家蚕胚胎细胞系BmE-SWU1的建立及其生物学特性

对家蚕反转期胚胎组织进行一年多的原代培养,分离筛选出了高繁殖细胞群体,建立了BmE-SWU1细胞系。该细胞系以梭形和近圆形细胞为主,杂以少量多突起和巨型细胞。细胞系的倍增时间在28℃时为57.6h,染色体呈短杆状或颗粒状,染色体数目异倍化,具有典型鳞翅目昆虫细胞系的染色体特征。BmE-SWU1细胞系对家蚕核型多角体病毒(BmNPV)高度敏感,半数组织培养感染剂量(TCID50)为2.92415×10-7。     

家蚕胚胎细胞系BmE-SWU2的建立及其生物学特性研究

家蚕反转期胚胎组织经过一年多的原代培养,建立了BmE-SWU2细胞系。该细胞系细胞呈短梭形或圆形,细胞较小,属上皮型贴壁生长细胞系;细胞生长速度快,铺平率高于80%,繁殖力强,细胞群体倍增时间为51.8 h。BmE-SWU2细胞系的二倍体细胞（2n=56）比例达到89.76％,属二倍体细胞系,其染色体具有鳞翅目昆虫染色体的典型特征,中期染色体呈短杆状或颗粒状。BmE-SWU2细胞系对BmNPV高度敏感,TCID₅₀为1.581×10^-7。     

粘虫胚胎细胞系的建立及对MsNPV敏感性的测定

用培养24h的粘虫受精卵成功地建立一株粘虫胚胎细胞后,命名为NEAU－Ms-980312,目前已传代201代,该细胞系主要含有圆形和纺锤形两种细胞,在28℃,120h的细胞群体倍增时间为47.82h。该细胞系对粘虫核型多角体病毒有较高的敏感性,用原代病毒以感染复数（m.o.i.)为0.08TCID50/细胞感染细胞,在接毒后10d,病毒感染率和每细胞产生的多角体数量分别是62．10％和33.59PIB。     

家蚕病毒的表面增强拉曼光谱研究

得到并分析了家蚕核型多角体病毒（ＢｏｍｂｙｘｍｏｒｉＮｏｃｌｅａｒＰｏｌｙｈｅｄｒｏｓｉｓＶｉｒｕｓ缩写为ＢｍＮＰＶ）和质型多角体病毒（ＢｏｍｂｙｘｍｏｒｉｃｙｔｏｐｌａｓｍｉｃＰｏｌｙｈｅｄｒｏｓｉｓＶｉｒｕｓ缩写为ＢｍＣＰＶ）包涵体（即核型多角体ＢｍＮＰＢ和质型多角体ＢｍＣＰＢ）在银胶溶液中的表面增强拉曼散射（ＳＥＲＳ）光谱。ＢｍＮＰＢ和ＢｍＣＰＢ通过氨基酸残基侧链的Ｓ原子、ＣＯＯ－（ＣＯＯＨ）和ＮＨ２（ＮＨ）基团以及蛋白质分子的Ｎ－末端与银表面相作用。Ｔｒｐ残基金部或大部处于疏水环境中。ＢｍＮＰＢ中蛋氨酸残基侧链的Ｓ－ＣＨ２和ＣＨ２－ＣＨ２链分别为扭曲和扭曲式构型。ＢｍＣＰＢ中的Ｓ－ＣＨ２和ＣＨ２－ＣＨ２链则分别属于反式和扭曲构型;Ｃ－Ｃ－Ｓ－Ｓ－Ｃ－Ｃ链为反式－扭曲－反式结构。     

家蚕胚胎细胞系的DNA指纹图谱分析

在建立可靠的家蚕细胞系基因组DNA制备和PCR扩增技术体系的基础上,筛选具有稳定多态性位点的RAPD和ISSR引物,建立家蚕细胞系基因组DNA的ISSR和RAPD分子标记技术体系,检测家蚕细胞系的DNA分子标记多态性,构建细胞系的DNA指纹图谱。筛选出了26个ISSR引物和43个RAPD引物,通过PCR扩增在家蚕胚胎细胞系和传代昆虫细胞系等9个样品中分别获得了797条和1205条多态性条带,多态性达到89．9%和76．6%,不同细胞系的DNA多态性有较大差异,三个家蚕胚胎细胞系具有各自特有的DNA标记。测定了9个样品间的Nei's相似系数和遗传距离,构建了系统发育树,结果表明本实验室建立的3个家蚕胚胎细胞系和家蚕“夏芳×秋白”聚为一簇,亲缘关系较近,而来自不同物种的五个传代昆虫细胞系聚为一簇,它们之间的遗传距离比3个家蚕胚胎细胞系之间的遗传距离更小。     

粘虫胚胎细胞系的建立及对MsNPV敏感性的测定

用培养24h的粘虫受精卵成功地建立一株粘虫胚胎细胞后,命名为NEAU-Ms-980312,目前已传代201代,该细胞系主要含有圆形和纺锤形两种细胞,在28℃、120h的细胞群体倍增时间为47.82h.该细胞系对粘虫核型多角体病毒有较高的敏感性,用原代病毒以感染复数(m.o.i.)为0.08TCID50/细胞感染细胞,在接毒后10d,病毒感染率和每细胞产生的多角体数量分别是为62.10%和33.59PIB.     

家蚕核型多角体病毒ORF 75基因的克隆和表达

河南农业大学植保学院尹新明、安世恒、江志伟、胡鹏等博士、教授根据GenBank序列L33180设计引物用PCR技术扩增了编码家蚕核型多角体病毒bombyx mori nueleopolyhedrovirus,BmNPVORF75基因的DNA片段,将其连接至PMD18-T载体上,获得了该基因的成熟蛋白阅读框序列,用设计的酶切位点将目的基因从克隆载体上切下,     

家蚕CPV感染离体细胞的电镜观察

本文报道了BmCPV感染家蚕细胞系后的电镜观察。病毒感染早期,细胞质内形成电子致密的病毒发生基质,由病毒发生基质形成BmCPV球状病毒粒子;病毒感染48小时后,多角体在病毒发生基质周围形成,大量的病毒粒子随机包埋在多角体内;病毒接种后96小时,多角体数目增多,其形状有三角,四角,五角及六角形,细胞质内充盈多角体致使细胞核被挤向细胞一侧并伴有形态的改变,受染细胞约为40％。     

家蚕核型多角体病毒基因polh的诱变分析

曾报道经化学诱变剂ＭＭＣ、９－ＡＡ和ＥＭＳ诱变的家蚕核型多角体病毒（ＢｍＮＰＶ）多角体形态出现异常,继代分离的诱变ＢｍＮＰＶ基因组对ＥｃｏＲＩ、ＢｇｌＩＩ和ＢａｍＨＩ的酶切谱发生变化。研究进一步揭示,诱变ＢｍＮＰＶ的多角体外层蛋白晶格排列呈现紊乱;多角体蛋白的ＳＤＳ－ＰＡＧＥ电泳谱与对照组比较有显著差异;对多角体蛋白基因ｐｏｌｈ的测序结果显示,３组诱变ＢｍＮＰＶ的ｐｏｌｈ基因发生了多处碱基（氨基酸     

Effects of overexpression and inhibited expression of thymosin,an actin‐interacting protein from Bombyx mori,on BmNPV proliferation and replication

Previous study showed that exogenously applied recombinant thymosin from Bombyx mori (BmTHY) reduces B. mori nucleopolyhedrovirus (BmNPV) proliferation in silkworm. Which stands to reason that BmTHY in B. mori is crucial for the defense against BmNPV. However, little is known about the effect of endogenously overexpressed or repressed BmTHY on B. mori resistance to virus infection. To study this issue, we constructed an overexpression and inhibited expression systems of BmTHY in BmN cells. The viral titer and the analysis from the quantitative real‐time polymerase chain reaction (PCR) revealed that overexpression of BmTHY decreased the copies of BmNPV gene gp41, which goes over to inhibit the proliferation of BmNPV in BmN cells, while the inhibited expression of BmTHY significantly enhanced viral proliferation in infected BmN cells. These results indicated that endogenous BmTHY can inhibit BmNPV proliferation and replication in infected BmN cells. Furthermore, Co‐IP showed that BmTHY could bind to actin in BmN cells. Also, the overexpression or inhibited expression of BmTHY shifted the ratio of F/G‐actin in infected BmN cells. Lastly, the BmTHY, an actin‐interacting protein, might be one of the key host factors against BmNPV, which inhibits viral proliferation and replication in BmN cells.     

Biochemical characterization of rab proteins from Bombyx mori

The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [(3)H]-GDP and [(35)S]-GTPgammaS with dissociation constants of 0.087 x 10(-6) M and 1.02 x 10(-6) M, respectively, whereas those of BRabN2 were 0.546 x 10(-6) M and 1.02 x 10(-6) M, respectively. Binding of [(35)S]-GTPgammaS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP-hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [(35)S]-GTPgammaS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [(35)S]-GTPgammaS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti-BRabN1 antibody revealed a single band in the testis of B. mori. These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP-bound state to the GDP-bound state by intrinsic GTP hydrolysis activity, and return to the GTP-bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.     

家蚕细胞中过表达家蚕核型多角体病毒核衣壳蛋白VP39抑制BmNPV的增殖

【目的】家蚕Bombyx mori核型多角体病毒（BmNPV）核衣壳蛋白VP39为病毒装配所必需。本研究旨在初探VP39在病毒侵染家蚕细胞过程中的功能及特征,以期为家蚕抗病毒研究提供研究基础。【方法】本研究通过构建原核表达载体,诱导原核表达得到多克隆抗体,以Western blot验证VP39表达时相;构建真核表达载体,转染细胞后以免疫荧光手段观测VP39表达定位及影响病毒增殖现象。【结果】制备了VP39多克隆抗体。VP39在病毒感染后大量定位于家蚕细胞核,部分定位于胞质,而过表达的VP39定位于家蚕细胞胞质;过表达VP39后抑制BmNPV感染家蚕细胞。【结论】在BmN-SWU1细胞中过表达VP39会影响BmNPV的扩散,导致BmNPV感染细胞数目大量减少。该结果为VP39调控宿主与病毒的相互作用提供了新的思路。     

家蚕浓核症病毒感染昆虫细胞系的电镜观察

本实验首次用中国株家蚕浓核症病毒感染三株昆虫细胞系:油桐尺蠖卵巢细胞系(BS-484)、甘兰夜蛾卵巢细胞系(NIAS—MB—19)和秋粘虫卵巢细胞系(IPLB—SF—21)。结果仅在BS—484细胞中观察到病毒感染引起的细胞病变效应。电镜观察发现在感染的BS—484中,细胞核明显膨大,其中核仁活性化,数目增多,同时还观察到核仁膨大和分裂现象。感染5—6天,可看到成熟病毒粒子于核内形成。在感染的细胞质中,线粒体肥大且失去脊,粗面内质网变成小泡体,其内积累大量核糖核蛋白体。许多细胞器空泡化或退化,细胞质中出现一些包含退化细胞器的大型自身吞噬体。     

一株棕尾别麻蝇胚胎细胞系的建立及其特性分析

双翅目昆虫细胞系广泛应用于遗传学、发育生物学、分子生物学、人和动物体病原学以及昆虫抗微生物肽的研究。本研究建立了一株新的棕尾别麻蝇Sarcophaga peregrina胚胎细胞系。该细胞系的原代培养始于2008年11月17日, 取材于棕尾别麻蝇晚期胚胎组织, 在Shields & Sang M3昆虫培养基中于28℃恒温培养, 在第26天进行第1次传代, 至今已历时21个月, 传代72次, 生长状态稳定, 被命名为Sp-E-HNU11。该细胞系的细胞形态主要呈梭形和近圆形, 杂以少量巨型细胞, 紧密贴壁生长。细胞群体倍增时间为42 h。染色体数目一般为10条或12条, 为二倍体或亚二倍体细胞系; 除一对颗粒状微型染色体外, 其他染色体呈短杆状。细胞系的β-萘酯酶和谷草转氨酶同工酶谱上分别显示出1条和3条酶带。随机引物扩增多态性 (random amplified polymorphic DNA, RAPD) 分析结果显示, 该细胞系与小菜蛾细胞系Px-E-HNU12、草地贪夜蛾细胞系IPLB-Sf-9和家蚕细胞系Bm-21E-HNU5呈现明显不同的带型特征。 Sp-E-HNU11细胞系的建立为昆虫抗微生物肽及其他相关的研究工作增添了新的研究工具和生产载体。     

Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus(BmNPV),a transfer vector was constructed which contained an Escherichia coli(E.coli)mini-F replicon and a lacZ:attTN7:lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene.B.mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo.The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E.coli DH10B.Recombinant bacmids were screened by kanamycin resistance,PCR and restriction enzyme(REN)digestion.One of the bacmid colonies,BmBacJS13,which had similar REN profiles to that of wild-type BmNPV,was selected for further research.To investigate the infectivity of BmBacJS13,the polyhedrin gene was introduced into the bacmid and the resultant recombinant(BmBacJS13-ph)was transfected to BmN cells.The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells.Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV.Bio-assays indicated that BmBacJS13-ph was also infectious to B.mori larvae.     

Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae.