截短型HCV NS5B RNA聚合酶在大肠杆菌中表达纯化及鉴定

为构建HCV截短型ns5bΔ21基因的原核表达质粒;在大肠杆菌中表达NS5B蛋白并纯化,制备其抗体。运用软件设计引物,以BB7复制子为模板PCR扩增ns5bΔ21基因;克隆入原核表达载体pRSETA。将重组表达质粒pRSETA-ns5bΔ21转化BL21大肠杆菌,并诱导表达、可溶性分析及纯化;用纯化的NS5B蛋白免疫小鼠制备多克隆抗体。结果酶切鉴定和序列测定表明成功的构建了重组表达质粒pRSETA-ns5bΔ21;其经IPTG诱导后行SDS-PAGE可见新生表达条带,Western-blot证实了其特异性和抗原性良好;利用镍离子亲和层析和电洗脱方法获得了纯化NS5B蛋白;用纯化蛋白免疫小鼠制备出了多价抗血清。说明表达和纯化的截短型HCV NS5B RNA聚合酶可用于下一步筛选单链抗体。     

Rapid Cloning and Expression of Glutaryl-7-Aminocephalosporanic Acid Acylase Genes from Soil Samples

A polymerase chain reaction （PCR）-based strategy was developed to rapidly obtain the gene encoding for an industrially important enzyme, glutaryl-7-aminocephalosporanic acid （GL-7-ACA） acylase.Different soil samples were cultured with a Pseudomonas selective medium to enrich specific microorganisms, and then the genomic DNA was extracted to serve as PCR templates. PCR primers for GL-7-ACA acylase gene amplification were designed on the basis of bioinformatics searches and analyses.The method was used to successfully amplify three GL-7-ACA acylase genes from different soil samples.The GL-7-ACA acylase genes were then cloned and overexpressed in Escherichia coil with a relatively high level of 266 unit · L^-1.     

DNA聚合酶Ⅲ全酶的功能和结构的发现

介绍了DNA聚合酶Ⅲ和聚合酶Ⅲ*的发现,DNA聚合酶Ⅲ全酶形式的提出以及全酶亚基的分离。同时,介绍了DNA聚合酶Ⅲ全酶的结构和功能,其中包括α核心的聚合酶功能,ε亚基的3′→5′外切核酸酶活性,[WTHZ]β[WTH1]亚基滑动夹子的功能以及γ复合物的结构与功能。     

RT—PCR检测草鱼呼肠孤病毒的方法研究

草鱼呼肠孤病毒（Grass carp reovirus,GCRV）为草鱼出血病的病原．本实验根据GenBank中GCRV和其他水生呼肠孤病毒毒株的第六基因片段,在其保守区设计了一对GCRV特异性引物,建立了快速检测GCRV的逆转录聚合酶链式反应（RT—PCR）方法．该PCR体系中,上下游引物的最适终浓度为120nmol／L,最适退火温度为52℃．PCR特异性试验表明：所设计的引物只能扩增GCRV的核酸,而不能扩增嗜水气单胞菌BSK-10、WSSV以及正常CIK细胞的DNA或RNA．敏感性试验表明,当GCRV的反转录模板稀释至5-7时,PCR结果还为阳性．用所建立的RT—PCR方法对5份样品进行检测,结果表明本研究建立的RT—PCR检测方法可靠且可行．     

Novel protein detection method based on proximity-dependent polymerase reaction and aptamers

In recent years, specific detection of proteins is one of the hot issues about aptamers in proteomics. Here we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thrombin was used as the model protein, and two aptamer probes with complementary sequence at 3′-end were designed for the two distinct epitopes of the protein. Association of the two aptamers with thrombin resulted in stable hybrids due to the proximity of 3′-end, then polymerase reaction was induced. The amount of obtained dsDNA was indicated using the fluorescence dye Sybr Green I. The results showed that the initial velocity of polymerase reaction had a positive correlation with concentration of thrombin. The advantages of this dual-aptamer-based approach included simple and flexible design of aptamer probes, high selectivity and high sensitivity. The detection limit was 6.9 pmol/L.     

Detection of Messenger RNA for Gonadotropin-Releasing Hormone (GnRH) but not for GnRH Receptors in Rat Pancreas

IntroductionGonadotropin- releasing hormone ( Gn RH) ,whichis synthesized in the brain and secreted into theanterior pituitary,plays a central role in thecontrol of mammalian reproduction[1,2 ] .Gn RH andGn RH m RNA have been reported to exist in non-hypothalamic tissues such as ovary[3,4 ] ,breast[5] ,testes,and placenta[6] .The Gn RH binding siteand its m RNA have also been found inextrapituitary tissues,including gonads andplacenta[7] .These findings indicate the biologicalsignifican…     

虾夷扇贝体内尼氏单孢子虫检测方法

为了解中国近海贝类受原生动物寄生虫--尼氏单孢子虫(Haplosporidium nelsoni)污染状况,利用显微镜观察、原位杂交方法(ISH)和聚合酶链式反应(PCR)方法对2007年采集于大连大窑湾和长海县养殖区的虾夷扇贝样品进行尼氏单孢子虫检测,并比较了浮筏式和底播式养殖的虾夷扇贝中尼氏单孢子虫的检测结果.结果表明,在2007年2月大窑湾浮筏式养殖的成体虾夷扇贝体内检出尼氏单孢子虫;在2007年5～8月长海县浮筏式养殖的成体扇贝体内普遍检测出尼氏单孢子虫,而在其他月份的成体扇贝体内以及全年浮筏式养殖的扇贝幼苗和底播养殖的扇贝体内未检出尼氏单孢子虫.     

应用PCR法检测庚型肝炎病毒感染

应用逆转录套式ＰＣＲ法检测了４８例丙型肝炎、２６例乙型肝炎及１２例甲乙丙丁戊型肝炎病毒感染标志均阴性的肝炎患者血清中的庚型肝炎病毒（ＨＧＶ）ＲＮＡ，结果表明我国肝炎患者中存在ＨＧＶ感染；ＨＧＶ可单独感染或与其它肝炎病毒混合感染，丙型肝炎患者中，ＨＧＶＲＮＡ的检出率为１０．４％，乙型肝炎患者中的检出率为３．８％，而在甲乙丙丁戊型肝炎标志均阴性的肝炎患者中的检出率为３３．３％，ＨＧＶ与其他肝炎病毒的混合感染可加重肝脏的损害，ＨＧＶ感染是甲乙丙丁戊型肝炎标志均阴性肝炎的重要病因之一     

拟南芥多聚ADP核糖聚合酶活性调控植物的抗旱性

为了观察植物多聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]的酶活对植物生长的作用,利用PARP抑制剂来抑制拟南芥PARP的体内活性,并观察表型.体外生化实验结果表明,3-氨基苯甲酰胺(3-Aminobenzamide,3-AB)可以抑制拟南芥PARP1和PARP2的多聚ADP核糖化修饰活性;利用3-AB来处理拟南芥,发现抑制剂处理过的植物表现出更强的耐旱能力.为了排除3-AB的非特异性作用,使用PARP的另外一个抑制剂苯甲酰胺(Benzamide,BA)对拟南芥进行处理,结果显示,BA处理后拟南芥也表现出相似的抗旱表型.另外,在正常生长条件下,3-AB或BA处理过的拟南芥生长量较大,植株鲜重明显增加.以上结果说明,抑制拟南芥PARP活性可以促进拟南芥抗旱能力的改善,其抑制剂具有潜在的应用价值.