果蝇中Ecp蛋白的表达、纯化与抗体制备

获得特异性抗Ecp多克隆抗体,为进一步研究ecp的功能奠定基础。利用特异引物,通过RT-PCR扩增出编码Ecp蛋白的全长cDNA,克隆至谷胱甘肽S转移酶融合蛋白表达载体pGEX-4T-1(His)6C中,转染大肠杆菌DH 5α,经诱导表达后,利用谷胱甘肽琼脂糖珠从细胞裂解物中特异吸附融合蛋白,经凝血酶裂解,释放出Ecp蛋白,再经Ni-NTA亲和层析,最终获得高纯度的Ecp蛋白。用纯化的Ecp蛋白免疫新西兰家兔,亲和层析纯化抗Ecp抗体。利用该抗体进行的Western Blot结果表明：Ecp蛋白在野生型黑腹果蝇胚胎、三龄幼虫神经系统、成虫、成虫头部组织中均有明显表达,提示ecp可能是一个重要的管家基因。     

重组人卵透明带ZP3蛋白在毕赤酵母中的表达

利用P.pastoris表达人卵透明带ZP3蛋白。设计特定引物从全长hZP3 cDNA上扩增含跨膜区序列的人卵透明带ZP3基因片段,并在N末端接上串联组氨酸编码序列的重组基因序列;扩增片段插入表达载体pPIC9K中;线性化后的重组质粒转入P.pastoris中,用高浓度G418筛选高拷贝菌株,然后甲醇诱导目的蛋白表达。用SDS-PAGE和Western blot分析表达产物。结果发现P.pastoris表达的人ZP3蛋白可以分泌到培养液中,并且可溶性好。纯化前后的重组人ZP3蛋白均能与兔抗猪ZP3蛋白抗体发生交叉反应,证实表达的目的蛋白具有反应原性。     

嗜水气单胞菌外膜蛋白基因ompTS的高效表达及其免疫原性

根据嗜水气单胞菌外膜蛋白基因ompTS的核苷酸序列设计引物,运用聚合酶链式反应（PCR）扩增出与预期大小相符的基因片段。将此基因片段克隆至质粒pRSET A的BamHI和EcoRI位点,构建重组质粒,转化大肠杆菌BL21（DE3）,经IPTG诱导获得高效表达,SDS-PAGE蛋白电泳表明在39.9kD处出现超强特异带,占总蛋白的51％。以 Ni-NTA-Conjugate抗体进行Western blot分析证明该399kD的蛋白为所表达的融合蛋白。纯化融合蛋白注射雄性新西兰大白兔可诱导产生特异抗体。ELISA和Western blot检测结果显示,该抗体与表达的融合蛋白和从嗜水气单胞菌中提取的36.9 kD外膜蛋白均呈阳性反应,表明所表达的融合蛋白仍保持原有外膜蛋白的免疫原性,为此融合蛋白作为疫苗的候选成份提供理论基础。     

液相芯片技术抗体修饰制备条件的优化

[目的]优化以琼脂糖水凝胶包裹的光子晶体为载体的液相芯片抗体修饰制备方法,提高抗体修饰密度及检测灵敏度。[方法]选取人Ig G作为待检靶蛋白,在琼脂糖水凝胶上首先固定羊抗人Ig G抗体,经过洗涤、封闭后,依次加入标准抗原、荧光标记的检测抗体,分别孵育、洗涤后,倒置荧光显微镜获取图像及荧光强度,根据荧光强度优化最佳琼脂糖水凝胶浓度、固定时间、氢氧化钠浓度、环氧氯丙烷浓度以及抗体浓度。[结果]在水凝胶浓度4%、Na OH浓度1.0mol/L、环氧氯丙烷浓度10%、反应时间3h、抗体浓度0.01mg/ml的条件下,荧光强度达到最强,即抗体修饰密度达到最大。[结论]该研究优化了以琼脂糖水凝胶包裹的光子晶体为载体的抗体修饰反应条件,提高了液相芯片检测的灵敏度。     

兔抗麻雀IgY酶标抗体的制备

目的制备辣根过氧化物酶（HRP）标记的兔抗麻雀IgY抗体,为禽类血清学检测体系的建立提供技术储备。方法硫酸铵盐析法粗提麻雀血清IgY,进一步在SDS-PAGE上分离后,切下带有目的条带的凝胶作为免疫原,免疫实验兔制备抗血清,Protein-A柱亲和纯化兔抗IgY血清IgG,,使用改良过碘酸钠法制备酶结合物。ELISA检测酶标抗体的工作浓度,western blotting检测酶标抗体的特异性。结果硫酸铵盐析法粗提IgY,可去除部分杂蛋白,SDS-PAGE上分离后切下带有目的条带的凝胶,可以得到足够纯度的抗原,将带有IgY的凝胶作为抗原免疫后获得的抗血清经Protein-A纯化后,二抗在SDS-PAGE上鉴定,纯度达到99%以上。改良的过碘酸钠法标记获得的抗体浓度为1.008 mg/mL,ELISA检测酶标抗体效价为1∶1000。Western blotting鉴定抗体具有特异性。结论获得了优质可靠的兔抗麻雀IgY酶标抗体。     

硫氧还蛋白抗体柱的制备

目的:探索硫氧还蛋白(Trx)抗体柱对Trx融合蛋白纯化的可行性。方法与结果:对含有Trx基因的质粒表达载体pTrxFus进行改造,在Trx读框之后加入6×His序列,并在大肠杆菌中表达C端带有6×His标签的Trx,经Ni2+柱亲和纯化后制备多克隆抗体;把经蛋白A纯化后的抗体偶联在溴化氰活化的琼脂糖凝胶上,制成Trx抗体柱;用此抗体柱纯化与Trx融合表达的豇豆胰蛋白酶抑制剂(CpTI),SDS-PAGE结果显示获得了纯度较高的Trx-CpTI。结论:用Trx抗体制成的免疫亲和层析柱可以有效纯化Trx融合蛋白。     

C19orf18蛋白的表达与纯化及多克隆抗体制备

旨在得到较高浓度和纯度的C19orf18蛋白,再将得到的目的蛋白用于制备其特异性的多克隆抗体。利用PCR技术扩增出C19orf18基因的胞内段和胞外段,中间用柔性肽连接,再将目的片段克隆到p ET28a(+)与p ET32a(+)载体上,通过诱导表达少量的目的蛋白,筛选出表达量较高的载体,再用筛选得到的表达量较高的载体大量表达目的蛋白,通过镍柱纯化得到较纯的C19orf18蛋白。用得到的蛋白免疫新西兰白兔,得到的抗血清先经过Protein A纯化,再进行抗原亲和纯化得到C19orf18蛋白多克隆抗体。结果显示,载体p ET28a-C19orf18蛋白表达量高于p ET32a-C19orf18,经过镍柱纯化后得到了较高浓度与纯度的目的蛋白,利用得到的目的蛋白作为抗原成功制备了其特异性的多克隆抗体。在原核细胞中成功表达了C19orf18重组蛋白,并得到了其特异性多克隆抗体,所制备的多克隆抗体可应用于ELISA、蛋白免疫印迹实验。     

大肠杆菌β-D-半乳糖苷酶的纯化与鉴定

大肠杆菌β-D-半乳糖苷酶的两种纯化方法:DEAE A50和sepharose 6B两次层析法、与底物类似物的亲和层析法均可获高纯度酶,后者方法简便,适于大量制备。酶在PAGE上显示六条区带;主区带与其余三条活性带迁移率分别为13.5,7.5,6.3和4.6mm。酶主区带分子量130000。酶与其相应抗体的免疫电泳呈两条沉淀线。酶米氏常数为4.348×10^-4M。其SH基含量18.5个/克分子蛋白。酶悬液于4℃保存稳定性较好。活力500u/mg蛋白。可达美国Sigma     

抗流感病毒单克隆抗体CR6261在糖基工程酵母中的表达及分析

目的:在糖基工程酵母中表达抗流感病毒单克隆抗体CR6261,并对其进行结构分析和活性测定。方法:从GenBank获得CR6261抗体的全基因序列,构建轻重链表达载体pPICZα-CR6261-H-L,电转入糖基工程酵母,利用甲醇诱导型启动子AOX1表达CR6261抗体蛋白,通过SDS-PAGE、Western印迹筛选表达菌株,用Protein A亲和层析、Superdex200凝胶过滤柱分离纯化抗体蛋白,应用ELISA方法对纯化后的蛋白进行活性鉴定。结果:CR6261抗体蛋白分泌在培养上清中,且纯化后的蛋白与糖基工程酵母表达的流感病毒血凝素蛋白HA5、HA7有结合活性,与商业化三价流感病毒裂解疫苗亦有结合活性。结论:实现了抗流感病毒单克隆抗体CR6261在糖基工程酵母中的表达,并初步检测了其广谱活性,可为酵母表达抗体蛋白药物制备提供参考,ELISA初步评价还显示了其在诊断上的应用前景。     

抗体在二氧化硅表面固定及活性测定

应用分子自组装技术,通过表面羟基化、氨基硅烷化和对苯二甲醛组装,在二氧化硅表面衍生活泼的醛基基团。利用抗体的氨基和醛基发生还原胺化反应将抗体固定在二氧化硅表面,通过抗原抗体反应定性检测固定抗体的生物活性。结果显示应用这种方法能够有效将抗体固定在二氧化硅表面,并保持抗体生物活性,该固定方法在蛋白质检测、分析和蛋白质芯片中有广泛应用价值。     

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.