表皮生长因子受体胞外区在哺乳动物细胞HEK293T中的分泌表达和鉴定

目的:融合表达表皮生长因子受体(EGFR)胞外功能区,为制备针对该分子的特异性抗体提供可用的靶标抗原。方法:通过PCR扩增EGFR胞外区基因,将其克隆入真核表达载体pABhFc中,重组质粒瞬时转染HEK293T细胞,进行EGFR的瞬时分泌表达,纯化获得电泳纯级的分泌蛋白,并通过ELISA、Western印迹、Biacore3000系统对融合蛋白进行鉴定。结果:经测序证实扩增得到了正确的EGFR胞外区基因序列,SDS-PAGE初步确认获得了单、双体的EGFR胞外区,ELISA检测证实双体融合蛋白hFc-EGFR可与商业化的EGF特异性结合,Western印迹检测证实单体融合蛋白His-EGFR可与商业化抗体特异性结合;经Biacore3000蛋白分子相互作用系统测定,双体融合蛋白hFc-EGFR与商业化抗体爱必妥的亲和力达0.5 nmol/L。结论:利用哺乳动物细胞HEK293T分泌表达系统获得了结构正确的单、双体2种类型的EGFR胞外区融合蛋白纯品,将用于抗EGFR特异性抗体的筛选。     

c-Fos不同结构域的真核表达及功能鉴定

目的：克隆c-Fos蛋白不同结构域基因片段,构建其含FLAG标签的真核表达载体,并鉴定其功能。方法：用PCR方法扩增获得c-Fos不同结构域片段基因,定向克隆至本实验室保存的FLAG-peDNA3．0载体中,瞬时转染293T细胞进行表达,并以本实验室保存的HA-e-Jun与FLAG-c-Fos不同结构域片段免疫共沉淀鉴定其相互作用区域,以佐证所构建的c-Fos结构域克隆的正确性。结果：测序结果表明c-Fos不同结构域序列正确,Western印迹显示可以在真核细胞中获得表达,并且免疫共沉淀结果显示c-Jun可以与c-Fos的bZIP区域特异性结合。结论：构建并表达了c-F0s不同结构域片段,为进一步研究与c-Fos相互作用的蛋白及其区域定位奠定了基础。     

重组抗癫痫肽二串体的表达、纯化与鉴定

目的：利用基因工程方法表达抗癫痫肽（AEP）串联体蛋白,并进行纯化、鉴定。方法：PCR扩增AEP及AEP—His基因片段,并分别克隆至测序载体中;测序正确后,利用基因重组技术获得二串体基因,构建毕赤酵母表达载体pPIC9K-AEP2-His,甲醇诱导AEP2-His在毕赤酵母中表达,用Ni—chelating SepharoseFF柱纯化表达的融合蛋白,并用抗His单克隆抗体进行Westernblot鉴定。结果：构建了AEP二串体酵母表达载体,获得了纯化的AEP2-His蛋白,其相对分子质量约20000。结论：用基因串联思路表达小分子多肽是一种可行的方法;AEP2-His蛋白的成功表达为其功能研究奠定了基础。     

炭疽毒素保护性抗原第四结构域在大肠杆菌中的可溶性表达

人工优化设计并合成炭疽毒素保护性抗原第四结构域基因,并与噬菌体gⅢ蛋白N端结构域基因融合,在大肠杆菌中可溶性表达融合蛋白。结果表明合成了炭疽毒素保护性抗原第四结构域基因,并在大肠杆菌中获得了高效可溶性融合表达,可溶性表达产物占细菌总蛋白量的36％左右;经亲和层析纯化获得了重组蛋白;Western印迹分析表明,表达产物能与His单抗（重组蛋白羧基端带有6xHis）发生特异性结合反应。以上结果表明获得了炭疽毒素保护性抗原第四结构域,为利用人抗体库进行筛选抗炭疽毒素的人源性中和抗体奠定了基础。     

重组人骨桥蛋白在哺乳动物细胞中的表达、纯化和活性研究

旨在运用哺乳动物表达载体的瞬时转染技术,转染人类胚胎肾细胞(Human Embryonic Kidney 293E,HEK293E),分泌性表达和纯化带His标签的重组人骨桥蛋白(Recombinant Human Osteopontin,rhOPN),并研究其促结肠癌以及非小细胞肺癌细胞增殖功能。合成和构建OPN融合6×His标签重组蛋白的表达载体pcDNA3.1-OPN,利用聚醚酰亚胺(Polyetherimide,PEI)瞬时转染法将pcDNA3.1-OPN转染到HEK293E细胞中,并用镍亲和层析柱对rhOPN进行纯化。SDS-PAGE电泳和Western Blot被用来检测纯化后的rhOPN蛋白在凝胶上的迁移率和纯度。此外,ELISA方法被用于测定rhOPN与抗OPN抗体间的结合能力,并用CCK-8(Cell Counting Kit-8)方法初步研究rhOPN蛋白对结肠癌及非小细胞肺癌细胞增殖的影响。结果显示,通过将pcDNA3.1-OPN瞬时转染HEK293E细胞,成功表达和纯化出纯度达95%的rhOPN,且rhOPN可以被抗OPN抗体很好的识别。rhOPN被证实在36μg/ml浓度时即可显著促进体外培养的HT29细胞增殖,在20μg/mL时能够促进体外培养的非小细胞肺癌H1299及HCC827细胞增殖。运用哺乳动物细胞瞬时转染技术,在HEK293E细胞中成功表达并纯化出高纯度的rhOPN蛋白,并发现rhOPN可以显著促进结肠癌及非小细胞肺癌细胞增殖,具有良好的生物学活性。     

人酪氨酸蛋白激酶Lyn的真核表达、纯化及鉴定

[目的]构建含人酪氨酸蛋白激酶Lyn基因的载体并进行真核表达、纯化和研究其对细胞增殖的影响。[方法]提取人Hela细胞总RNA,用RT-PCR方法获得Lyn基因并克隆至pcDNA3.1(-)载体。经双酶切、PCR和测序方法鉴定后,将重组质粒瞬时转染HEK 293T细胞表达目的蛋白,应用组氨酸标签镍离子螯合磁珠纯化融合蛋白,通过Western Blot检测蛋白的表达及纯化,并用CCK-8法检测过表达Lyn后细胞增殖能力的变化。[结果]成功构建真核表达质粒pcDNA3.1(-)-Lyn并进行瞬时表达和蛋白纯化,CCK-8法检测过表达Lyn的HEK 293T细胞的增殖能力显著性下降(P0.01)。[结论]Lyn在HEK 293T细胞中成功瞬时表达及纯化,并可以使细胞的增殖能力受到明显抑制,为稳定表达和深入研究其生物学功能及作用机制奠定基础。     

突变型人TRBP 基因的构建及表达

目的:探讨RNAi过程中TRBP传递双链RNA的机制,构建RNA结合结构域突变的人TRBP基因真核表达载体。方法:设计突变引物,通过拼接PCR将突变型TRBP基因克隆入pFLAG-CMV4真核表达载体,经双酶切和测序鉴定正确后,命名为pFLAG-CMV4-TRBPm。瞬时转染HEK-293细胞,western-blot检测目的蛋白表达。结果:成功扩增了突变序列,构建了突变型TRBP基因的真核表达载体,转染HEK-293细胞后检测到带标签的目的蛋白表达。结论:突变型TRBP基因真核表达载体的成功构建,为进一步研究TRBP的生物学效应奠定了基础。     

一种人源抗狂犬病病毒单克隆抗体的瞬时表达及性质分析

目的瞬时表达人源重组抗狂犬病病毒单克隆抗体,并对抗体性质进行分析。方法 PCR法扩增抗体轻、重链可变区并分别构建真核表达质粒;瞬时转染HEK293 EBNA1细胞;抗体经亲和纯化后,CE-SDS法分析纯化后抗体单体比例,用快速荧光灶抑制试验(RFFIT)分析抗体体外抗狂犬病病毒中和活性,ELISA分析抗体与3aG、CTN、PV及CVS株的结合。结果提取瞬时表达质粒的A260/280 nm比值在2.0～2.1之间纯化后抗体非还原CE-SDS单体纯度为74.4%;还原CE-SDS抗体轻、重链峰合计占比超过95%;抗体RFFIT体外中和活性为293.37 IU/mL,比活达628.21 IU/mg,抗体与3aG、CTN、PV及CVS株交叉反应均为阳性。结论该株抗体具有较高体外抗狂犬病病毒中和活性,与主要疫苗株均有较强结合。     

单纯疱疹病毒I型糖蛋白D胞外区的真核表达及生物学活性分析

单纯疱疹病毒 (Herpes simplex virus,HSV) 包膜糖蛋白D (glycoprotein D,gD) 是HSV的结构蛋白之一,具有重要抗原表位,是目前疫苗研究的热点。为了分离纯化HSV gD1糖蛋白胞外区片段并对其生物学活性进行分析,本研究将化学合成的gD1胞外区基因片段克隆至真核表达载体pCEP4,重组质粒转染HEK293细胞进行瞬时表达,产物经Western blotting检测后用亲和层析法进行分离纯化,ELISA检测其抗原性。以纯化的重组蛋白作为抗原免疫小鼠,ELISA测血清特异性抗体效价以评价其免疫原性。构建的重组质粒经测序显示基因序列完全正确。表达产物的Western blotting分析发现,在相对分子量约46 kDa处有外源蛋白表达,与预期蛋白带一致。用Ni柱得到了纯化的重组gD1蛋白,ELISA检测显示其具有良好的抗原性,免疫小鼠7周后血清中抗体效价达到5×103。重组gD1蛋白的抗原性及免疫原性分析为HSV检测试剂和基因重组亚单位疫苗的研制提供了实验依据。     

大鼠脑特异基因rRP58的克隆及定位分析

EST（AW055733）是在SD大鼠脑内一特异表达基因cDNA的3′端片段,与人和小鼠的一个转录抑制因子基因（RP58）同源,利用RT-PCR的方法,从SD大鼠脑中得到rRP58基因两个不同长度的转录本,全长的rRP58基因编码产物为C2H2型锌指蛋白,含两个保守结构域;氨基端的POZ结构域及羧基端的锌指结构域（ZFD）。在POZ和ZFD结构域之间的序列,BLAST搜索未发现保守结构。但是富含酸性氨基酸残基,将编码ZFD结构域（nt1033-1569)及POZ结构域（nt1-372)的序列克隆到原核表达载体pQE30中,表达并分离纯化了His6-ZFD和His6-POZ（融合了6个组氨酸标签的蛋白质产物）。用His6-ZFD和His6-POZ免疫兔子后,得到了ZFD和POZ两者的多克隆抗体,并利用所制备的抗体进一步分析了rRP58基因多转录本产物在大鼠中的组织分布,免疫组织化学实验结果显示rRP58蛋白主要分布在小脑颗粒细胞中。     

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.