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

将乙肝病毒融合表面抗原基因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,筛选到二倍体酵母工程菌Y95xY19/YFD158。对两种工程菌的研究表明：二倍体工程菌发酵密度为单倍体工程菌的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胞中的平均拷贝数略低于单倍体工程菌。     

酵母ADH2—SUC2融合启动子的构建及其对基因表达的调控

将ＡＤＨ２基因的ＵＡＳ与带有不同长度缺失上游区的ＳＵＣ３基因融合,构建成４种具有不同融合启动子的ＳＵＣ２基因的表达质粒ＹＲＤ１１０１．ＹＦＤ１１０△９．ＹＦＤ１１０△１７、ＹＦＤ１１０△１１。将这些质粒及对照表达质粒ＹＦＤ２６△１．ＹＦＤ２５转化酵母菌Ｙ３３,在阻遏与去阻遏培养条件下,对各种转化子所产生的蔗糖酶进行了活性测定和组分分析。结果表明：在葡萄糖去阻遏生长条件下,ＹＦＤ１１０△１的启动子组合中ＵＡＳｓｕｃ２和ＵＡＳＡＤＨ２对ＳＵＣ２基因的表达有协同激活作用。在阻遏条件下Ｙ３３／ＹＦＤ１１０△１与Ｙ３３／ＹＦＤ１１０△９、Ｙ３３／ＹＦＤ２６△１、Ｙ３３／ＹＦＤ２５一样,均表达很低的糖基化蔗糖酶,３种去阻遏培养条件比较说明,在低糖培养基中对糖基化蔗糖酶表达的去阻遏效果最佳     

虹鳟生长激素cDNA在酵母中的表达

采用聚合酶链式反应（ Ｐ Ｃ Ｒ） 技术对虹鳟生长激素ｃ Ｄ Ｎ Ａ 进行改造。将改造后的基因克隆到含酵母 Ｐ Ｇ Ｋ 启动子的大肠杆菌酵母穿梭质粒ｐ Ｍ Ａ９１ ,转化酿酒酵母 Ｙ３３ ,构建表达鱼生长激素的酵母工程菌 Ｙ３３（ｐ Ｍ Ａｒ Ｇ Ｈ１６） ,并在酵母中获得表达,表达量约占细胞可溶性蛋白总量的３ ％ 。表达产物作为饲料添加剂投喂罗非鱼,具有明显的促进生长作用     

重组人肿瘤坏死因子纯化方法的探索

ＴＮＦ是一种具有多种生物学作用的细胞因子,高纯度ＴＮＦ的获得是进一步研究其作用机理和探讨防治方法的基础,对克隆后工程菌表达的纯化进行研究,经盐析,透析,离子交换等步骤,成功地制备了高纯度的ｒｈＴＮＦ,经ＳＤＳ－ＰＡＧＥ测定,分子量１７ｋＤ,纯度〉９５％,比活性为３．４４×１０＾７Ｕ／ｍｇ,与国外同产品相似。     

基因工程变链菌防龋疫苗的研制

本文采用我们自己构建的基因工程龋齿疫苗菌株,大罐发酵培养３批、菌体收量平均湿重为２．６７ｇ／Ｌ,表达率为４４．２７％。菌体经超声裂解,硫酸铵和链霉素粗提,再经吸附层检,凝胶过滤和离子交换柱层析等步骤纯化精提ＧＴＦ抗原３批。提纯的ＧＴＦ抗原,平均比活,提纯倍数和收率,分别为５８．３４ｕ／ｍｇ、６．０８倍和１９．２５％。此抗原经ＳＤＳ－ＰＡＧＥ电泳,分子量为５５ＫＤ,纯度达到９６．７％,并对湿度具有较好的稳定性能。且此抗原制备的疫苗免疫家兔具有较好的免疫原性     

高生物量富铁酵母菌的选育及其发酵条件的研究

对402株不同种属的酵母菌株进行初筛、复筛,筛选到一株生物量较高的二倍体菌株ZY-46(Saccharomyces cerevisiae)和一株铁富集量较高的二倍体菌株ZY-173(Saccharomyces kluyveri)。然后以它们为出发菌,分别进行单倍体分离、硫酸二乙酯(DES)诱变,并通过原生质体融合,得到一株高生物量富铁酵母融合菌株ZYF-15。在优化的发酵条件下,该融合菌株生物量可达11．2g／L,细胞铁含量达24．5mg／g干细胞,细胞总铁含量分别比原始亲株ZY-46和ZY-173提高了2．6倍和1．9倍。     

利用IL—3／GM—CSF融合蛋白（PIXY—321）扩增脐血造血细胞方…

利用单克隆抗体免疫磁珠吸附方法脐血ＣＤ３４＋细胞,并观察了ＩＬ－３／ＧＭ－ＣＳＦ融合蛋白（ＰＩＸＹ－３２１）对脐血ＣＤ３４＋细胞的刺激作用。ＰＩＸＹ－３２１对脐血ＣＤ３４＋细胞扩增作用大于ＩＬ－３和ＧＭ－ＣＳＦ单独及联合应用,在液体培养条件下,每毫升２０ｎｇＰＩＸＹ－３２１可有效地扩增脐血造血祖细胞,适宜扩增时间为５－８天,扩增后造血细胞的数量可达扩增前的８－１０倍,从而初步建立了一种简单可行的脐     

利用IL－3／GM－CSF融合蛋白（PIXY－321）扩增脐血造血细胞方法的初步建立

利用单克隆抗体免疫磁珠吸附方法分离脐血ＣＤ３４＋细胞,并观察了ＩＬ３／ＧＭＣＳＦ融合蛋白（ＰＩＸＹ３２１）对脐血ＣＤ３４＋细胞的刺激作用。ＰＩＸＹ３２１对脐血ＣＤ３４＋细胞扩增作用大于ＩＬ３和ＧＭＣＳＦ单独及联合应用。在液体培养条件下,每毫升２０ｎｇＰＩＸＹ３２１可有效地扩增脐血造血祖细胞,适宜扩增时间为５－８天,扩增后造血祖细胞的数量可达扩增前的８－１０倍,从而初步建立了一种简单可行的脐血造血细胞扩增方法。     

粒细胞—巨噬细胞集落刺激因子／白细胞介素—3融合蛋白的表达研究

利用ＰＣＲ扩增得到粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）、白细胞介素－３（ＩＬ－３）完整基因片段,将其分别克隆ｐＧＥＭ－Ｔ构建成ＧＭ－ＣＳＦ／ＩＬ－３融合蛋白基因,ＤＮＡ序列与设计预期一致。将得到的融合蛋白基因克隆对７２ＲＮＡ聚合酶表达载体ｐＴ７ｚｚ,得到表达质粒ｐＦｕ,经转化至表达宿主Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）,在ＩＰＴＧ诱导下获得融合蛋白目的产物的直接表达。经ＳＤＳ－ＰＡＧＥ电泳鉴     

产甘油假丝酵母胞浆3-磷酸甘油脱氢酶编码基因的克隆

当酵母细胞处于高渗压环境时,甘油被诱导合成以提高其胞内渗透压,这一过程受ＨＯＧ途径的调控。ＧＰＤ１基因为ＨＯＧ途径的重要靶基因,高效表达使胞内３磷酸甘油脱氢酶酶活水平提高可极大地提高甘油的产量。本研究将产甘油假丝酵母（Ｃａｎｄｉｄａｇｌｙｃｅｒｏｌｏｇｅｎｅｓｉｓ）染色体ＤＮＡ经Ｓａｕ３ＡＩ部分酶解后的５～１０ｋｂＤＮＡ片段与经ＢａｍＨＩ线性化及ＣＩＰ处理过的酵母大肠杆菌穿梭质粒ＹＥｐ５１连接,以大肠杆菌ＤＨ５α为受体,构建产甘油假丝酵母的染色体基因文库。通过遗传互补法,在含５０ｇ／Ｌ氯化钠的培养基上筛选出１５个转化子,对转化子０６０１进行了进一步鉴定,转化子０６０１所含质粒ＹＥｐ０６０１带有ＹＥｐ５１的标记并可以消除Ｓａｃｃｂａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ６４２菌株由于其ＧＰＤ１,ＧＰＤ２两基因的缺失突变而表现出的渗透压敏感性,表明已克隆到产甘油假丝酵母的编码胞浆３磷酸甘油脱氢酶的基因     

Haploidy influences Bak and Bcl-xL mRNA expression and increases incidence of apoptosis in porcine embryos

The objective of this study was to determine developmental pattern, total cell number, apoptosis and apoptosis-related gene expression in haploid and diploid embryos following parthenogenetic activation. In vitro-matured porcine oocytes were activated by electrical pulses and cultured in the absence or presence of cytochalasin B for 3 h. Zygotes with two polar bodies (haploid) and one polar body (diploid) were carefully selected and were further cultured in NCSU 23 medium containing 0.4% bovine serum albumin (BSA) for 7 days. The percentage of development to blastocyst stage was higher (p < 0.01) in the diploid than in the haploid parthenotes. In haploid blastocysts, average total cell number was significantly reduced (p < 0.05) and apoptosis was increased at day 7. The relative abundance of Bcl-xL and Bak mRNA in the diploid blastocysts was similar to that of in vivo-fertilized embryos. However, Bcl-xL was significantly decreased, and Bak mRNA was significantly increased (p < 0.05) in haploid parthenotes compared with the diploid parthenotes. These results suggest that the haploid state affects apoptosis-related gene expression which results in increased apoptosis and decreased developmental competence of haploid parthenotes.     

Effects of an acridine half-mustard (ICR 191) on growth and ploidy of frog cells in culture

The effects of an acridine half-mustard, ICR 191, on the growth rate and ploidy of four haploid and two diploid lines of Rana pipiens cells in culture were studied. Growth curves indicate that the haploid and diploid cell lines were equally resistant to a 4-hour exposure of this drug (0.1 micrometer to 10 micrometer. ICR 191 treatment induced the haploid cell cultures to become diploid. The proportion of diploid cells increased progressively with respect to time after the 4-hour exposure period. The greater the concentration of ICR 191 applied, the more rapid the rate of conversion. Autoradiographic determinations of percent labelled nuclei indicate that DNA synthesis was not inhibited in haploid or in diploid cells. Therefore, the increased proportion of diploid cells did not originate from the small percentage of diploid cells in the initial population. Instead the haploid cells were converted to diploid cells. Time lapse cinematography indicated that the conversion mechanism was other than cell fusion. Conversion to higher ploidy did not occur when diploid cell cultures were exposed to ICR 191.     

Karyotype Characterization of In Vivo- and In Vitro-Derived Porcine Parthenogenetic Cell Lines

Mammalian haploid cell lines provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19–38 chromosomes were the predominant karyotype (59.48–60.91%). The diploid cells were the second most observed karyotype (16.17%–22.73%). Although a low percentage (3.45–8.33%) of cells with 19 chromosomes were detected in 18.52% of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels. We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo-derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes. The porcine PA embryo-derived diploid fibroblasts enriched from sorting might be candidate cells for paternally imprinted gene research.     

Construction and Characterization of Isogenic Series of Saccharomyces cerevisiae Polyploid Strains

Tetraploid cells of Saccharomyces cerevisiae are generated spontaneously in a homothallic MATa/MATα diploid population at low frequency (approximately 10⁻⁶ per cell) through the homozygosity of mating-type alleles by mitotic recombination followed by homothallic switching of the mating-type alleles. To isolate tetraploid clones more effectively, a selection method was developed that used a dye plate containing 40 mg each of eosin Y and amaranth in synthetic nutrient agar per liter. It was possible to isolate tetraploid clones on the dye plate at a frequency of 1 to 3% among the colonies colored dark red in contrast to the light red of the original diploid colonies. Isogenic series of haploid to tetraploid clones with homozygous or heterozygous genomic configurations were easily constructed with the tetraploid strains. No significant differences in specific growth rate or fermentative rate were observed corresponding to differences in ploidy, although the haploid clones showed a higher frequency of spontaneous respiratory-deficient cells than did the others. However, a significant increment in the fermentative rate in glucose nutrient medium was observed in the hybrid strains constructed with two independent homozygous cell lines. These observations strongly suggest that the polyploid strains favored by the brewing and baking industries perform well not because of the physical increment of the cellular volume by polyploidy but because of the genetic complexity or heterosis by heterozygosity of the genome in the hybrid polyploid cells.     

Evolution of karyotype in haploid cell lines of Drosophila melanogaster

Seven continuous cell lines have been established in vitro from lethal embryos produced by the female sterile mutant mh 1182 of Drosophila melanogaster. Six lines show haploid metaphases. Karyotype analysis revealed a high level of aneuploid cells with frequent chromosome fragments. In three lines, haploid cells were quickly overgrown by diploid cells. Two lines were more stable but the proportion of haploid cells decreased with time. One line was stable, showing 80-90% of haploid cells for over 1 000 cell generations. Stable haploid clones have been isolated from two lines. Crossing of mh 1182/mh 1182 females with males bearing a ring X chromosome shows that the haploid genome retained in the cells is of maternal origin and that the diploid cells derive from pre-existing haploid cells. The appearance of the diploid cells and the conditions of karyotypic stability are analysed.     

Growth, peroxidase activity, and protein content in stem pith and callus tissues from haploid and diploid Nicotiana tabacum plants

Pith segments isolated from haploid and diploid Nicotiana tabacum cv. IAC-70 plants from five different heights in the stem, were cultured in vitro on MS-62 medium supplied with 0.5 mg/l IAA and 0.02 mg/l kinetin. Pith tissues of haploid plants showed greater growth potential than those of diploids; peroxidase activity was higher in the calluses of diploid tissues whereas protein content was higher in those of haploids. After three subcultures the growth pattern was inversed, as were the results for peroxidase activity and protein content. After the onset of culture, peroxidase activity in haploid explants dropped sharply, reaching values lower than in diploid tissues 40 d later, but after three subcultures these values were higher than those for diploid calluses. The results are discussed from the viewpoint of a possible relationship between greater polyploidization in haploid tissues on the one hand and growth decrease and peroxidase activity increase on the other.     

Proteome comparative analysis of gynogenetic haploid and diploid embryos of goldfish (Carassius auratus)

Recently, it was found that in the gynogenetic haploid and diploid embryos of goldfish, which have exactly the same genome, the haploid condition results in obstruction of gene expression and abnormal development while the diploid embryos have normal gene expression and development. A diploid-dependent regulatory apparatus was proposed to regulate gene expression. To study the difference at the protein expression level of the embryos of haploid and diploid in development, we extracted the total proteins of both the gynogenetic haploid and diploid embryos of goldfish in the same eye formation stage. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. The stained gel images were analyzed with the PDQUEST software. A part of protein spots that were differentially expressed in haploid and diploid embryos were identified by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry and database analysis. Sixteen protein spots that were absolutely different (only expressed in diploid embryos but not in haploid embryos or vice versa) and 16 protein spots that were up- and downregulated were identified unambiguously, which include some proteins that are correlative with eyes development, nerve development, developing regulation, cell differentiation, and signal transduction. The different significantly gene expression during embryos developing between diploid and haploid is demonstrated.     

Cleavage rate of haploid and diploid parthenogenetic mouse embryos during the preimplantation period.

The lack of a paternal genome in parthenogenetic embryos clearly limits their postimplantation development, but apparently not their preimplantation development, since morphologically normal blastocysts can be formed. The cleavage rate of these embryos during the preimplantation period gives a better indication of the influence of their genetic constitution than blastocyst formation. Conflicting results from previous studies prompted us to use a more suitable method of following the development of haploid and diploid parthenogenetic embryos during this period. Two classes of parthenogenetic embryos were analysed following the activation of oocytes in vitro with 7% ethanol: 1) single pronuclear (haploid) embryos and 2) two pronuclear (diploid) embryos. Each group was then transferred separately during the afternoon to the oviducts of recipients on the 1st day of pseudopregnancy. Control (diploid) 1-cell fertilised embryos were isolated in the morning of finding a vaginal plug, and transferred to pseudopregnant recipients at approximately the same time of the day as the parthenogenones. Embryos were isolated at various times after the HCG injection to induce ovulation, from each of the three groups studied. Total cell counts were made of each embryo, and the log mean values were plotted against time. The gradient of the lines indicated that 1) the cell doubling time of the diploid parthenogenones was 12.25 +/- 0.34 h, and was not significantly different from the value obtained for the control group (12.74 +/- 1.17 h), and that 2) the cell doubling time of the haploid parthenogenones (15.25 +/- 0.99 h) was slower than that of the diploid parthenogenones and the control diploid group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolome analysis of differential responses of diploid and haploid yeast to ethanol stress

Metabolomic analysis was carried out to investigate the metabolic differences of diploid (α/a) and homogenous haploid (α,a) yeasts, and further assess their response to ethanol stress. The dynamic metabolic variations of diploid and haploid caused by 3 and 7% (v/v) ethanol stress were evaluated by gas chromatography coupled to time-of-flight mass spectrometry combined with statistical analysis. Metabolite profiles originating from three strains in presence/absence of ethanol stress were distinctive and could be distinguished by principal components analysis. Results showed that the divergence among the strains with ethanol stress was smaller than without it. Furthermore, the levels of most glycolytic intermediates and amino acids in haploid were lower than these in diploid with/without ethanol stress, which was considered as species-specific behaviors. The increases of protective metabolites including polyols, amino acids, precursors of phospholipids, and unsaturated fatty acids under ethanol stress in three strains revealed the ethanol stress-specific responses. Higher fold change in most of these protectants in haploid indicated that haploid was more susceptible to ethanol stress than diploid. These findings provided underlying basis for better understanding diploid and haploid yeasts, and further breeding tolerant strains for efficient ethanol fermentation.