Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation

A mouse-human somatic cell hybrid clone, deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) and containing a structurally normal inactive human X chromosome, was isolated. The hybrid cells were treated with 5-azacytidine and tested for the reactivation and expression of human X-linked genes. The frequency of HPRT-positives clones after 5-azacytidine treatment was 1000-fold greater than that observed in untreated hybrid cells. Fourteen independent HPRT-positive clones were isolated and analyzed for the expression of human X markers. Isoelectric focusing showed that the HPRT expressed in these clones is human. One of the 14 clones expressed human glucose-6-phosphate dehydrogenase and another expressed human phosphoglycerate kinase. Since 5-azacytidine treatment results in hypomethylation of DNA, DNA methylation may be a mechanism of human X chromosome inactivation.     

Mitogens and oncogenes can block the induction of specific voltage-gated ion channels

The mechanisms underlying the ontogeny of voltage-gated ion channels in muscle are unknown. Whether expression of voltage-gated channels is dependent on mitogen withdrawal and growth arrest, as is generally true for the induction of muscle-specific gene products, was investigated in the BC3H1 muscle cell line by patch-clamp techniques. Differentiated BC3H1 myocytes expressed functional Ca2+ and Na+ channels that correspond to those found in T tubules of skeletal muscle. However, Ca2+ and Na+ channels were first detected after about 5 days of mitogen withdrawal. In order to test whether cellular oncogenes, as surrogates for exogenous growth factors, could prevent the expression of ion channels whose induction was contingent on mitogen withdrawal, BC3H1 cells were modified by stable transfection with oncogene expression vectors. Expression vectors containing v-erbB, or c-myc under the control of the SV40 promoter, delayed but did not prevent the appearance of functional Ca2+ and Na+ channels. In contrast, transfection with a Val12 c-H-ras vector, or cotransfection of c-myc together with v-erbB, suppressed the formation of functional Ca2+ and Na+ channels for greater than or equal to 4 weeks. Potassium channels were affected neither by mitogenic medium nor by transfected oncogenes. Thus, the selective effects of certain oncogenes on ion channel induction corresponded to the suppressive effects of mitogenic medium.     

TK基因重组缺陷山羊痘病毒构建及其生物学特性鉴定

[目的]利用同源重组技术构建TK基因缺陷的山羊痘病毒（GTPV）毒株,为研制出更安全、高效的GTPV弱毒疫苗和病毒载体提供备选毒株.[方法]采用PCR克隆GTPV AV41株TK基因（ORF56）及其侧翼基因组片段,在TK基因内部插入报告基因EGFP抗性基因gpt达盒,构建GTPV重组转移载体pTK-Eg.将重组转移载体pTK-Eg与GTPV AV41株共转染Vero细胞,通过空斑纯化筛选阳性重组病毒,鉴定其生长特性和遗传稳定性,并接种山羊评价其安全性和免疫原性.[结果]成功构建获得一株TK基因缺陷的重组病毒vTK-Eg.与亲本毒株GTPV AV41相比,vTK-Eg的生长特性及其形成的细胞病变均未发生显著改变,只是毒价略微下降100.5个数量级;在原代牛睾丸（BT）细胞上连续传代,至少在10代内保持遗传性状和病毒滴度稳定.接种vTK-Eg的山羊精神和食欲均正常,接种后体温升高和局部反应的严重程度均较接种亲本毒株GTPV AV41的山羊有所降低;vTK-Eg与GTPV AV41株诱导山羊产生的GTPV中和抗体水平无明显差异（P＞0.05）.[结论]构建的TK基因重组缺陷病毒vTK-Eg具有良好的遗传稳定性和免疫原性,较亲本毒株其安全性也有所提高,可作为研制GTPV基因工程弱毒疫苗和活载体疫苗的备选毒株.     

枣树ZjAPX基因的原核表达

为了解枣树抗氧化系统中抗坏血酸过氧化物酶基因的作用和功能,将从枣树结果枝cDNA文库中筛选获得的ZjAPX cDNA序列,连接到原核表达载体pGEX-4T-2,导入E.coli体内进行了表达产物鉴定。SDS-PAGE电泳检测表明,1.0 mmol/L的IPTG诱导4 h表达了蛋白产物;IPTG的浓度和诱导时间优化表明,1.2 mmol/LIPTG诱导5 h时,蛋白的表达量最大。研究为枣抗坏血酸过氧化物酶基因生物学功能的深入研究奠定了基础。     

赤点石斑鱼神经坏死病毒MCP基因原核表达条件优化

[目的]优化赤点石斑鱼神经坏死病毒的主衣壳蛋白（MCP）基因的原核表达条件。[方法]采用RT-PCR技术扩增出赤点石斑鱼神经坏死病毒MCP基因,构建重组表达载体pRSETA-MCP,以其转化大肠杆菌BL21（DE3）plysS,在不同培养基、温度、pH值条件下进行诱导表达。[结果]重组菌在SOB和LB培养基、pH值7.0、37℃条件下表达量最高,所得的融合蛋白分子量约为44.5 kD。[结论]该研究为RGNNV-MCP疫苗研制奠定了基础。     

反义AcInv基因转化马铃薯方法的研究

马铃薯反义AcInv基因,采用农杆菌介导法,选用生产上推广的9个普通四倍体栽培种,对影响转化的各个因素进行了优化研究。结果表明:在愈伤组织诱导过程中,卡那霉素(Km)的适宜浓度为100 mg/L;生根过程中为50 mg/L。外植体的预培养为:茎段2 d,叶盘3 d效果较好;农杆菌浓度当OD600值为0.5时对茎段和叶盘的转化效果均较佳;茎段和叶盘侵染时间分别达8 min和10 min时转化率较高;外植体与农杆菌共培养时间分别为2 d和3 d时,茎段和叶盘的转化率较高;各个品种的外植体在卡那霉素抗性培养基上均能形成愈伤组织,但只有1号(“大西洋”)最后从愈伤组织上分化成苗,形成正常植株,植株再生率为11.2 %,PCR扩增检测表明大部分转基因植株为阳性。     

红鳍东方鲀抗苗勒氏管激素(AMH)基因在不同发育时期的组织表达

为研究抗苗勒氏管激素(AMH)基因在红鳍东方鲀Takifugu rubripes各组织和不同发育时期的表达情况以及在性别分化和性腺发育等过程中的作用,利用荧光实时定量PCR(qRT-PCR)技术对红鳍东方鲀成鱼的肾脏、脑、心脏、脾脏、肝脏、精巢和卵巢等组织进行了amh基因的表达分析以及不同发育时期(23、30、40、60、80日龄及2龄和3龄鱼)红鳍东方鲀个体水平上amh基因的表达分析。结果表明:红鳍东方鲀3龄成鱼脾脏、肾脏和肝脏中的amh基因表达量极显著高于其他组织(P0.01),在精巢和心脏中的表达量极显著高于卵巢和脑(P0.01),其中脾脏中表达量最高,脑中表达量最低;红鳍东方鲀23日龄、30日龄、2龄和3龄鱼中,雄鱼amh基因的表达量极显著高于同期雌鱼(P0.01),而60日龄和80日龄时雌鱼表达量显著高于雄鱼(P0.05),40日龄时雌、雄鱼的表达量无显著性差异(P0.05),3龄时雌、雄个体性腺中amh基因的表达量均显著高于2龄鱼(P0.05);雄性幼鱼amh基因的表达量从23~30日龄呈增长趋势,且在30日龄时达到最高,随后逐渐降低,而雌鱼整体amh基因的表达量也呈现先升高后降低的趋势,80日龄时雌鱼amh基因表达量最高。研究表明,amh基因在红鳍东方鲀的性腺发育和性别分化过程中发挥着一定的作用。     

赤点石斑鱼神经坏死病毒MCP基因原核表达条件优化(英文)

[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The recombinant expression vector pRSET A-MCP was constructed and transformed into BL21(DE3)plysS to express proteins with induction in different media, at different pH, or at different temperatures. [Result] The expression level of recombinant bacteria reached a peak with induction under the following condition: SOB or LB medium, pH 7.0, 37 ℃ while the fusion protein was about 44.5 kD in molecular weight. [Conclusion] This study provided a basis for the development of RGNNV-MCP vaccine.     

牛病毒性腹泻病毒E0基因原核表达载体的构建及表达

采用RT-PCR方法,利用牛病毒性腹泻病毒(BVDV)C24V株接种MDBK细胞,提取病毒RNA,扩增出BVDV-E0基因,将所得片段与pET-28a表达载体连接,转化至BL-21大肠杆菌中,筛选阳性克隆,鉴定后证明pET-28a-E0原核表达载体构建成功。经IPTG诱导后收集菌体进行SDS-PAGE和Western-blot鉴定,鉴定结果表明,目的蛋白在大肠杆菌中得以表达。     

Identification of DNA sequence responsible for 5-bromodeoxyuridine-induced gene amplification

Bromodeoxyuridine (BrdUrd) treatment of the prolactin nonproducing subclone of GH cells (rat pituitary tumor cells) induces amplification of a 20-kilobase DNA fragment including all of the prolactin gene coding sequences. This amplified DNA segment, which is flanked by two unamplified regions, thus designates a unit of BrdUrd-induced amplified sequence. Cloned DNA segments, 10.3 kilobases long, from the 5' end of the rat prolactin gene of BrdUrd-responsive and -nonresponsive cells, were ligated to the thymidine kinase gene of herpes simplex virus type 1 (HSV1TK), and the hybrid DNA was transferred to thymidine kinase-deficient mouse fibroblast cells by transfection. The HSV1TK gene and the rat prolactin gene were amplified together in drug-treated transfectants carrying the hybrid DNA HSV1TK gene and rat prolactin gene of BrdUrd-responsive GH cells. These results suggest that the 10.3-kilobase DNA segment at the 5' end of the rat prolactin gene of BrdUrd-responsive GH cells carries the information for drug-induced gene amplification (amplicon) and that another gene, such as the HSV1TK gene, is also amplified when the latter is placed adjacent to this segment.     

Reversibility of progression of the transformed phenotype in Ad5-transformed rat embryo cells

The carcinogenic process is extremely complex and is affected by diverse environmental and host factors. The mechanism for the gradual development of the transformed phenotype (a process termed "progression") was studied in type 5 adenovirus (Ad5)-transformed rat embryo cells. Progression was not correlated with major changes in the pattern of integration of viral DNA sequences. Instead, it was associated with an increased methylation of integrated viral sequences other than those corresponding to the E1 transforming genes of Ad5. A single exposure of progressed cells to the demethylating agent 5-azacytidine (Aza) resulted in a stable reversion to the unprogressed state of the original parental clone. A further selection of cells after growth in agar allowed the isolation of Aza-treated clones that had regained the progressed phenotype. These observations indicate that progression is a reversible process and suggest that progression may be associated with changes in the state of methylation of one or more specific genes.     

猪日本乙型脑炎病毒NS5原核表达和多克隆抗体制备

提取猪日本乙型脑炎病毒(JEV)上海分离株的基因组RNA,反转录合成cDNA,扩增JEV-NS5基因片段,亚克隆到原核表达载体pET-28(a)上,构建重组原核表达质粒pET-28(a)-JEV-NS5,转化大肠埃希氏菌BL21(DE3)菌株,IPTG诱导表达重组JEV-NS5蛋白,获得分子质量为103 ku的重组蛋白.该蛋白主要以包涵体形式表达,表达量占总菌体蛋白的35%以上,His-band Ni+纯化后,获得高纯度重组蛋白占总蛋白的比例达75%以上.以纯化的蛋白免疫小鼠,制备小鼠抗JEV-NS5抗体,抗体效价达到3×104,并能与病毒感染的样本反应,证明该蛋白具有较好的特异性.     

鸭瘟病毒TK基因及其编码蛋白的生物信息学分析

运用生物信息学方法,对鸭瘟病毒TK基因的基本元件和TK蛋白的理化性质、结构、功能进行分析。明确了TK基因启动子、转录起始位点、编码区P、olyA的位置;发现TK蛋白的二级结构以α螺旋为主,属于混合型蛋白,在结构和功能上与疱疹病毒胸苷激酶高度相似。生物信息学分析结果说明,鸭瘟病毒TK蛋白具有疱疹病毒胸苷激酶的活性,鸭瘟病毒TK基因可能为鸭瘟病毒的非必需毒力基因。     

免疫增强剂对猪伪狂犬灭活疫苗的免疫增强作用

为了评价免疫增强剂VA5对猪伪狂犬病毒灭活疫苗的免疫增强作用,将VA5与灭活疫苗混合并制成疫苗。通过常规Bartha-K61株灭活疫苗、含免疫增强剂灭活疫苗和Bartha-K61活疫苗3组免疫效力对比试验,首次免疫后14和35 d对猪进行采血,并且进行中和实验以及对相关细胞因子进行检测。结果表明,该免疫增强剂能够显著地提高猪伪狂犬灭活疫苗血清中抗体产生,同时能显著提高猪外周血淋巴细胞(PBMC)中IL-2和IFN-γ mRNA表达。交叉中和试验表明,VA5能够显著地提高猪伪狂犬灭活疫苗对伪狂犬流行变异株的中和作用。试验表明,VA5能够显著提升猪伪狂犬病毒灭活疫苗体液与细胞免疫,为研制免疫效果更好的灭活疫苗提供了依据。     

粉和平月季植株再生体系建立研究

以试管苗的幼嫩小叶为外植体,探讨了粉和平月季通过诱导愈伤组织建立植株再生体系的方法。结果表明:2,4-D能有效诱导无菌小叶产生愈伤组织,经5.0mg/L的2,4-D光下诱导愈伤20d后,愈伤组织诱导率最高可达91.11%,且其愈伤质量最优。将生长良好的愈伤组织转至TDZ浓度为0.5～2.0mg/L的MS培养基中发现,暗培养8～16d均能诱导愈伤组织产生不定芽,但暗培养时间超过16d愈伤组织容易褐化死亡。在含1.2mg/LTDZ的MS培养基上暗培养14d后其不定芽诱导率可达30%。将生长良好的不定芽转入壮苗培养基MS+6-BA1.0mg/L+NAA0.01mg/L中培养25d左右,组培苗芽枝叶粗壮,生长旺盛;将长至2～3cm长的健壮小苗转入生根培养基,生根率最高仅为8%。     

伪狂犬病毒TK基因缺失通用转移载体的构建及初步应用

以伪狂犬病毒为载体表达其他病原体抗原蛋白是伪狂犬病疫苗研究的一个重要方向。根据已发表的伪狂犬病毒（PRV）Ra株TK基因序列设计2对引物,用PCR方法得到同源左右臂,克隆入PUC19载体中。利用平末端连接的方法将绿色荧光蛋白（EGFP）的基因表达盒插入到缺失位点,并在下游引入1个多克隆位点,构建缺失通用转移载体PUC19TK-EGFP。用脂质体转染试剂盒将PUC19TK-EGFP和PRV-Ra株的基因组共转染BHK细胞,以EGFP为标记基因,用噬斑法得到纯化的重组病毒,命名为TK/EGFP,为研制以伪狂犬病毒为载体的二价或多价基因工程疫苗奠定了物质基础。     

影响大豆遗传转化主要因素及相关条件优化

以浙春3号,华春6号,0428三个大豆基因型的胚尖、子叶节和下胚轴为受体材料,运用农杆菌介导法转化GUS基因,通过对GUS基因瞬时表达率的比较,探讨大豆遗传转化的主要影响因素（基因型、外植体等）,并进一步对遗传转化条件进行优化。结果表明：以胚尖为受体时,3个基因型的GUS基因瞬时表达率都很低,在丛生芽的再生部位没有瞬时表达。以下胚轴为受体时,萌发5 d,共培养3 d后,浙春3号、华春6号、0428在丛生芽再生部位的GUS瞬时表达率达到最高,分别为4392%,5445%,5847%。而以子叶节为受体时,按照常规方法（萌发5 d）,浙春3号、华春6号、0428分别在共培养5,4,4 d时,其丛生芽再生部位的GUS瞬时表达率最高,分别为2305%,1379%,478%;利用浙春 3号对子叶节法的萌发时间进行了进一步的优化,发现萌发时间为3 d时在丛生芽再生部位的GUS瞬时表达率、芽诱导率较萌发5 d都有较大幅度的提高。