TALENs介导的CCR5△32/△32突变赋予CD4+ U87细胞抵抗HIV-1感染的能力

CCR5Δ32/Δ32(C C chemokine receptor type 5,CCR5)基因型的骨髓干细胞移植,可以治愈感染人免疫缺陷病毒-1（HIV-1）的患者。本研究运用TALENs结合同源重组技术产生纯合子的CCR5△32/△32突变,并赋予CD4+ U87 细胞抵抗HIV-1感染的能力。首先,采用重叠延伸PCR合成 CCR5△32 donor DNA。构建CCR5-TALENs 和CCR5△1-TALENs质粒,将CCR5-TALENs 及CCR5△1-TALENs质粒分别结合CCR5△32 donor DNA,共转染野生型CD4+ U87 细胞。其次,用T7E1酶切分析转染后的TALENs和CCR5△1-TALENs打靶效率。通过单克隆培养筛选CCR5△32/△32突变的单克隆细胞。最后,将CCR5△32/△32 CD4+ U87 细胞和野生型CD4+ U87细胞进行Bal HIV-1病毒攻击实验,采用ELISA方法检测上清中P24抗原含量。测序结果表明,通过重叠延伸PCR成功获得1 602 bp CCR5△32 donor DNA;CCR5-TALENs 质粒第1轮、第2轮和第3轮转染,打靶效率分别为14.80%, 38.20%和50.40%;从29个单个细胞培养的克隆中成功筛选出1个CCR5△32/△1基因型CD4+ U87细胞,CCR5与CCR5△32 donor DNA之间同源重组效率为1.7%; CCR5△1-TALEN质粒第2轮转染,打靶效率为23.5%,从34个单细胞培养的克隆中筛选出3个CCR5△32/△32 基因型CD4+ U87细胞,CCR5与CCR5△32 donor DNA之间同源重组效率达到8.8%。Bal HIV-1攻击实验表明,野生型CD4+ U87细胞培养上清第2、4、6、8、10、12 d,平均P24抗原含量分别为58.47±2.35、162.23±4.78、458.78±27.34、613.35±26.78、580.35±24.73、483.34±30.85 pg/mL。而CCR5△32/△32基因型CD4+ U87细胞的培养上清平均P24抗原含量分别为11.30±1.76、5.13±0.88、3.43±0.44、3.84±0.69、3.21±0.44、4.24±0.46 pg/mL。本研究表明,TALENs 结合同源重组技术无缝隙地介导了CCR5△32/△32突变,并赋予了CD4+ U87细胞抵抗HIV-1感染的能力。     

一种拮抗HIV-1并靶向DC的融合基因的设计及表达鉴定

艾滋病病毒 (Human immunodeficiency virus,HIV) 通过与靶细胞膜的融合感染宿主细胞,研究表明阻断HIV与受体靶分子的结合可以阻止HIV进入宿主细胞,抑制HIV病毒的感染。设计合成了一个包含CD4和CCR5与HIV-1结合的主要功能结构区,及Flt3-L和Mip-3α分子的融合基因,构建了2个融合基因的真核表达载体pABK-CKR5-CD4/Flt3L-Mip3α (pABK-HIV-MF) 和pABK-CKR5-CD4 (pABK-HIV-MT),在人胚肾293细胞中进行了表达。RT-PCR、细胞免疫荧光技术、ELISA和Western blotting检测结果表明融合基因在真核细胞中获得了正确的表达,这为进一步研究其对于HIV-1的拮抗并靶向树突状细胞 (DC) 清除研究奠定了基础。     

人类免疫缺陷病毒入侵的新受体-- CD8分子

已知人类免疫缺陷病毒(HIV)感染通过包膜糖蛋白gp120与CD4分子结合,以及在细胞因子CDCR4和CCR5受体协同作用下完成吸附和穿入.本文报道从2例艾滋病病人体内分离到HIV-1变异株:AD3.v22和AD3.v6,可通过CD8受体侵犯CD8+细胞.     

打靶恒河猴CD4~+ T细胞的TRIM5α基因影响其感染HIV的能力

目的:探讨打靶恒河猴CD4~+ T细胞的TRIM5α基因对其感染HIV-1能力的影响。方法:从恒河猴的外周血中通过磁珠分选获得CD4~+ T细胞,并采用流式检测阳性率。构建打靶TRIM5α基因的TALEN质粒,通过电转导入CD4~+ T细胞,流式分选出转染TALEN质粒的细胞,提取基因组T7E1酶切检测打靶效率。HIV-1病毒感染打靶TRIM5α的CD4~+ T细胞,并通过ELASA检测病毒感染的情况。结果:成功地从恒河猴的外周血中分选出了CD4~+ T细胞,流式检测阳性率为99.5%。打靶TRIM5α基因的TALEN质粒转染CD4~+ T细胞的转染效率约为24.8%,并可成功打靶TRIM5α,打靶效率约为40%。ELASA检测结果表明打靶TRIM5α的恒河猴CD4~+ T细胞对HIV病毒的感染能力增强。结论打靶恒河猴CD4~+ T细胞的TRIM5α基因可使其易感HIV病毒,为进一步建立恒河猴HIV-1感染动物模型奠定基础。     

基于调控趋化性细胞因子受体CCR5抗HIV-1的研究进展

获得性免疫缺陷综合征(AIDS),又称"艾滋病",是威胁着人类生命健康的重大传染性疾病。趋化性细胞因子受体5(Chemotactic cytokine receptor 5,CCR5)作为嗜巨噬细胞性1型人类免疫缺陷病毒(Human immunodeficiency virus type 1,HIV-1)进入宿主细胞的必需的辅助受体自发现以来被广泛关注。CCR5在HIV靶细胞表面的表达水平与靶细胞的病毒载量呈正相关且和艾滋病的进程强相关。因此,通过干预CCR5在靶细胞表面的表达能在一定程度上阻止HIV进入靶细胞。因此,本文从分子水平阐述干预CCR5的主要生物学机制,并对近几年的研究进行综述。     

人类免疫缺陷病毒与细胞凋亡

人类免疫缺陷病毒1型(HIV-1)感染可使被感染者体内CD4细胞数量减少,最终导致艾滋病。关于HIV-1如何杀死免疫细胞的精确机制仍是1个争论的问题。现已知道,细胞凋亡为HIV-1诱导细胞死亡的一个重要机制。HIV可直接诱导细胞凋亡,也可以通过活化作用,同源被感染的细胞的介导,以及CD^8 T细胞诱导细胞凋亡。且细胞因子在HIV诱导细胞凋亡的过程中发挥着重要的作用。本综述主要从以上几个方面总结HIV-1诱导细胞凋亡的机制。     

性病艾滋病感染者HIV-1相关基因CCR5△32、CCR2b-64I、SDF1-3'A多态性分布的研究

研究HIV-1相关等位基因CCR5△32、CCR2b-64I、SDF1-3'A在性病艾滋病感染人群中的突变频率和多态性分布的特点,为我国艾滋病的预防和未来的基因治疗提供初步依据.收集198例汉族性病艾滋病感染者的血液标本,提取基因组DNA,经PCR检测,并用统计学方法分析.198例性病艾滋病人群CCR.5△32、CCR2b-64I、SDF1-3'A的突变频率分别为0.25%、16.16%、25.00%,与中国普通汉族人的结果一致.CCR5△32的突变频率较低,而CCR2b-64I、SDF1-3'A的突变频率较高,提示本地性病艾滋病汉族人群对性传播的HIV-1(R-5)毒株有较大的易感性.     

细胞内表达CCR5Delta32蛋白对HIV-1感染抑制作用的研究

人CCR5Delta32突变个体能有效抵制HIV-1感染,主要是由于该个体淋巴细胞内表达的CCR5Delta32突变蛋白能通过反式显性失活效应(TDN)抑制细胞表面HIV-1辅受体CCR5和CXCR4的产生．通过构建CCR5Delta32慢病毒载体,体外转染人外周血单个核细胞(PBMCs),研究细胞内表达CCR5Delta32蛋白对HIV-1感染的抑制作用．结果表明,表达CCR5Delta32蛋白的人PBMCs对HIV-1 R5、X4及R5X4毒株感染均具有显著的抑制作用．这些工作为后续的AIDS基因治疗研究奠定了基础．     

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.