含G0S2基因启动子的荧光素酶报告基因载体的构建

目的:克隆人G0S2基因启动子并构建荧光素酶报告基因载体,为进一步研究G0S2基因转录调控提供质粒。方法:利用PCR技术从人胚肾293A细胞基因组DNA中克隆获得G0S2基因启动子的DNA片段,将其克隆至pGL3-basic表达载体中,并转化人大肠杆菌DH5α,经限制性内切酶酶切、PCR及测序鉴定得到确认;将重组载体质粒与半乳糖苷酶表达质粒psV-β-Galactosidase共转染至大鼠血管平滑肌细胞(VSMC),检测细胞中荧光素酶的活性。结果:pGL3-G0S2-Promoter重组质粒插入片段和相邻序列正确,克隆的G0S2基因片段有启动子活性(P0.05)。结论:成功构建了pGL3-G0S2-Promoter报告基因质粒,为进一步研究G0S2基因的表达奠定了基础。     

表达超抗原SEA基因的溶瘤腺病毒载体的构建与鉴定

目的:构建表达超抗原SEA基因的溶瘤腺病毒载体并鉴定.方法:采用PCR技术,从产SEA的葡萄球菌标准菌株ATCC13565基因组DNA中获得SEA全长基因序列,酶切后克隆入pCA13质粒,构建重组病毒质粒pCA13-SEA.将鉴定正确的pCA13-SEA与含有腺病毒右臂的质粒pBHGE3通过Lipofectamine2000共转染HEK293细胞,经同源重组产生重组腺病毒Ad-SEA.Ad-SEA在293细胞中大量扩增并通过氯化铯密度梯度离心法纯化、测定其滴度.结果:经PCR扩增、酶切鉴定、序列测定证实,SEA基因成功克隆到溶瘤腺病毒载体中,可实现SEA基因的表达.结论:成功构建了表达超抗原SEA基因的溶瘤腺病毒载体,为进一步研究该病毒对膀胱肿瘤靶向治疗的作用奠定了基础.     

麦迪霉素产生菌酮基还原酶基因在大肠杆菌中的表达

用PCR法扩增麦迪霉素产生菌酮基还原酶（MKR）基因,得到约0．8kb的DNA片段,扩增片段重组到利用依赖T7RNA聚合酶的高效表达载体pT7－7中,在大肠杆菌中表达出28．9kD的蛋白质。表达的蛋白质具有生物活性。     

牛呼吸道合胞体病毒G蛋白的截短表达与鉴定

经生物学软件DNA Star分析,将牛呼吸道合胞体病毒G基因截短成2个片段G1和G2。然后用人工合成的牛呼吸道合胞体病毒G基因为模板,用PCR分别扩增G1和G2基因片段,其大小分别为570 bp和308 bp。将目的片段定向克隆到pET30a表达载体中,酶切及测序鉴定均正确后,转化BL21表达菌,经IPTG诱导后G1和G2基因片段都获得了表达,且都为可溶性表达。用Ni柱亲和层析法在非变性条件下纯化重组蛋白,经免疫印迹试验鉴定证明纯化的重组蛋白G1具有良好的抗原性和特异性,而重组蛋白G2无反应性。应用纯化的重组蛋白G1进行的间接ELISA与免疫印迹试验在国内牛血清中检测到了BRSV血清抗体。本研究所表达的重组蛋白G1为基于牛呼吸道合胞体病毒G蛋白的血清学诊断方法的建立与牛呼吸道合胞体病毒G蛋白生物学功能的研究奠定了基础。     

2型登革病毒ns1基因克隆及其表达产物的生物学特性研究

目的 克隆并表达2型登革病毒非结构蛋白ns1基因片段,初步鉴定重组蛋白的生物学特性.方法 利用登革热2型病毒重组质粒,经PCR方法扩增出ns1全长基因片段,在pQE30表达系统中表达,表达产物用Ni柱亲和层析纯化后,用鼠抗登革病毒免疫血清对重组蛋白进行Western Blot及ELISA鉴定.结果 构建的重组质粒pQE-30/NSl,pQE-30/NS1-N,pQE-30/NS1-80-200aa和pQE-30/NS1-C经IPTG诱导,重组蛋白高效表达并纯化成功,经Western Blot及ELISA证实重组蛋白可以被免疫血清特异识别.结论 2型登革病毒结构蛋白表达载体在大肠杆菌SG13009中高效表达.纯化产物具有较强的免疫原性,为进一步研究NS1的生物学特性和血清学检测奠定了基础.     

相思豆毒蛋白—a A链在大肠杆菌中的表达及体外生物活性的测定

用RT PCR方法克隆了相思豆毒蛋白 aA链 (ABRaA)的cDNA编码序列 ,并将其重组到原核表达质粒 pET2 8b中。成熟的ABRaA在大肠杆菌中得到高效表达 ,可溶性重组蛋白质的获得率达 4mg/L培养物 ,而且具有较好的纯度。重组蛋白质体外生物活性的测定结果表明 ,其对兔网织红细胞裂解液的体外蛋白质生物合成具有较强的抑制作用 ,IC50 为 0 .0 8nmol/L ,与天然ABRaA的 (0 .0 6nmol/L)相差不大 ;重组ABRaA蛋白表现出较强的N 糖苷酶活性 ,其可切割大鼠肝脏核糖体 2 8SrRNA的A4 32 4位点 ,释放出一条约 4 2 0nt的小片段RNA。这些结果提示重组的ABRaA具有有效的生物活性 ,可以作为一种潜在的肿瘤化疗药物而用于制备免疫毒素。     

核苷酸切除修复基因XPA反义RNA表达载体的构建及其抑瘤功能

采用RT PCR方法扩增出 4 2 6bp着色性干皮病A(xerodermapigmentosumgroupA ,XPA)cDNA片段 (2～ 4 2 7bp) ,反向插入pcDNA3 1质粒构建XPA反义RNA表达载体 .经测序证实 ,该片段序列与XPAmRNA对应片段完全互补 .通过脂质体Lipofectamine 2 0 0 0将重组质粒转染肺癌A5 4 9细胞 ,RT PCR检测表明转染XPA反义RNA重组质粒能够抑制肺癌细胞XPAmRNA表达 ;MTT实验表明转染XPA反义RNA的肺癌细胞对顺铂敏感性增强 .本研究为深入探讨NER途径基因功能及临床克服肿瘤耐药提出了一个新的思路     

重组质粒二级结构对丙型肝炎病毒E2蛋白基因表达的影响

将含HCV/E2基因的重组质粒 pUC18/HCV -E2的BamHI酶切基因片段和NcoⅠ +HindⅢ酶切基因片段分别克隆到原核表达载体pRSETHis中 ,构建两株重组HCV/E2重组表达质粒 pRSET·C/E2和 pRSET·A/E2。在诱导表达中 pRSET·A/E2的外源基因获得了高效表达 ,而 pRSET·C/E2的外源基因完全未得到表达。     

金黄色葡萄球菌肠毒素A Asp227Ala基因的克隆及表达

目的：金黄色葡萄球菌肠毒素A Asp227ala基因的克隆及表达。方法：利用错配PCR方法,从含有金黄色葡萄球菌肠毒素A（Staphylococcal enterotoxn A,SEA)基因的质粒中扩增出约720bp的DNA片段,将其克隆到表达载体7ZTS中,并转化于JM109（DE3）。结果：重组质粒的测序结果表明,它含有702bp（不包括N端72bp的信号肽编码区）,其核苷酸序列与文献报道完全一致,推导的氨基酸序列显示227位的天冬氨酸已突变为丙氨酸。结论：该基因所表达的蛋白为可溶性蛋白,表达量占总蛋白51.5%。表达的蛋白与天然肠毒素A产生的抗体能发生凝集作用,具有与天然SEA类同的抗原活性。     

犬瘟热病毒核衣壳蛋白基因片段的克隆和表达

目的　构建pMal N重组表达载体 ,转化E .coliDH5α ,诱导表达犬瘟热病毒 (CDV)重组核衣壳 (N)蛋白。方法　采用RT PCR技术 ,从CDVRNA中扩增编码N蛋白的基因片段 ,通过连接反应 ,构建重组克隆载体和重组表达载体 ,转化感受态。E .coliDH5α细胞。通过IPTG诱导表达CDV重组N蛋白。结果　扩增出约 1 7kbCDV全长的主要结构蛋白N蛋白基因 ,通过PCR获得 5 36bpN蛋白基因片段。将N蛋白基因片段克隆入原核表达载体pMal C2 ,表达产物麦芽糖结合蛋白 (MAP)与N蛋白的融合蛋白的相对分子质量约 6 0× 10 3,与预期大小一致。结论　构建的pMal N重组载体所表达的CDVN蛋白为进一步研究CDV的特异、敏感的抗体检测方法打下基础     

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.