产物经Ｗｅｓｔｅｒｎｂｌｏｔ验证,能分别特异地与ｈＴＮＦα抗体与ｐｒｅＳ１抗体结合,稀释复性后,该融合蛋白还具有ＴＮＦ的生理功能（对Ｌ９２９细胞的细胞毒活性）。经ＤＮＡ序列测定,ｐｒｅＳ１（１－６５）肽基因正确地融合在ｈＴＮＦα基因之后。该结果提供了一种制备ｐｒｅＳ１的新方法,为进一步开展治疗肝癌和乙肝的导向药物打下基础。     

乙肝病毒表面抗原持续表达的新致病机制

乙型肝炎表面抗原(HBsAg)持续阳性是控制乙肝中难以解决的重大问题。本研究通过揭示HBsAg致病机制的基础研究,寻找抑制或清除HBsAg的新途径。通过建立有可比性的HBsAg转基因鼠和稳定表达细胞系及相应对照,进行比较转录组学和蛋白质组学研究,发现了HBsAg在HBV慢性感染中的一些新致病机制。其中包括:HBsAg促进肝细胞内CypA分泌,后者可趋化炎症细胞在HBsAg阳性灶周围浸润;在细胞模型中,HBsAg分泌可引起胞内GRP78蛋白下降,导致肝细胞抗凋亡能力减弱;发现HBsAg在细胞中可上调截短的LEF1基因的表达,缺乏活化全长LEF1促成瘤和增殖活性;而肝癌组织中LEF1则倾向于核内分布,并活化Wnt下游基因Cyclin D1与c-myc,有促肿瘤活性。在转基因鼠和细胞模型中都发现了物质和能量代谢相关的基因发生变化,并与临床慢性乙肝患者表现相符。研究中有关CypA的发现提供了抑制HBsAg的新途径;有关代谢的变化提出了改变饮食内容与习惯可能有利于HBsAg阳性感染者的预后。     

乙型肝炎病毒表面抗原preS2+S基因在Pichia Pastoris酵母系统的分泌表达

拟获得adw2亚型乙型肝炎(乙肝)病毒表面抗原preS2 S基因在PichiaPastor妇酵母分泌型表达系统(pPIC9K)的高效表达。实验首先将adw2亚型乙肝病毒表面抗原preS2 S基因重组到分泌型酵母表达载体(pPIC9K)形成表达质粒,电转化酵母细胞：KM71,G418筛选多拷贝整合克隆。经甲醇诱导表达并用SDS-PAGE电泳及酶免疫法检测表达产物。经100个克隆筛选获得了表达量较高的表达菌株WC4。该菌株甲醇诱导后细胞上清10倍浓缩SDS—PAGE电泳检测显示,细胞上清中有特异蛋白条带,且第6天表达量最高, 表达产物单体分子量为31kD左右。用美国雅培公司AUZYME MONOCLONAL试剂盒估算表达量为2μg／100OD600细胞。上述结果表明,乙肝病毒表面抗原preS2 S基因在本系统中获得了分泌表达。同时检测了酵母细胞裂解液中特异蛋白质的表达,结果发现,自甲醇诱导后第一天即可检测到表达产物,而且除了第6天细胞外表达量高于细胞内外,其余各天的表达水平均表现为细胞内高于细胞外。以上结果提示,利用分泌型酵母表达系统表达乙肝病毒表面抗原在技术上可行,但表达产量偏低,一些蛋白滞留在细胞内未能分泌到培养基中。     

乙肝病毒表面抗原基因在花生中的遗传转化及免疫原性检测

首次用花生半胚作为外植体,与农杆菌EHA105(含质粒p1301HBs)共培养5d,再生培养基中通过加入潮霉素进行抗性筛选,得到抗潮霉素的芽,经芽的伸长、诱导生根获得转化植株。经PCR、PCR-Southern杂交、Southern点杂交等分子检测,证实目的基因已整合到花生基因组中,ELISA检测证实了在花生中表达的HBsAg具有较好的活性。经初步定量,花生小芽的蛋白初提液中可溶性蛋白含量1.044g/L,HBsAg小蛋白的含量约占总可溶性蛋白的0.032%,每克转化植株小芽鲜重含HBsAg小蛋白约2.4×10-7g。转基因花生植株初提重组蛋白经HPLC纯化、浓缩后,注射初免一次的小鼠,有明显的特异性抗体产生。口服饲喂已免疫但抗体下降至0.025(HBsAbELISAD值)Balb/c小鼠,发现有较强的抗体回升,达3.54,表明转基因花生疫苗可以加强口服免疫。     

Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.     

Vitreoscilla Hemoglobin (VHb) Gene Expression and Function in Genetically Engineered Bacteria for Production of Phenylalanine

人工设计合成密码子优化的 VHb 基因及其天然的低氧启动子序列,并进行融合 T7 终止子克隆至 L-Phe 的高表达质粒中,构建高产 L-phe 的抗贫氧高密度发酵基因工程菌.高密度发酵过程中的低氧情况可诱导 VHb 基因的表达,含VHb 的工程菌较对照工程菌发酵结果显示:菌株的稳定期延长约4h,提高菌株的产酸周期,L-phe 产量提高约14％.     

基于基因突变的基因工程抗体亲和力成熟研究

基因工程重组抗体可通过对基因序列的分析、修饰与修改,从而改进抗体特性。对基于基因突变的基因工程抗体亲和力成熟的方法进行总结,主要包括小分子有机物-抗体相互作用机制分析、抗体序列数据库及计算机模拟技术,以及新兴的分子模拟与对接技术。     

Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction.     

Genetically Engineered Saccharomyces Yeast Capable of Effective Cofermentation of Glucose and Xylose

Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose. However, Saccharomyces spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose. We developed recombinant plasmids that can transform Saccharomyces spp. into xylose-fermenting yeasts. These plasmids, designated pLNH31, -32, -33, and -34, are 2μm-based high-copy-number yeast-E. coli shuttle plasmids. In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a xylitol dehydrogenase gene (both from Pichia stipitis), and a xylulokinase gene (from Saccharomyces cerevisiae). These xylose-metabolizing genes were also fused to signals controlling gene expression from S. cerevisiae glycolytic genes. Transformation of Saccharomyces sp. strain 1400 with each of these plasmids resulted in the conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth. Furthermore, the resulting recombinant yeasts also have additional extraordinary properties. For example, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium. These properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently coferment glucose and xylose present in the same medium to ethanol simultaneously.     

Genetically Engineered Human Cancer Models Utilizing Mammalian Transgene Expression

Cancer models are vital to cancer biology research, and multiple cancer models are currently available that utilize either murine or human cells, each with particular strengths and weaknesses. The ability to transform primary human cells into tumors through the expression of specific transgenes offers many advantages as a cancer model, including genetic malleability and the ability to transform specific cell types. Until recently, the conversion of primary human cells into tumors through transgene expression required the use of viral genetic elements, which unfortunately adds uncertainty regarding which cancer pathways are affected and how they are affected. In recent years multiple reports have described the transformation of primary human cells into tumors using only mammalian transgenes. This review focuses on these five cancer models, comparing the different cell types which were transformed into tumors and which transgenes were expressed, as well as the cancer pathways affected in the disparate models. These genetically-engineered human cancer models offer a valuable tool to complement existing cancer models and further cancer research.     

Characterization of Chromosome Stability in Diploid,Polyploid and Hybrid Yeast Cells

Chromosome instability is a key component of cancer progression and many heritable diseases. Understanding why some chromosomes are more unstable than others could provide insight into understanding genome integrity. Here we systematically investigate the spontaneous chromosome loss for all sixteen chromosomes in Saccharomyces cerevisiae in order to elucidate the mechanisms underlying chromosome instability. We observed that the stability of different chromosomes varied more than 100-fold. Consistent with previous studies on artificial chromosomes, chromosome loss frequency was negatively correlated to chromosome length in S. cerevisiae diploids, triploids and S. cerevisiae-S. bayanus hybrids. Chromosome III, an equivalent of sex chromosomes in budding yeast, was found to be the most unstable chromosome among all cases examined. Moreover, similar instability was observed in chromosome III of S. bayanus, a species that diverged from S. cerevisiae about 20 million years ago, suggesting that the instability is caused by a conserved mechanism. Chromosome III was found to have a highly relaxed spindle checkpoint response in the genome. Using a plasmid stability assay, we found that differences in the centromeric sequence may explain certain aspects of chromosome instability. Our results reveal that even under normal conditions, individual chromosomes in a genome are subject to different levels of pressure in chromosome loss (or gain).