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     

异源多角体蛋白对家蚕核型多角体病毒粒子的包装

利用PCR方法从AcMNPV基因组DNA中分离出多角体蛋白基因 ,将该扩增片段克隆到转移载体pBacPAK8中 ,得到重组转移载体pOAc。将该质粒DNA与线性化的Bm BacPAK6病毒基因组DNA共传染BmN细胞 ,得到了能形成多角体且不产生蓝色空斑的重组病毒hp BmNPV。纯化该重组病毒的多角体颗粒 ,并对多角体蛋白、病毒核酸及多角体病毒颗粒进行分析 ,发现AcMNPV的多角体蛋白能在家蚕细胞中大量表达且能在细胞内识别家蚕核型多角体病毒并组装成多角体颗粒 ;病毒基因组DNA因部分交换 ,其酶切行为发生了相应的变化 ;电镜观察发现经AcMNPV多角体蛋白包装的家蚕核型多角体病毒的多角体颗粒大小为1 2 μm～ 2 9μm ,明显小于野生型家蚕核型多角体病毒的多角体颗粒     

家蚕核型多角体病毒水平转移基因分析

为了探讨杆状病毒基因组的遗传进化模式,文章利用家蚕核型多角体病毒(BmNPV)和其宿主家蚕全基因组数据,进行了全基因组的同源性搜索和系统进化分析,结果显示,BmNPV的几丁质酶(Chi)基因、凋亡抑制蛋白3(IAP3)基因和尿苷二磷酸葡萄糖转移酶(UGT)基因为水平转移基因。这3个基因都来源于其宿主昆虫。通过核苷酸组成、密码子偏好性、选择压力等基因特征分析,发现BmNPV水平转移基因与其基因组序列存在明显差异,进一步验证水平转移基因的外源性。对3个水平转移基因的功能分析发现它们有利于杆状病毒在宿主昆虫中的侵染与繁殖,并提高杆状病毒在昆虫中的生存能力。     

家蚕中肠组织抗核型多角体病毒病的相关蛋白分析

家蚕中肠上皮是病毒经口侵入遇到的第一个组织。昆虫幼虫抵御杆状病毒的感染,可通过选择性的使感染的中肠上皮细胞发生调亡并在释放病毒粒子进入血淋巴之前使感染的细胞从中肠脱落。为研究家蚕抗核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)病的机制,通过对BmNPV高度抗性和高度敏感性的家蚕品系杂交和回交构建了近等基因系。本文对家蚕高抗,敏感及近等基因系5龄起蚕中肠组织的蛋白质表达谱进行了二维电泳 (two-dimensional gel electrophoresis,2-DE) 分析,并利用基质辅助激光解吸电离飞行时间 (matrix-assisted laser desorption/ionization-time of flight, MALDI-TOF) 质谱对差异蛋白进行鉴定。结果发现了5个差异表达的蛋白。推测这些蛋白可能与家蚕中肠对BmNPV的抗性或感性有关。     

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,     

家蚕抗核型多角体病分子标记筛选

对家蚕抗核型多角病毒病以不同的杂交方式,构建3种近等基因系,用RAPD技术筛选出抗病主基因连锁分子标记OPA-18700和感病连锁分子标记OPY-11400。同时在F2群体中验证了抗性分子标记的有效性。     

家蚕核型多角体病毒蛋白组份检测方法建立

研制了兔抗家蚕核型多角体病毒蛋白组份的多克隆抗体,用间接法建立了家蚕核型多角体病毒蛋白的dot-ELISA检测方法。此法检测多角体病毒蛋白的灵敏度达10ng,与人血清和兔血清无交叉反应,可用于家蚕生物反应器生产的目的蛋白中多角体病毒蛋白组份的检测。     

家蚕核型多角体病毒中国株和日本株vp39基因的比较研究

以苜蓿丫纹夜蛾核型多角体病毒的核衣壳蛋白基因ｖｐ３９设计引物,用ＰＣＲ技术扩增了家蚕核型多角体病毒中国株（ＢｍＮＰＶ－Ｃｈ）－１．３ｋｂ片段并测定了其全序列长１２３０ｂｐ。推导的氨基酸３５１个,其与ＢｍＮＰＶ日本株（ＢｍＮＰＶ－Ｊａ）ｖｐ３９基因核苷酸序列同源性达９７．５％,氨基酸同源性达９７．１％。该片段在Ｅ．ｃｏｌｉＢＬ２１中诱导表达能产生分子量约为３８ｋＤ的特征性蛋白带,证明所扩增片段为Ｂｍ     

Functional analysis of Dicer-2 gene in Bombyx mori resistance to BmNPV virus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.     

家蚕性别决定研究进展

作为模式生物和经济昆虫,家蚕的性别决定向来受到广泛的关注,近年来更得到深入的研究.主要在分子水平上概述了国内外有关家蚕性染色体方面最新的研究状况,通过对比,探讨了家蚕的几个可能决定性别的基因及其作用过程,以及可能的性别决定靶基因,并就目前性别决定研究现状中的几个突出的问题进行了探讨.     

Screening of Bombyx mori brain proteins interacting with protein tyrosine phosphatase of BmNPV

In this study, glutathione-S-transferase pull-down combined with mass spectrometry techniques were used to identify the candidate proteins interacting with protein tyrosine phosphatase of the Bombyx Mori nucleopolyhedrovirus in the B. mori (BmNPV-PTP) brain. A total of 36 proteins were identified from BmNPV-PTP coprecipitate samples by searching the NCBI_Bombyx Mori database with the original mass spectrum data. Among those proteins, the interaction between BmNPV-PTP and B. mori cyclophilin A may accelerate the apoptosis of certain nerve cells involved in regulating behavior, and thus may be an inducer of enhanced locomotor activity (ELA). After the BmNPV invasion, BmNPV-PTP binding to peripheral-type benzodiazepine receptors may initiate a series of abnormal cascades of the nervous system, which results in abnormal hyperactive behavior in B. mori. Besides this, vacuolar ATP synthase catalytic subunit A, annexin, and several enzymes for energy conversion were identified, which may play a role in enhancing viral entry and infectivity and provide energy for enhancing the locomotor activity of B. mori. In general, the results of this study will facilitate the understanding of the molecular mechanisms underlying the ELA of B. mori larva induced by BmNPV.     

The effects of BmNPV on biochemical changes in primary cultures of Bombyx mori embryonic tissue

The effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) on biochemical changes of TC-100 medium containing 10% fetal bovine serum (FBS) in embryonic primary cultures of silkworm was investigated. The primary cultures that reached 60% confluence were infected by 0.5, 1, and 2-ml viral inoculums (diluted with TC-100 medium representing multiplicity of infection (MOI) of 0.25, 0.5, and 1). Glucose, uric acid, urea, total protein, cholesterol, and alkaline phosphatase were measured in the medium of BmNPV-infected primary cultures. All biochemical compounds showed significant changes. Glucose decreased considerably by about 55 mg/ml, while different concentrations of the virus inoculums did not demonstrate significant differences among them. Total protein had only increased in 2 ml concentration and there were no changes in other concentrations. Uric acid as a by-product accumulated dramatically in all concentrations, while the amount of urea reduced in all treatments and this reduction was more evident in lower concentrations. Cholesterol consumption was high in cultures postinfection, while alkaline phosphatase (ALP) activity decreased in infected cells.     

Transgenic breeding of anti-Bombyx mori L. nuclear polyhedrosis virus silkworm Bombyx mori

Silkworm strains resistant to Bombyx mori L. nuclear polyhedrosis virus were obtained through transgenic experiments. piggyBac transposon with an A3 promoter were randomly inserted into the silkworm, driving the enhanced green fluorescent protein (EGFP) reporter gene into the silkworm genome. Polymerase chain reaction results verified the insertion of the extraneous EGFP gene, and fluorescence microscopy showed that the EGFP was expressed in the midgut tissue. The morbidity ratio of the nuclear polyhedrosis decreased from 90% in the original silkworm strain to 66.7% in the transgenic silkworm strain. Compared with the resistance to the Bombyx mori L. nuclear polyhedrosis virus in the Qiufeng strain, which is commonly used in the production, there was an increase of 33 centesimal points in the transgenic silkworms. The antivirotic character in the Chunhua x Qiuyue strain, which was bred from a different transgenic family, was about 10 centesimal points higher than that in the Qiufeng x Baiyu, another crossbreed used in production. Our results indicated a good application value of the transposon-inserted mutation in the breeding of anti-BmNPV silkworm strain.     

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。     

Construction of a BmNPV polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, Bombyx mori

The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.     

Standard method for detecting Bombyx mori nucleopolyhedrovirus disease-resistant silkworm varieties

Bombyx mori nucleopolyhedrovirus (BmNPV) disease is one of the most serious silkworm diseases, and it has caused great economic losses to the sericulture industry. So far, the disease has not been controlled effectively by therapeutic agents. Breeding resistant silkworm varieties breeding may be an effective way to improve resistance to BmNPV and reduce economic losses. A precise resistance-detection method will help to accelerate the breeding process. For this purpose, here we described the individual inoculation method (IIM). Details of the IIM include pathogen BmNPV preparation, mulberry leaf size, pathogen volume, rearing conditions, course of infection, and breeding conditions. Finally, a resistance comparison experiment was performed using the IIM and the traditional group inoculation method (GIM). The incidence of BmNPV infection and the within-group variance results showed that the IIM was more precise and reliable than the GIM.     

家蚕核型多角体病毒遍在蛋白及结合肽的研究

从家蚕核型多角体病毒镇江株(Bombyx mori nuclear polyhedrosis virus Zhenjiang strain, BmNPV-ZJ)基因组DNA中克隆遍在蛋白（BmVUB）基因.序列分析结果显示,BmVUB基因长234 bp,编码77个氨基酸.BmVUB氨基酸序列内有一致HTH序列,遍在蛋白保守序列LRLRGG,参与遍在蛋白-蛋白酶复合体形成的4个保守性功能位点(Lys-29、Cys-48、Cys-63、Gly-76)及保守的Gly-Gly-X（X是疏水氨基酸残基）蛋白酶切信号序列,在遍在蛋白保守的Gly-Gly后多出由一个氨基酸组成的延伸肽.原核诱导表达表明BmVUB主要以可溶性形式存在,表达量占总菌蛋白的50%以上.纯化的遍在蛋白浓度为1.03～2.46 g/L,制备抗体,效价在3.2×10^-5以上.用噬菌体表面展示技术筛选遍在蛋白结合肽,所筛选的结合肽可与遍在蛋白特异性结合,其中结合肽VAPHHAYAPMRT对细胞增殖有明显的浓度调控作用,即低浓度促进细胞生长,高浓度强烈抑制细胞的生长.