无标签人源性脂联素球状结构的表达、纯化与活性检测

目的:利用基工程方法构建无标签人性脂联素球状结构(gAd)基的核表达载体,并对重组蛋白进行诱导表达、纯化及鉴定.方法:从正常人脂肪组织里提取总 RNA,反转录合成 cDNA,经 PCR 扩增、酶切后连入pET-22b(+)载体构建重组质粒 pET-22b(+)-gAd,转化大肠杆菌 BL21(DE3)感受态细胞,经低温、低浓度 IPTG 诱导使其可溶性表达,采用硫酸铵沉淀、凝胶过滤层析和离子交换层析三步分离纯化,得到不带任何标签的人性gAd;运用 SDS-PAGE、Western 印迹、HPLC 对重组蛋白进行鉴定,通过对 AMP 激活的蛋白激酶(AMPK)的磷酸化水平检测纯化蛋白的生物学活性.结果:构建了核表达载体 pET-22b(+)-gAd,实现了人性 gAd 在核细胞中的可溶性表达,纯化的蛋白经 SDS-PAGE 和 Western 印迹分析证实为 gAd,HPLC 分析蛋白纯度达到95%以上;通过对 AMPK磷酸化水平的检测,证明纯化的 gAd 具有高生物学活性.结论:重组表达和纯化了无标签、高生物学活性的人性脂联素球状结构,为其进一步的理论研究、生产开发奠定了基础.     

重组A型产气荚膜梭菌α毒素C端片段的分离纯化

押在构建出高表达、高活性的A型产气荚膜梭菌α毒素保护性抗原工程菌株pBV/cpa408的基础上,对表达产物进行了分离纯化研究。诱导表达菌液离心后,超声破碎沉淀细菌,上清用80%饱和硫酸铵沉淀,沉淀蛋白经透析,上凝胶过滤层析柱分离纯化,得到纯度达95%以上的表达目的蛋白,经SDS-PAGE测定,相对分子质量为15.5×103,序列与文献报道的相符。     

重组干扰素-τ在大肠杆菌中的表达与纯化

目的:探讨干扰素(IFN)-τ在大肠杆菌中的表达及纯化。方法:含有IFN-τ基因的pBV220表达质粒转化大肠杆菌BL21,于42℃温控诱导重组菌表达IFN-τ。经过包涵体溶解、DEAE离子交换层析、硫酸铵沉淀及梯度透析,使重组IFN-τ获得纯化和复性。结果:诱导后的表达产物经SDS-PAGE分析,有相对分子质量约21000的条带。纯化复性后目的蛋白纯度可达90%。结论:工程菌可稳定高效地表达IFN-τ。硫酸铵沉淀结合阴离子交换是一种简便高效的纯化方法,可获得较高纯度的IFN-τ。     

洋葱伯克霍尔德氏菌产邻苯二酚2,3-双加氧酶的研究

对洋葱伯克霍尔德氏菌L 68生长及产邻苯二酚2,3 双加氧酶(C23D)的条件进行了研究,其最适产酶pH7.2;最适生长温度30～35℃;最适培养时间48h;苯酚浓度0.09%最有利于菌体产酶.对菌株L 68产生的C23D酶进行了纯化,超声波破碎后的细胞提取液经硫酸铵分级沉淀、DEAESepharoseFastFlow层析、Hydroxyapatite层析、SephadexG 150层析后,收率为20%,酶比活力提高了230倍.SDS PAGE检测得到了分子量为(34±1)kDa的蛋白.     

特异识别HIV-1序列的锌指蛋白的表达纯化及其在Biacore CM-5芯片上的固定

目的:为了更好地利用Biacore 3000研究锌指与核酸的相互作用,将特异性识别HIV-15′端一段保守序列的三锌指蛋白固定在CM-5芯片上。方法:将特异性识别HIV-15′端一段保守序列5′-CTGTGTTTG-3′的三锌指基因克隆到表达载体pET-22b( )中,转化大肠杆菌BL21(DE3)菌株,经IPTG诱导表达重组三锌指蛋白,超声碎菌进行SDS-PAGE分析;包涵体形式的表达产物用盐酸胍溶解后,经一步凝胶柱复性并纯化;随后摸索适宜固定的pH值并通过化学方法进行固定。结果:表达的重组蛋白主要以包涵体形式存在于超声沉淀中,纯化及柱复性后的蛋白纯度为98.8%,并在CM-5芯片上成功固定。结论:本研究为利用Biacore实时定量研究锌指蛋白与其识别DNA的相互作用进行了尝试。     

重组A型产气荚膜梭菌α毒素保护性抗原的制备及初步免疫保护作用研究

诱导表达重组工程菌Pbv/cpa408后,将表达菌体超声破碎,上清经80%饱和硫酸铵一次沉淀,经透析,上凝胶过滤层析柱进行分离纯化,薄层凝胶扫描结果显示,纯化的蛋白纯度达95%以上;用纯化蛋白免疫昆明小鼠,以1.0MLD100腹腔进行攻击,被免疫小鼠获得了100%的保护。     

融合干扰素-BLA（IFN-BLA）的构建、表达、纯化及抗病毒活性测定

干扰素-BLA(IFN-BLA)由干扰素-beta-1b(IFN-beta-1b)和干扰素-alpha-2b（IFN-alpha-2b）通过连接肽-GGGS-融合而成。优化了其在大肠感菌BL21 CodonPlus (DE3)-RIL中的实验室表达条件,表达的目的蛋白占菌体总蛋白的35％以上并且主要以包涵体的形式存在。对包涵体的复性条件进行了摸索,建立了IFN-BLA的复性及纯化方法,纯化后的蛋白产量约为45 mg/L, 纯度在90％以上。抗病毒活性分析表明这一新的融合蛋白可能具有协同或加成活性。     

人纤溶酶原饼环区5(hPK5)基因的分泌型表达

构建人纤溶酶原饼环区5(hPK5)基因的原核可溶性表达载体并进行表达和纯化,获取大量高纯度、具有生物活性的hPK5蛋白。以纤溶酶原cDNA为模板,PCR扩增了hPK5基因,经过适当酶切后构建表达载体pET22b(+)hPK5,转入大肠杆菌BL21(DE3)进行表达并经组氨酸亲和层析获得纯化。带有重组质粒pET22b(+)hPK5的大肠杆菌经IPTG诱导后以可溶性形式表达16kDa的蛋白,其表达量占菌体总蛋白的30%以上,纯化后目的蛋白纯度达95%以上,Western印迹表明重组蛋白具有Histag抗原活性。构建了pET22b(+)hPK5重组质粒并成功地在大肠杆菌中获得可溶性表达,为获得大量hPK5基因工程产品奠定了实验基础。     

头孢菌素脱乙酰酶的重组表达及固定化

本文构建了利用trp启动子表达头孢菌素脱乙酰酶(CAH)的重组大肠杆菌DH5α-pCAH。重组菌在7L发酵罐(装液量2L)中发酵28 h,发酵液OD_(600)达到27,产酶313 kU/L发酵液,粗略估算重组蛋白占细胞总蛋白的70%。发酵生产的重组CAH粗酶液经过硫酸铵分级沉淀分离纯化和超滤除盐浓缩两步操作,纯化倍数为1.44,总酶活回收率56%,聚丙烯酰胺凝胶电泳检测纯化后蛋白没有明显杂蛋白条带出现。纯化后的CAH共价结合固定在环氧基载体LX-1000EP(c)上,通过对固定化条件的优化最终得到固定化酶比活443 U/g。该固定化酶重复催化50 mL 5%7-ACA底物100次后,酶活没有降低。     

B型肉毒毒素轻链的高效制备及活性鉴定

目的:在大肠杆菌中高效表达B型肉毒毒素轻链(BoNT/BLC)并纯化,研究其生物学活性。方法:根据报道的BoNT/B LC基因序列设计引物,从肉毒梭菌中扩增BoNT/BLC基因片段,将其克隆至原核表达载体pET-22b中,重组质粒转化大肠杆菌BL21(DE3)Rosetta感受态细胞,构建重组大肠杆菌pET-22b BoNT/BLC/BL21(DE3)Rosetta,在20℃条件下用IPTG诱导目的蛋白表达,表达产物经His Trap FF柱纯化,用SDS-PAGE对目的蛋白进行鉴定,并利用相应底物对纯化产物进行生物活性分析。结果与结论:构建了重组大肠杆菌pET-22b BoNT/BLC/BL21(DE3)Rosetta,BoNT/BLC表达量达到了细菌总蛋白的30%左右,通过一步亲和纯化目的蛋白后经SDS-PAGE检测其纯度在95%以上,制备的重组LC的酶活略高于B型肉毒毒素全毒素,可作为试剂用于BoNT/BLC抑制剂高通量体外检测方法的研究。     

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.