ZO-1基因的慢病毒载体构建

目的：相邻两心肌纤维的连接处称心肌闰盘,它在心肌的机电活动中扮演重要角色。目前有关闰盘相关基因Cx43的研究已被广泛关注,而在不同心肌病理状态下,其变化及功能都与另一闰盘相关基因ZO-1有极为密切的联系。目前有关ZO-1的研究比较少,此次欲将猪ZO-1基因克隆到慢病毒表达载体并进行鉴定,为进一步研究其功能奠定基础。方法：利用聚合酶链反应（PCR）扩增猪ZO-1基因,并克隆到慢病毒表达载体,通过转化、抽提质粒、双酶切和测序鉴定构建的Lenti-EFlct—EGFP-TRE—ZO-1重组载体。结果：猪ZO-1基因片段重组到慢病毒载体;PCR和双酶切鉴定,电泳结果显示均能得到与理论大小相符的片段;经测序证实成功构建Lenti-EF1α—EGFP—TRE-ZO-1慢病毒表达载体。结论：通过一系列实验证实Lend．EF1α—EGFP—TRE—ZO—1慢病毒表达载体构建成功,可利用其感染相关细胞或注入动物体内,为后续的体外及在体研究奠定基础,明确其在不同心肌病理状态下所发挥的独特作用。     

乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒的构建及鉴定

目的：构建乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒,为探讨HPSE在在肿瘤浸润转移过程中的分子机理奠定基础。方法：乙酰肝素酶cDNA全长扩增和最佳siRNA干扰片段筛选分别采用PCR和Real-time PCR方法,慢病毒系统载体分别使用pWPI和siRNA pSico系统,采用限制性内切酶快速连接方法联接目的基因和最佳最佳siRNA干扰片段,表达载体鉴定均采用核苷酸序列测定,HPSE重组慢病毒表达质粒和siRNA片段细胞转染采用脂质体转染法。结果：成功扩增乙酰肝素酶全长并连接入pWPI载体构建成重组表达载体HPSE-pWPI,重组质粒测序结果显与HPSE基因的同源性达99%。转染293T后有HPSE基因的表达。筛选出最佳siRNA干扰片段为HPSE-1222并成功插入pSico载体,构建成重组表达载体HPSE-siRNA pSico,重组载体测序显示与构建的shRNA结构序列完全一致。结论：成功采用慢病毒载体系统构建了乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒,为探讨HPSE在在肿瘤浸润转移过程中的分子机理奠定基础。     

Netrin-1慢病毒表达载体的构建及鉴定

目的:构建携带有Netrin-1基因的逆转录病毒载体,为研究Netrin-1在神经发育中的作用奠定基础。方法:PCR扩增Netrin-1基因片段后,将其克隆入慢病毒表达载体pLXSN;通过PCR、酶切、测序鉴定重组质粒。重组质粒转染PA317包装细胞后获得包装的病毒颗粒。病毒颗粒感染人脑胶质瘤细胞SW038-C2,经Western blot证明重组病毒在真核细胞内表达Netrin-1的情况。结果:经PCR扩增、酶切和测序验证,重组质粒构建正确,命名为pLX-NT。Western blot证明在感染细胞泳道有一特异性条带。结论:成功构建了能表达Netrin-1的慢病毒载体。     

以减毒沙门氏菌为载体山羊痘病毒P32基因真核表达载体的构建与鉴定

目的：构建以减毒沙门氏菌为载体山羊痘病毒P32基因真核表达载体。方法：以pMD-18T-P32/LD为模板,通过聚合酶链反应（PCR）扩增出山羊痘病毒P32基因片段,定向插入真核表达载体pVAX1中,再重组表达质粒pVAX1-P32/LD。转入DH5α,再转入减毒鼠伤寒沙门氏菌,通过双酶切、PCR及插入片段序列测序对质粒进行鉴定。结果：携带山羊痘病毒P32基因片段的重组减毒鼠伤寒沙门氏菌疫苗S7207/pVAX1-P32/LD已成功构建。结论：为山羊痘基因工程疫苗的研制及诊断方法的建立奠定了基础。     

介导大鼠结缔组织生长因子基因沉默的慢病毒载体转移质粒的构建及鉴定

目的：构建介导大鼠结缔组织生长因子（CTGF）基因沉默的慢病毒载体转移质粒pGCL-CTGF,为进一步包装慢病毒载体奠定基础。方法：以大鼠CTGF基因为靶基因,根据RNA干扰（RNAi）序列设计原则,设计4对有小发夹结构的RNAi靶点序列,退火形成双链DNA,双酶切后定向克隆到慢病毒载体转移质粒pGCL-GFP中,构建4个含靶基因片段的重组慢病毒载体转移质粒pGCL-CTGF,并对质粒进行PCR及测序鉴定。结果：CTGF的短发夹RNA（shRNA）片段被成功克隆到慢病毒载体转移质粒pGCL-GFP中,4个插入序列与设计的靶基因片段完全一致。结论：构建了能够表达4个含CTGF靶基因片段的慢病毒载体转移质粒,为进一步包装介导CTGF基因沉默的慢病毒载体奠定了基础。     

重组表达小鼠MIP-1α和B7-1基因的慢病毒载体的构建及鉴定

目的构建重组表达小鼠MIP-1α和B7-1基因的慢病毒载体,为淋巴瘤基因治疗的实验研究奠定基础。方法设计引物扩增获得目的基因小鼠MIP-1α和B7-1基因的全长编码序列cDNA,将目的基因与经酶切线性化的慢病毒载体进行定向连接,其产物转化感受态细胞,对长出的阳性克隆进行PCR鉴定和直接测序序列分析。MIP-1α和B7-1目的基因质粒转染293T细胞,观察绿色荧光蛋白（GFP）表达,采用Western Blot法检测其蛋白表达,实时荧光定量PCR,检测慢病毒浓缩液的滴度。结果成功构建了重组表达小鼠MIP-1α和B7-1基因的慢病毒载体,实时荧光定量PCR证实MIP-1α、B7-1基因重组慢病毒载体的滴度均达2.00E＋8 TU/mL。结论本研究成功构建并包装出高滴度的小鼠MIP-1α和B7-1基因重组慢病毒载体,为淋巴瘤基因治疗的实验研究奠定了基础。     

小鼠resistin基因慢病毒表达载体构建及在KMB17细胞中的表达

[目的]构建携带小鼠resistin基因的慢病毒表达载体,并高效转导靶细胞。[方法]从小鼠脂肪组织提取总RNA,经PCR扩增、酶切、连接、转化后构建重组质粒;通过293T细胞包装为重组慢病毒颗粒,然后感染KMB17细胞,检测小鼠resistin基因在靶细胞中的表达。[结果]PCR扩增到预期的342 bp大小的resistin基因目的片段;构建的重组质粒经酶切得到了342 bp大小的目的片段,测序鉴定为小鼠resistin基因序列;重组慢病毒包装可观察到很强的绿色荧光,说明具有较高的质粒转染效率;重组慢病毒感染KMB17后可检测到小鼠resistin基因转录水平和蛋白水平的表达,证明resistin基因得到有效转导并可在靶细胞中高水平表达。[结论]成功构建了携带小鼠resistin基因的慢病毒表达载体并可在KMB17靶细胞中获得高效表达。     

CXCR4慢病毒载体的构建及其在人脐带血间充质干细胞的表达

目的：构建趋化因子CXC亚族CXCR4的慢病毒表达载体并观察其转染人脐带间充质干细胞后的表达。方法：用逆转录PCR方法获取CXCR4基因编码区片段,将构建的慢病毒载体质粒pLVTHM-EGFP-CXCR4与包装质粒psPAX2和包膜质粒pMD2．G共转染293T细胞,包装生产慢病毒。用相同滴度的慢病毒转导等量间充质干细胞（Mesenchymal Stem Cell,MSCs）,后采用Real time PCR检测CXCR4 mRNA、Western Blot方法检测蛋白质的表达。结果：PCR、酶切和测序结果表明成功的构建了CXCR4基因重组慢病毒载体。同时用该慢病毒载体转染MSCs后可有效地增加MSCs中CXCR4的表达。结论：成功构建了CXCR4的慢病毒表达载体并能在MSCs中表达,为进一步研究其在干细胞移植中的应用奠定基础。’     

携带人二氢叶酸还原酶基因慢病毒表达载体构建及鉴定

目的:构建携带人二氢叶酸还原酶(DHFR)基因的慢病毒表达载体pWPI。方法:采用PCR方法扩增二氢叶酸还原酶cDNA全长,与EZ-T克隆载体连接,HindIII及BamHI-HF限制性内切酶双酶切回收的PCR片段并补平其缺口。慢病毒系统载体使用pWPI系统,采用PmeI酶切载体后回收片段,将其磷酸化,T4酶连接载体与目的基因。表达载体鉴定均采用核苷酸序列测定,重组质粒采用脂质体转染293T包装细胞后获得包装的病毒颗粒。结果:成功扩增二氢叶酸还原酶全长并连接入pWPI载体构建成重组表达载体DHFR-pWPI,重组质粒测序结果显与DHFR基因的同源性达100%,按标准生产程序转染293T后有DHFR基因的表达。结论:成功采用慢病毒载体系统构建了二氢叶酸还原酶重组慢病毒转基因,为探讨DHFR在肿瘤多药耐药过程中的分子机理奠定基础。     

脂肪储存小滴蛋白5 重组腺病毒的制备

目的:利用AdEasy腺病毒表达系统构建含有小鼠脂肪储存小滴蛋白5(LSDP5)基因的重组腺病毒。方法:从小鼠肝脏cDNA克隆出LSDP5基因全长,克隆至pMD18-T载体中,酶切测序。回收酶切产物,连接到腺病毒穿梭载体pShuttle-CMV,构建pShuttle-CMV-LSDP5重组质粒,经PmeI酶切线性化后转化至含有腺病毒骨架质粒pAdEasy-1的BJ5183中。筛选阳性克隆,提取重组质粒,PacI酶切线性化并转染AD293细胞进行包装,提取病毒DNA,鉴定重组病毒并检测病毒滴度。结果:LSDP5基因克隆经测序证实与Genebank公布一致,双酶切重组pMD18-T载体得到1400 bp左右的片段。重组穿梭载体经Kpn I和Sal I双酶切后得到预期片段。PacI酶切得到30 Kb大片段和4.5 Kb小片段。转染AD293细胞后收集病毒,经PCR鉴定,获得理想的目的片段。取病毒上清反复感染AD293细胞以扩增病毒,最后所得病毒滴度为2.5×109pfu/ml。结论:成功构建了携带脂肪储存小滴蛋白5基因的重组腺病毒载体,为进一步研究LSDP5基因功能奠定基础。     

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.