人Leptin基因的克隆及其在大肠杆菌中的表达

目的克隆Leptin基因,构建其重组表达菌株,并对表达产物进行免疫学鉴定。方法利用RT-PCR方法从脂肪细胞RNA中扩增出人瘦素前体(pre-Leptin)的cDNA片段和去信号肽序列的Leptin成熟基因片段,并插入pET32a中,转化大肠杆菌BL21(DE3),获得表达重组人瘦素(rh-Leptin)的菌株。结果DNA序列分析显示获得的pre-Leptin和成熟基因片段的序列与预计序列一致,菌株诱导后经SDS-PAGE检测,rh-Leptin表达量达菌体总蛋白的40%以上。Western blot分析显示重组Lep-tin具有免疫学活性。结论已获得高效表达Leptin的重组菌株。     

传染性法氏囊病病毒疫苗B87株基因组B节段的克隆和序列分析

目的获得传染性法氏囊病病毒(IBDV)疫苗B87株B节段全长cDNA,并对其序列进行分析,为进一步在分子水平上研究病毒基因组的功能、抗原变异和毒力变异奠定基础。方法使用蛋白酶K和酚/氯仿抽提法提取病毒基因组RNA,用LiCl分级沉淀方法纯化病毒基因组dsRNA,通过RT-PCR一步扩增获得B节段的全长cDNA。将其克隆到pGEM-T载体上,然后测序,并用DNAStar软件进行序列分析。结果测序结果表明,克隆的B87株基因组B节段全长为2827bp,与超强毒参考株UK661和HK46的同源性分别为89.8%和89.3%,与强毒参考株Harbin-1和ZJ2000的同源性分别为90.2%和89.5%;与变异毒株GLS的同源性为97.8%;与弱毒参考株CEF94和Gt株的同源性均为99.8%,与P2株的同源性达100%。结论通过对9株IBDV编码氨基酸序列进行分析、比较,推测B节段上10个独特的氨基酸位点可能与毒力相关。     

纤溶酶原饼环区5蛋白重组大肠杆菌高效表达体系的建立

目的　建立纤溶酶原饼环区 5 (Humanplasminogenkringle 5 ,hPK 5 )蛋白重组大肠杆菌高效表达体系 ,为高密度发酵创造条件。方法　观察一级、二级种子的生长状态 ,比较表达hPK 5蛋白的 4种重组工程菌株在相同条件下的表达情况 ,选出首选发酵种子 ;对其培养时间、诱导时间、培养基种类、pH等条件进行优化 ;并用凝胶成像分析系统对SDS PAGE结果进行分析。结果　经过筛选 ,JM10 9 pBV2 2 0 hPK 5 (简称JP5 )是获取hPK 5蛋白的首选工程菌株 ,其最佳表达条件是LB培养基 (pH 7.4、溶解氧充足 )、30℃培养 3h、4 2℃诱导 6h。在此条件下 ,JP5表达目的蛋白占菌体总蛋白的 38%左右。结论　为高密度发酵获取hPK 5蛋白奠定了实验基础     

人白细胞介素-24基因的克隆、表达和纯化

目的对人白细胞介素-24(hIL-24)进行克隆、表达和纯化。方法分离人外周血单个核细胞,ConA刺激培养,提取细胞总RNA,应用RT-PCR技术从外周血单个核细胞中扩增hIL-24成熟肽编码区,定向克隆至融合表达载体pGEX-4T-1,重组载体pGEX-4T-1/hIL-24经DNA测序鉴定后,转化E.coliBL21(DE3),IPTG诱导表达,阴离子交换层析纯化融合蛋白,SDS-PAGE和Westernblot鉴定。结果所得hIL-24基因经序列分析,与GenBank公布一致。所构建融合表达载体pGEX-4T-1/hIL-24测序正确,转化BL21(DE3)后,37℃诱导表达相对分子质量约为44000的融合蛋白,主要以包涵体形式存在,占菌体总蛋白的30.63%。经纯化后,纯度达85%以上。Westernblot检测能与兔抗人IL-24的多克隆抗血清发生结合反应。结论已成功构建了hIL-24的融合表达载体pGEX-4T-1/hIL-24,并在大肠杆菌中高效表达,纯化后获得了高纯度的融合蛋白,为hIL-24的功能及活性研究奠定基础。     

钩端螺旋体黄疸出血群赖株LipL41基因的克隆表达及鉴定

目的　对钩体黄疸出血群赖株LipL4 1基因进行克隆、表达及鉴定。方法　采用高保真PCR ,从我国钩体黄疸出血群赖株基因组中扩增全长LipL4 1基因片段 ,并构建原核表达系统。结果　所克隆的LipL4 1基因核苷酸和氨基酸序列与已发表LipL4 1基因序列 (GenbankL4 6 794 )同源性分别为 96 2 5 %和 98 87% ;所表达的目的蛋白经Westernblot证实为具有良好免疫反应性的广谱抗原。结论　可作为研制基因工程疫苗的候选抗原     

Anti-cloning protocol suitable to EPCglobal Class-1 Generation-2 RFID systems

Radio frequency Identification (RFID) systems are used to identify remote objects equipped with RFID tags by wireless scanning without manual intervention. Recently, EPCglobal proposed the Electronic Product Code (EPC) that is a coding scheme considered to be a possible successor to bar-code with added functionalities. In RFID-based applications where RFID tags are used to identify and track tagged objects, an RFID tag emits its EPC in plaintext. This makes the tag inevitably vulnerable to cloning attacks as well as information leakage and password disclosure. In this paper, we propose a novel anti-cloning method in accordance with the EPCglobal Class-1 Generation-2 (C1G2) standard. Our method only uses functions that can be supported by the standard and abides by the communication flow of the standard. The method is also secure against threats such as information leakage and password disclosure.     

Genetic science, animal exploitation, and the challenge for democracy

As the debates over cloning and stem cell research indicate, issues raised by biotechnology combine research into the genetic sciences, perspectives and contexts articulated by the social sciences, and the ethical and anthropological concerns of philosophy. Consequently, I argue that intervening in the debates over biotechnology requires supra-disciplinary critical philosophy and social theory to illuminate the problems and their stakes. In addition, debates over cloning and stem cell research raise exceptionally important challenges to bioethics and a democratic politics of communication.

外源小鼠核酸促电离辐射损伤的同系小鼠肠腺细胞修复的相关基因克隆

为研究克隆外源核酸促电离辐射损伤的小鼠肠腺细胞修复的相关基因。建立BALB／c小鼠电离辐射后给予外源RNA与生理盐水治疗的6、12、24h,4d和8d的模型,收集空肠组织标本后采用消减杂交基础上的LD－PCR技术,获取与受照射小鼠肠腺损伤修复相关的基因克隆,对其进行全自动测序与GeneBank检索,新基因递交给基因库。获取了90个与肠腺修复相关的基因,杂交证实在核酸治疗组与生理盐水治疗组之间呈差异表达,其中18个是新基因,GeneBank接受号为AF240164－240181。获取了90个可能与外源核酸促肠腺修复相关的基因克隆,其中18个是新的相关基因。     

Nitrification efficiency and nitrifying bacteria abundance in combined AS-RBC and A2O systems

This study makes a comparison between the nitrification performance of TNCU-I (a combined activated sludge-rotating biological contactor process) and A2O systems by the use of a pilot plant and batch experiments. The nitrifier abundance in both systems was determined, using cloning-denaturing gradient gel electrophoresis (DGGE) and fluorescent in-situ hybridization (FISH), to investigate the role of rotating biological contactor in the TNCU-I process. The stability of the nitrification performance and the specific nitrification rate were found to be greater in TNCU-I system than in the A2O system. RBC biofilm promoted nitrifying activity that contributed to the nitrification performance, especially at a low SRT. By using the cloning-DGGE method, the genera Nitrosospira and Nitrospira were found to be present in all the samples, while the genus Nitrosomonas was observed only in the TNCU-I RBC biofilm. In addition, the proportions of ammonia oxidizer in the TNCU-I RBC biofilm, the TNCU-I activated sludge and the A2O activated sludge were 11.4%, 13.2%, and 4.1%, respectively, higher than the nitrite oxidizer fractions of 3.3%, 5.7% and 2.1%, respectively, according to the cloning-DGGE method. On the other hand, the proportions of ammonia oxidizers in the afore-mention materials were 10.3%, 13.7%, and 5.2%, higher than the nitrite oxidizer fractions of 2.5%, 3.6% and 2.3%, according to the FISH experiments. This implies that the proportion of ammonia oxidizer in the TNCU-I process was 3.2 and 2.6 times that in the A2O process, determined by the cloning-DGGE and FISH methods, respectively. These amounts are also close to the ammonia oxidization rate of 2.9 times. All the data show that RBC added to the aerobic zone of TNCU-I process would increase the nitrifier abundance and enhance the nitrification performance of the system.     

睫状神经营养因子突变体的克隆、表达、纯化及生物学活性

目的设计具有更高生物学活性的睫状神经营养因子突变体,并对其进行表达、纯化及生物学活性检测。方法以计算机分子模拟系统设计突变体,重叠延伸PCR方法获得突变体的编码区DNA序列,克隆入表达载体pThioHisA,转化E.coliBL21,以IPTG诱导表达。复性纯化后,用鸡胚背根神经节无血清培养法和小鼠减重法进行生物学活性测定。结果目的蛋白以包涵体形式存在,表达量在35%以上,纯化后的纯度达95%以上,能有效地促进鸡胚背根神经节的生长,并能使正常小鼠的体重降低,脂肪指数下降。结论所设计的突变体经表达及纯化后,具有良好的生物学活性,为进一步研究突变体蛋白的促神经生长和减肥作用奠定了基础。