人IGF-1在大肠杆菌中的可溶表达和纯化

目的:在大肠杆菌中的可溶表达和纯化人胰岛素样生长因子1(hIGF-1)。方法:根据hIGF-1的氨基酸序列和大肠杆菌密码子偏爱性,利用重叠延伸PCR的方法合成hIGF-1DNA序列,构建表达载体,在大肠杆菌OrigamiB(DE3)中与硫氧还蛋白TrxA融合表达,并通过盐析和镍柱亲合层析进行纯化。结果:SDS-PAGE分析显示,重组融合蛋白以可溶形式存在,分子量约为28kDa,占上清总蛋白的50%以上。经盐析和镍柱亲合层析进行纯化,目标蛋白纯度可达到90%左右。结论:复合干扰素在大肠杆菌中的高效可溶表达。     

蛇毒纤溶酶Alfimeprase在大肠杆菌中的可溶表达和纯化

Alfimeprase是Fibrolase的突变体,是一种蛇毒纤溶酶,有纤溶活性而无出血性。根据Alfimeprase的氨基酸序列和大肠杆菌密码子偏爱性,利用PCR的方法合成Alfimeprase DNA序列,分别融合在NusA和MBP的C端,与分子伴侣FkpA在大肠杆菌Origami B(DE3)中共表达,融合蛋白NusA/Alfimeprase以部分可溶的形式存在,可溶部分占上清总蛋白的25%左右,通过镍柱亲合层析纯化和肠激酶切割得到具有纤溶活性的重组蛋白Alfimeprase。本研究是首次报道在大肠杆菌中可溶表达Alfimeprase,为以后深入研究其功能及应用奠定了基础。     

LSECtin-CRD-GST融合蛋白的原核表达、纯化及复性

目的:构建C型凝集素LSECtin主要功能结构域CRD的原核表达载体,在大肠杆菌中表达LSECtin-CRD-GST融合蛋白。方法:根据Gen Bank发布的LSECtin基因序列设计引物,利用基因重组技术将获得的LSECtin-CRDc DNA定向克隆至C端带GST蛋白标签序列的融合表达载体p GEX-6p-1中,转化大肠杆菌Origami(DE3)进行重组蛋白的诱导表达,用GST柱亲和纯化融合蛋白。结果:获得了原核表达载体p GEX-6p-1-LSECtin-CRD,诱导表达出大量相对分子质量约40×103的包涵体融合蛋白,经纯化、复性获得可溶蛋白,经Western印迹鉴定为目的蛋白。结论:获得足量的LSECtin-CRD-GST融合蛋白,为进一步研究CRD蛋白结构域的动态构象变化提供了实验材料。     

庚型肝炎病毒NS5 cDNA片段的表达及其免疫原性的研究

一段长度为880bp的庚型肝炎病毒cDNA在大肠杆菌BL21（DE3）菌株中得到表达。此cDNA被插入到表达质粒pGEX-5X-1中,位于编码日本血吸虫谷胱甘肽硫转移酶（GST）的DNA序列下游,并与GST处于同一阅读框。用乳糖在37℃下诱导表达出以体形式存在的GST－NS53融合蛋白,并用脲溶法提取了该蛋白;在20℃诱导时,表达出的蛋白大部分可溶,用谷胱甘肽Sepharose－4B亲和层析柱对可溶性的融合蛋白进行了纯化。免疫印迹实验证明,此融合蛋白能这臾型肝炎病人的血清和自制的扩GST在汪有特异性地识别。用PCgene软件对NS53氨基酸序列的亲水性和抗原决定簇进行了分析,本研究为庚型肝炎ELISA诊断试剂研制打下了基础。     

次级淋巴组织趋化因子（SLC）基因克隆及在大肠杆菌中的表达

次级淋巴组织趋化因子(SLC)是通过搜索表达序列标签(EST)数据库克隆出来的一CC类趋化因子。以人SLC序列为蓝本,利用重叠PCR(SOE-PCR)的方法获得了适宜在大肠杆菌中表达的SLC基因,将此序列分别克隆至表达载体pTMF和pALM中,转化大肠杆菌,诱导表达。Western Blotting鉴定结果表明目的蛋白以可溶蛋白和包涵体两种形式表达,两种形式的蛋白所占比例依培养和诱导条件的不同而变化。对两种形式的表达产物分别用Ni-NTA金属亲和层析和包涵体复性方法纯化在实验中还对纯化条件进行了探索。对纯化蛋白的电泳结果显示:纯化样品的分子量比预期的分子量要大。     

人源抗菌肽LL-37的原核表达及活性鉴定

目的:实现大肠杆菌高效可溶表达人源抗菌肽LL-37。方法:LL-37基因克隆至原核载体pET32a,于大肠杆菌BL21(DE3)中诱导表达。运用相关生物信息学软件分析重组蛋白Trx-LL-37的理化性质、亲/疏水性、蛋白质二级结构及其可溶表达概率。实验还考察了不同诱导温度对重组蛋白可溶表达比例的影响。结果:生物信息学分析显示,Trx-LL-37分子量21.5kD,理论等电点6.3,物理性质稳定,二级结构简单,具有可溶表达倾向。重组蛋白最佳诱导温度为17℃,与37℃相比,可溶表达比例由37.2%提高至50.2%,并且总表达量也提高了5%左右。抑菌结果显示纯化产物对多种常见细菌的生长具有抑制作用。结论:可采用融合方式通过原核系统高效可溶表达LL-37,为LL-37的功能研究打下基础。     

Zif268的锌指DNA结合区在大肠杆菌中的功能性表达

锌指蛋白是最大的蛋白家族,是识别核酸最常见的、最有效的结构元件。通过选择合适的表达载体及诱导表达条件,实现了小鼠转录因子Zif268的锌指DNA结合区在大肠杆菌中的部分可溶性表达。凝胶迁移率移动试验证实纯化的可溶部分锌指DNA结合区可以特异性识别、结合其天然靶序列,提示锌指DNA结合区在大肠杆菌中得到了功能性表达。锌指DNA结合区在大肠杆菌中的功能性表达成功为锌指蛋白DNA相互作用的胞内遗传筛选模型的建立奠定了基础。     

一个新的产黄青霉谷胱甘肽转移酶基因的克隆和鉴定

以产黄青霉(Penicillium chrysogenum Thom)cDNA为模板,克隆得到一个新的谷胱甘肽转移酶基因PcgstB,其开放阅读框长651bp,编码216个氨基酸的蛋白质。与已知序列进行BLASTp比较显示,该蛋白具有保守的GST结构域,与烟曲霉GstB的序列一致性最高,达65%。将PcgstB与原核表达载体pTrc99A连接得到表达质粒pTrc-gstB,转化大肠杆菌DH5α,经IPTG诱导后获得以可溶形式表达的重组PcGstB蛋白。以1-chloro-2,4-dinitrobenzene(CDNB)为底物检测,确认该蛋白具有GST活性。     

重组人干细胞因子的纯化

干细胞因子 (SCF)是一种重要的造血生长因子 ,它在自体造血干细胞动员、肿瘤放疗化疗的辅助治疗、贫血和其它血液病的治疗上具有良好的应用前景 .为建立一种实用的rhSCF制备工艺 ,从表达rhSCF的工程菌分离包涵体 ,用尿素变性 ,低浓度尿素溶液稀释复性 .复性液中的rhSCF经离子交换层析吸附、浓缩 ,凝胶排阻层析分离去除共价二聚体和疏水层析精纯化 ,获得纯化的rhSCF .纯化的rhSCF纯度大于 95 % ,回收率为 4 7% ,可促进人红白血病细胞TF - 1的生长 ,比活性为 0 .9× 10 6U mg ,与英国NIBSC的rhSCF标准品相似 .上述结果表明 ,所建立的制备高纯度rhSCF工艺高效简便 ,产物具有良好的生物活性     

人类肠道病毒68型3A蛋白可溶区的表达及其结构初步研究

为了解人类肠道病毒68型3A蛋白可溶区的结构,本研究构建了EV-D68 3A蛋白可溶区的原核表达载体,在大肠杆菌中表达、纯化并对其结构进行了初步研究。首先,采用PCR的方法扩增EV-D68 3A(1~61)基因片段,克隆至原核表达载体pET-28a-His-SUMO中,转化进入BL21(DE3)感受态细胞,诱导表达出His-SUMO-3A(1~61)融合蛋白。经Ni-NTA树脂亲和层析初步纯化后得到大量融合蛋白,利用ULP酶酶切去除His-SUMO标签,通过Ni-NTA树脂亲和层析、阴离子交换层析柱、分子筛层析等方法分离标签并进一步纯化目的蛋白。最后通过化学交联反应检测目的蛋白的多聚化状态。结果显示,利用pET28a-His-SUMO-3A(1~61)原核表达载体能够在大肠杆菌中成功表达出大量可溶性的融合蛋白;经过多步纯化方法得到了大量高纯度的目的蛋白,平均每升大肠杆菌可得到约5mg目的蛋白,纯度达95%以上;通过化学交联反应证明了该蛋白的多聚化存在形式。本研究成功建立了EV-D68 3A蛋白可溶区的表达和纯化系统,为进一步获得3A蛋白晶体、研究3A蛋白功能以及设计以3A为靶点的抗病毒药物奠定了基础。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.