犬瘟热病毒水貂分离株F基因的克隆及原核表达

根据GenBank中登录的犬瘟热病毒F基因序列设计了1对引物,以从水貂犬瘟热病毒中提取的RNA为模板,扩增出一约1 000 bp的F基因片段。将PCR产物按相应的阅读框架克隆到原核表达载体pET-32a中,并将重组质粒转化E.coliBL21(DE3),用1.0 mmol/L IPTG在30℃下诱导表达。结果显示,F基因的表达量约占细菌总蛋白的35%。SDS-PAGE电泳显示,表达产物的分子质量约为55 ku,与预计大小相符;经Western-blotting试验进一步证实,该基因获得了正确表达。     

牛朊蛋白单氨基酸变异体的原核表达

应用pGEX原核表达载体构建了牛朊蛋白的1个正常重组蛋白和3个单氨基酸变异体的原核表达质粒,即pGEX BoPrP(93~241)、pGEX BoPrP(93~241)(S154N)、pGEX BoPrP(93~241)(A129V)和pGEX BoPrP(93~241)(Q234R)。经转化E.coli BL21 (DE3),均表达了预期大小约为42.4 ku的融合蛋白。在优化的诱导表达条件下,各重组菌可产生表达量占细菌总蛋白量30%~45%的融合蛋白。经免疫印迹检查,所有融合蛋白均可与抗牛单克隆抗体4C11 反应。表明,单氨基酸突变并不影响单抗4C11的识别表位。     

乳牛L-选择素在中性粒细胞中表达与分布的定位研究

采用间接荧光标记及胶体金标记方法通过流式细胞仪和低压扫描电子显微镜,检测了L-选择素在健康泌乳中期乳牛(A组)及患金黄色葡萄球菌型临床型(B组)和亚临床型乳腺炎乳牛(C组)中性粒细胞上表达的基本规律。结果表明:三者表达L-选择素的中性粒细胞阳性率分别为97.90%、97.69%和96.67%(收集5 000个细胞),组间差异不显著(P>0.05),但患亚临床型乳腺炎乳牛的阳性率从3次测定的结果看是逐渐增加的;L-选择素表达的间接荧光强度组间差异极显著(P<0.01);而患亚临床型乳腺炎的乳牛组内差异也极显著(P<0.01);扫描电镜观察到的中性粒细胞表面脊样结构的分布及形态与流式细胞仪检测的结果具有相似性。     

The Rule in Wilkinson v Downton: Conduct,Intention, and Justifiability

The decision in OPO v MLA [2014] EWCA Civ 1277 causes confusion to the rule in Wilkinson v Downton. A strong line of authorities indicates that the defendant must either have an actual intention to cause physical injury or be reckless as to the causing of such harm, the latter being determined by the likelihood of harm being caused by the defendant's act. ‘Imputed intention’ does not form a separate category of mental state. There was also a missed opportunity to develop a ‘justifiability’ criterion, by which policy considerations can be taken into account to preclude an application of the tort. This criterion ought to be developed in a principled manner, in line with the existing jurisprudence concerning human rights and with the policy limitations as developed in the context of other torts.     

著作权法中思想与表达两分法原则含义

思想与表达两分法原则被称为著作权法的基本原则。我国目前仅在《计算机软件保护条例》中对思想与表达两分法原则进行了规定,而著作权法却没有确立这一原则。有必要在著作权法中确立这一原则,并在司法审判中使用与之相配套的方法论。     

猪生殖与呼吸综合征病毒非结构蛋白nsp4基因的表达及其产物的纯化

根据GenBank中已登录的猪生殖与呼吸综合征病毒(PRRSV)全基因组序列,设计合成了1对特异性引物,对PRRSV非结构蛋白nsp4基因进行了RT-PCR扩增,将回收的目的基因片段克隆到大肠埃希氏菌表达载体pET28a中,构建了重组质粒pET-nsp4,测序结果证实了重组质粒pET-nsp4的可靠性。将pET-nsp4转化至大肠埃希氏菌表达菌株BL21(DE3),用IPTG诱导表达,SDS-PAGE结果表明,重组菌可表达分子质量约23 ku的蛋白。经镍离子亲和层析柱(Ni-NTA)纯化,获得了高纯度的重组蛋白,Western-blot分析结果表明,nsp4重组蛋白在大肠埃希氏菌系统中获得了正确表达。     

口蹄疫病毒P1 2A和3C基因重组逆转录病毒表达载体的构建

以A型口蹄疫病毒(FMDV)AV88(L)和XJ99株的P12X基因和AV88(L)3C基因的阳性克隆质粒为模板,用PCR扩增各基因片段,酶切后分步定向亚克隆至逆转录病毒表达载体pBABE-puro上经PCR、酶切鉴定及测序。结果表明,获得了2个含不同RGD基序的A型FMDV衣壳蛋白和蛋白酶基因的重组逆转录病毒表达载体pBABE-AV88(L)P1 2X3C和pBABE-XJ99 P1 2X3C,为研究安全、高效、低成本的FMDV空衣壳亚单位疫苗奠定了基础。     

Disputes about corporate expression absorption and their legal remedies

Corporate expression is the expression that a company gives to the outside in its capacity as a legal entity. Often referring to resolutions made by shareholder meetings and the board of directors, based on good faith and bound by contractual spirit, a company must be held liable for its expression. Corporate expression absorption refers to the corporate behaviors and situations wherein the majority voting shareholders and directors replace the will of the minority voting shareholders and directors within their own will. Among them, the majority voting shareholders at a shareholders’ meeting (shareholders’ general meeting) are decision-making shareholders, and directors, managers and other senior management staff that decide corporate affairs are called decision-making members. Corporate expression absorption consists of two sorts: absorption by shareholders’ meeting and absorption by the board of directors. Shareholders’ meeting is a company’s authoritative organization; when the voting rights of some shareholders exceed the statutory limit, they will be able to manipulate the expression of shareholders’ meetings and replace the will of other shareholders with that of their own. The expression absorption by the board of directors refers to the practice wherein the majority directors decide on important corporate matters in accordance with the majority rule. Thus, it can be seen that the corporate expression absorption is a double-edged sword, not only capable of uplifting operational efficiency but also likely to help decision-making shareholders achieve personal gains and transfer corporate interests. As for the disputes of corporate expression absorption, the following legal remedies might be adopted: (1) Limit the voting rights of decision-making shareholders. (2) Provide shareholders with veto power over specific events. (3) Ask the chambers of commerce (industry associations) to arbitrate specific events. (4) Preserve the market value of shares held by dissenting directors. (5) Expand cumulative voting; (6) Provide shareholders the right to exit. (7) Legal remedies for corporate deadlock. (8) Shareholders’ derivative lawsuits. __________ Translated from China Law, No. 4, 2005     

肉鸡胫骨软骨发育不良分子机理的研究进展

对参与肉鸡胫骨软骨发育不良的酶、生长因子、维生素D及其受体,基因表达水平进行了综述,它们形成一个复杂的网络调控系统。阐述了它们在肉鸡胫骨软骨发育不良中所起的作用和变化。认为对参与干扰软骨细胞分化的各种基因的深入研究将为基因选择、消除机能紊乱、防治胫骨软骨发育不良的发生开辟新的途径。     

猪传染性胸膜肺炎放线杆菌外膜蛋白基因的克隆和表达

以猪传染性胸膜肺炎放线杆菌(APP)血清7型25-4株基因组DNA为模板,用PCR扩增外膜蛋白(OMP)基因特异片段,并克隆于pMD 18-T中,经酶切及核苷酸序列分析鉴定后,亚克隆于原核表达载体pGEX-6P-1,成功构建了重组表达载体pGEX-omp;以此转化大肠埃希氏菌BL21(DE3),经SDS-PAGE鉴定,表达的可溶性融合蛋白分子质量约为61 ku,命名为GST-OMP。以GST亲和层析柱纯化并利用Xa因子酶解,获得切掉标签的OMP。经ELISA检测,该OMP蛋白能够与兔抗APP的阳性血清反应,具有很好的免疫活性。GST-OMP蛋白的成功表达为APPOMP相关分子生物学功能的研制奠定了基础。