棉铃虫核型多角体病毒p35基因的PCR扩增和克隆及其在原核细胞中的 …

用苜蓿银纹夜蛾核型多角体病毒凋亡抑制基因ｐ３５的合成引物,从棉铃虫核型多角体病毒基因组中用ＰＣＲ扩增到１ｋｂ片段。采用ＰＣＲ－双脱氧核苷酸链终止银染法,测定了所扩增到的１０１０ｂｐ全序列,发现其开放阅读框完整,长８９７ｂｐ,编码２９９个氨基酸。     

棉铃虫核型多角体病毒p35基因的PCR扩增和克隆及其在原核细胞中的表达

用苜蓿银纹夜蛾核型多角体病毒（ Ａｃ Ｎ Ｐ Ｖ） 凋亡抑制基因ｐ３５ 的合成引物,从棉铃虫核型多角体病毒（ Ｈａ Ｎ Ｐ Ｖ） 基因组中用 Ｐ Ｃ Ｒ 扩增到了１ｋｂ 片段。采用 Ｐ Ｃ Ｒ－ 双脱氧核苷酸链终止银染法,测定了所扩增到的１ ０１０ｂｐ 全序列,发现其开放阅读框完整,长８９７ｂｐ ,编码２９９ 个氨基酸。用 Ｄ Ｎ Ａ Ｓ Ｉ Ｓ 和 Ｐ Ｒ Ｏ Ｓ Ｉ Ｓ 软件分析发现,此片段与 Ａｃ Ｍ Ｎ Ｐ Ｖ ｐ３５ 基因同源区核苷酸同源性为９１ ％ ,氨基酸同源性也达８１ ％ ,证明了所扩增的片段是 Ｈａ Ｎ Ｐ Ｖ 的ｐ３５ 基因。为了研究此ｐ３５ 基因的功能及其效应机制,克隆了这一基因并在大肠杆菌细胞中实现了表达。     

杆状病毒凋亡抑制基因的研究进展

杆状病毒(bacu lovirus)感染昆虫细胞能引起细胞凋亡,但在长期进化过程中,杆状病毒可通过自身编码凋亡抑制基因的表达,抑制细胞凋亡以利于自己的增殖。目前在杆状病毒基因组中已发现两种不同类型的细胞凋亡抑制基因p35/p49和iap,这两类凋亡抑制基因分别作用于细胞凋亡途径的不同位点,以抑制细胞的凋亡。近年来人们对这两种基因的蛋白结构及作用机制等方面进行了大量的研究,这些为今后研究昆虫细胞凋亡,扩大杆状病毒宿主范围等方面奠定了基础。     

粘虫核型多角体病毒凋亡抑制基因的定位、序列和启动子结构

以AcNPV凋亡抑制基因p35为探针,与LsNPV DNA的限制性片段和LsNPV DNA EcoRV片段杂交,发现EcoRV 5.5kb片段有强烈的杂交信号.将此片段亚克隆后,测定了1244bp序列,发现一个完整的ORF,推导的302个氨基酸与AcNPV p 35蛋白有70.4%的氨基酸同源性,证明所测ORF为LsNPV的p 35基因.结构分析发现其5′端有早期基因启动子元件GC、ACGT和TATA box.有22 bp的顺向重复序列,包括由两个重叠的TATA box和上下游两个ACGT motif组成的两套启动子元件,这些结构特征与AcNPV的凋亡抑制基因十分相似.     

粘虫核型多角体病毒凋亡抑制基因的定位,序列和启动子结构

以ＡｃＮＰＶ凋亡抑制基因ｐ３５为探针,与ＬｓＮＰＶＤＮＡ的限制性片段和ＬｓＮＰＶＤＮＡＥｃｏＲＶ片段杂交,发现ＥｃｏＲＶ５．５ｋｂ片段有强烈的杂交信号。将此片段亚克隆后,测定了１２４４ｂｐ序列,发现一个完整的ＯＲＦ,推导的３０２个氨基酸与ＡｃＮＰＶｐ３５蛋白有７０．４％的氨基酸同源性,证明所测ＯＲＦ为ＬｓＮＰＶ的ｐ３５基因。结构分析发现其５′端有早期基因启动子元件ＧＣ、ＡＣＧＴ和ＴＡＴＡｂｏｘ。有２２ｂｐ的顺向重复序列,包括由两个重叠的ＴＡＴＡｂｏｘ和上下游两个ＡＣＧＴｍｏｔｉｆ组成的两套启动子元件,这些结构特征与ＡｃＮＰＶ的凋亡抑制基因十分相似。     

杆状病毒凋亡抑制基因

杆状病毒(baculoviruses)感染昆虫细胞会引发细胞凋亡,然而病毒为了确保自身的复制和繁殖会抑制宿主细胞的凋亡.杆状病毒在长期进化过程中获得了凋亡抑制基因,如苜蓿银纹夜蛾核型多角体病毒(Autographa californica MNPV, AcMNPV)中的p35基因,棉铃虫核型多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus, HaSNPV)基因组中的IAP基因家族,以及莲纹夜蛾核形多角体病毒(Spodoptera littoralis multicapsid nucleopolyhedrovirus, SpliMNPV)的p49基因等.尽管这些基因都具有抑制细胞凋亡的功能,但是作用途径和方式却各有差异.对杆状病毒3种抗凋亡基因的结构和功能作一简单的介绍和评述.     

美国白蛾核型多角体病毒p35基因的克隆及序列分析

对美国白蛾核型多角体病毒（HycuNPV,Hyphantria cunea nucleopolyhedrovirus）p35基因的序列分析表明：HycuNPV p35编码序列900?bp, 编码299氨基酸。同源性分析表明：HycuNPV p35与BomoNPV T3、AucaNPV、SpliNPV、LeseNPV、HearNPV在核苷酸水平上为99.9%、95.7%、93.6%、80.2%和87.2%,在氨基酸水平上为99.7%、90.3%、77%、64.9%和73.2%,显示了杆状病毒p35基因在进化上的保守性。BomoNPV T3中位的H122,在HycuNPV中被R取代。推测HycuNPV p35蛋白的功能及抑制细胞凋亡的能力与BomoNPV T3 p35蛋白的相似。     

棉铃虫核型多角体病毒p40基因序列分析

克隆了棉铃虫(Helicoverpaarmigera)单粒包埋型核型多角体病毒(HaSNPV)基因组HindⅢ-H、J片段,其长度分别为10kb和6.7kb.通过对克隆片段末端测序,得到HaSNPVp40基因全序列.p40基因编码区全长966bp,预计可编码36.kD的多肽.HaSNPVp40基因核苷酸序列与HzSNPVp40基因有98%的相同性.这两种基因与BmNPVp40(GenBank-L33180)和AcMNPVgp41(GenBank-L22858)的氨基酸序列相似性43%左右,但四种蛋白对应于HzSNPVp40、HaSNPVp40的28～277氨基酸区域,同源性高达62%,并且存在一个保守的亲水性结构域.进一步将在基因组中相邻的HindⅢ-H、J两片段用BamHⅠ、EcoRⅠ、HindⅢ、PstⅠ四种限制性内切酶进行了分析.     

家蚕核型多角体病毒山东株p35基因的克隆及序列分析

根据家蚕核型多角体病毒T3株p35基因非编码区序列设计一对特异性引物,应用PCR技术从家蚕核型多角体病毒山东株中扩增得到一条1 080 bp的片段。测序结果表明,该片段含有p35基因,开放阅读框长900 bp,编码299个氨基酸,分子量为34.91kDa,pI=6.4。序列分析表明,BmNPV山东株的p35基因与T3株存在差异。 Abstract:According to the 5′ and 3′ untranslated region sequences of p35 gene from Bombyx mori nuclear polyhedrosis virus isolate T3,a pair of primers was designed employing computer analysis.With the primers,polymerase chain reaction (PCR) was performed and a 1 080 bp fragment was obtained from BmNPV strain Shandong.Sequencing result showed that it was p35 gene (GenBank accession number：AY157746),and it included an open reading frame of 900 bp,encoding 299 amino acids with Mr=34.91kDa and pI=6.40.BLAST analysis indicated that there were seven bases and three amino acids difference between p35 from BmNPV strain Shandong and that from BmNPV isolate T3.     

棉铃虫核型多角体病毒p40基因序列分析

克隆了棉铃虫（Helicoverpa armigera）单粒包埋型核型多角体病毒（HaSNPV）基因组HindⅢ-H,J片段,其长度分别为10kb和6．7kb。通过对克隆片段末端测序,得到HaSNPV p40基因全序列。p40基因编码区全长966bp,预计可编码36.kD的多肽。HaSNPVp40基因核苷酸序列与HzSNPVp40基因有98％的相同性,这两种基因与BmNPVp40（GenBank-L33180）和AcMNPVgp41(GenBank-L22858)的在酸序列相似性43％左右,但四种蛋白对应于HzSNPVp40,HaSNPVp40的28－277氨基酸区域,同源性高达62％,并且存在一个保守的亲水性结构域,进一步将在基因组中相邻的Hind Ⅲ-H、J两片段用BamHⅠ、EcoRⅠ、HinⅢ、PstⅠ四种限制性切酶进行了分析。     

Expression of the Baculovirus p35 Gene Inhibits Mammalian Neural Cell Death

Expression of the apoptosis suppressor genep35, derived from the baculovirus Autographa californica nuclear polyhedrosis virus, markedly inhibited the cell death of stably transfected mammalian neural cells whether the cell death was induced by glucose withdrawal, calcium ionophore, or serum withdrawal. The p35 protein, which is required to block virus-induced apoptosis of cultured insect cells, is only the second gene product shown to block mammalian neural cell death, with Bcl-2 being the first. Because there is no apparent homology between p35 and Bcl-2, the existence of a cellular death program that may be modulated at multiple points is suggested/Furthermore, these findings demonstrate that the putative cellular death program is conserved across species and cell types.     

Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells

Baculovirus has many advantages as vectors for gene transfer.We demonstrated that recombinant baculovirus vectors expressing p35(Ac-CMV-p35) and eGFP(Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently.The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter.MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells.Cell growth curve showed the Ac-CMV-p35 and Ac-CMV-GFP transduced and non-transduced cells had similar proliferation rate,so baculovirus-mediated p35 expression had no adverse effect on cell proliferation.In addition,baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D,UV or serum-free media.These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.     

Baculovirus-mediated expression of p35 confers resistance to apoptosis in human embryo kidney 293 cells

Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac-CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35 expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy. Foundation items: National Nature Science Foundations of China (30325002, 30670077) and Innovative Foundations Wuhan Institute of Virology, CAS (020208)     

杆状病毒AcMNPV p35基因的克隆表达及多抗制备

用PCR方法扩增得到苜蓿丫纹夜蛾核多角体病毒(Autographa californic anucleopolyhedrovirus,AcM N-PV)p35基因,将其克隆至质粒pET-32a( )上,构建得到重组质粒pET-p35,转化大肠杆菌BL21(DE3),经IPTG诱导,表达了1条约为55 ku的蛋白带。以Ni2 -NTA偶连抗体检测证明所表达的蛋白为带有组氨酸的融合蛋白。采用割胶回收的方法纯化融合蛋白,以纯化的融合蛋白制备多克隆抗体,效价为1/1 024。免疫印迹分析表明,该抗血清能与感染苜蓿丫纹夜蛾核多角体病毒的细胞蛋白样品发生特异性反应。     

苜宿银纹夜蛾核型多角体病毒vp39基因的克隆及其对S_f9细胞形态的影响

最近的研究发现：ＡｃＮＰＶ的ｖｐ３９基因与侵染密切相关［１］．在侵染过程中,ＶＰ３９蛋白与宿主的肌动蛋白结合,使其重排形成缆索（ｃａｂｌｅ）．导致细胞骨架发生变化有利于病毒编码的蛋白酶的水解．最后,子代病毒颗粒大量形成,宿主昆虫体全部液化成为脓水．可...     

p35Nck5a基因重复性打靶载体的构建和ES细胞基因打靶研究

为了在小鼠胚胎于细胞（ＥＳ）中引起神经细胞ｃｄｃ２类激酶调节亚基ｐ３５Ｎｃｋ５ａ基因的定点 重复,采用常规的分子克隆技术,构建得到长约１２．２ｋｂ的基因重复性打靶载体ｐＧＤＴＶ。用电 穿孔法将线性化的ｐＧＤＴＶ载体转入ＥＳ细胞,经过Ｇ４１８和ＧＡＮＣ分组药物选择,获得２４５个 双药物抗性的细胞克隆,细胞存活率为６．２２ × １０－５。经ＰＣＲ和基因组Ｓｏｕｔｈｅｒｎ杂交鉴定,２个 ＥＳ细胞克隆发生了ｐ３５Ｎｃｋ５ａ基因的重复,同源重组率为５．０８×１０－７、负向选择系统的应用使 同源重组事件的富集效率提高了７倍。为建立Ａｌｚｈｅｉｍｅｒ病的转基因小鼠模型打下了基础。