DpNPV多角体蛋白拉曼光谱的研究

研究了马尾松毛虫核型多角体病毒多角体蛋白的拉曼光谱。结果表明，ＤｐＮＰＶ多角体蛋白的二级结构主要为α螺旋和无规卷曲，后者含有大量的β回析，多角体蛋白的侧链Ｃ－Ｃ－Ｓ－Ｓ－Ｃ－Ｃ－构型为反式－扭曲－反式。     

昆虫核多角体病毒的垂直传播

对昆虫核型多角体病毒在宿主体内的垂直传播方式、诱发因素、生物防治应用以及检测方法等方面进行了综述.     

杆状病毒多角体蛋白的A蛋白-金胶体免疫电镜标记

本文报导葡萄球菌A蛋白-金胶体免疫技术,成功地应用于杆状病毒多角体蛋白细胞内超薄切片的定位标记,并讨论了它的特点.     

SlNPV多角体蛋白基因的序列分析

ＳｌＮＰＶ多角体蛋白基因的开放读码框为７５０ｂｐ,编码２４９个氨基酸的蛋白质,是目前所发现的最长的多角体蛋白基因．ＳｌＮＰＶ多角体蛋白Ｍｒ＝２９２３６,其氨基酸序列与其它核多角体病毒的多角体蛋白氨基酸序列有高度同源性．     

斜纹夜蛾核型多角体病毒日本株(C3)p10基因的克隆及序列分析

从斜纹夜蛾核多角体病毒(Spodoptera litura mukieapsid nucleopolyhedrovirus，Splt MNPV)日本株(C3)基因组中克隆了p10基因．核苷酸序列分析表明，该克隆片断涵盖了318bp的读码框及5’端启动子区191bp和3’端终止区62bp的序列．在起始密码子ATG上游-63～-59bp处有一杆状病毒晚期和晚晚期启动子基序TAAG．联配分析表明，Splt MNPV日本株(C3)的核苷酸序列和氨基酸序列与其它9种核多角体病毒的同源性有较大差异．以p10基因为基础绘制的杆状病毒分子进化树将家蚕核多角体病毒(Bombyx mori nuchopolyhedrovirus，BmNPV)与苜蓿丫纹夜蛾核多角体病毒(Autographa californica multicapcid nucleopolyhedrovirus，AcMNPV)归到同一分枝，这与以gp37基因和egt基因为基础绘制的杆状病毒分子进化树结果一致．     

斜纹夜蛾核型多角体病毒单克隆抗体的制备和分析

用纯化的斜纹夜蛾核型多角体病毒（Spodoptera litura nucleopolyhedrovirus)的病毒核衣壳免疫小鼠,取脾脏细胞与骨髓瘤细胞融合,经筛选得到5株稳定分泌单克隆抗体的杂交瘤细胞株。它们所分泌的单抗分属IgG1,IgG2a,IgG2b和IgG3等4种抗体亚类,其中1株单抗识别分子质量为41ku的病毒蛋白,其余4株单抗均识别31ku的病毒蛋白。胰蛋白酶部分酶解分析发现,31ku的蛋白的4株单抗分别识别至少3个不同的抗原决定簇。所有5株单抗均不与其他4种核型多角体病毒发生交叉反应。利用所制备的单抗进行了病毒在虫体中增殖动态的检测。这些单克隆抗体可用来深入研究病毒的流行规律和复制机制。     

柳毒蛾核型多角体病毒内蒙株的形态学研究

从内蒙古太仆寺旗自然死亡的柳毒蛾虫尸中分离提纯多角体。其平面图呈不规则形状，有三角形、四￣六边形和近圆形。大小相差悬殊，直径在０．９４￣２．４７μｍ之间，约有５６．４％的多角体在１．６０￣２．１０μｍ之间，平均为１．７８μｍ。多角体硷解后释放出短杆状病毒束，大小为２０４￣４１６ｎｍ×１０４￣２９４ｎｍ之间，有４２％的病毒束在３４５￣３６５ｎｍ×１６１￣１８５ｎｍ之间，平均为３５８×１７０ｎｍ。约７     

栗黄枯叶蛾核型多角体病毒病毒粒子的拉曼光谱研究

通过对栗黄枯叶蛾核型多角体病毒TvNPV(Trabala vishnou Nuclear Po-lyhedrosis Virus)的病毒粒子的拉曼光谱研究,讨论了它的蛋白质分子空间构象.其蛋白质主要为β折叠结构、α螺旋结构以及少量无规卷曲(其中包括β回折)结构.蛋白质中酪氨酸残基多数暴露于分子表面,少数埋藏在分子内部;色氨酸残基大多埋藏在疏水环境中.蛋白质分子侧链C—C—S—S—C—C构型为反式—扭曲—反式.     

大袋蛾核型多角体病毒,病状及毒力的生物测定

报道了对大袋蛾核型多角体病毒、病状的观察。林间因感染大袋蛾核型多角体病毒而死亡的幼虫可分为两类：一类为纯病毒感染，另一类为病毒和细菌混合染。用不同浓度的大袋蛾核型多角体病毒感染大袋蛾卵，得多角体浓度与死亡率的直线回归方程：ｙ＝０．７８＋０．８２３ｘ，ＬＣ５０为１．１８×１０＾５ＰＩＢ／ｍＬ。用１０＾５，５×１０＾５，１０＾６ ＰＩＢ／ｍＬ感染卵，得感染时间与死亡率的关系和ＬＴ５０，分别为：ｙ＝６．     

AcMNPV bro基因的表达和作用研究

bro基因(baculovirus repeated orf)是杆状病毒编码的一个独特的基因家族,一些病毒的基因组中编码有多个该基因家族的成员.研究分析了苜蓿银纹夜蛾核型多角体病毒(AcMNPV)基因组仅有的一个bro基因,结果表明:AcMNPVbro基因(ac-bro)在感染后6h就开始转录,并在8h时检测到BRO表达;ac-bro基因敲除的重组病毒感染后子代病毒的效价略低于野生型病毒,且其DNA复制明显推迟.利用大肠杆菌中重组表达的Ac-BRO蛋白进行的体外实验提示其具有结合DNA的能力.这些结果提示ac-bro基因在病毒的复制中有一定的作用.     

Nucleotide sequence of polyhedrin gene of LsNPV and analysis of baculovirus polyhedron proteins

The intact 741 bp polyhedrin gene of LsNPV was sequenced by Silver, Sequencing System, and shares 90.6% and 97.0% nucleotide identity, 97.2% and 97.6% amino acid identity with PfNPV and MdNPV polh genes respectively. The 14 bp conservative sequence with the core element GTAAG, is located in the 5′untranslated region of the gene. The polh gene was predicted to encodes a 246 amino acid residures with molecular weight of 29.0 kd, in which the number of acidic amino acids and alkaline amino acids was roughly equal resulting in almost no charges in polyhedrin protein molecule and hence occlusion body. It gives a valuable implication that ionic bonds as well as hydrophobic bonds and hydrogen bond may play an important role in the crystallization of polyhedrin, by comparing amino acid variation of twenty—one polyhedrin. The comparsion of promoter regions of polyhedrin gene and class II gene shown that they are very similar, but also have differences in GC content. This could explain that both categories of gene are highly expressed, and polyhedrin genes are expressed more higher than class II gene. Wang Jiawang: born in 1962, Doctor of science. Present address: Cancer Institute, CAMS, Beijing 100005     

利用空间诱变选育辅酶Q10高产菌

 以空间搭载为诱变手段,筛选可用于工业化生产的辅酶Q10高产菌株。以类球红细菌（Rhodobacter sphaeroides）为出发菌株进行空间搭载,根据菌落形态进行初筛,摇瓶发酵进行复筛,HPLC检测含量等方法筛选辅酶Q10高产菌株。最终得到名为Shenzhou6的突变株产量比对照菌株提高30％,产量可达（0.8+0.02）g/L,经过优化培养可满足工业生产需要。同时也说明空间诱变因其复杂的空间环境条件,可以作为一种诱变手段进行工业化菌样的筛选。     

DNA甲基转移酶抑制剂DAC对AcMNPV感染的影响

通过DNA甲基转移酶抑制剂DAC(地西他滨,decitabine)改变DNA甲基化水平,研究杆状病毒苜蓿银纹夜蛾核型多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus,AcMNPV)DNA的甲基化与病毒复制和基因表达调控的关系。通过流式细胞术和病毒mRNA的Real-time PCR检测发现,低浓度DAC(30nmol/L)处理细胞后会使报告基因绿色荧光蛋白基因(Green fluorescent protein gene,gfp)的表达提前,且表达水平明显上升。对低浓度DAC处理组和对照组产生的病毒效价监控表明,低浓度DAC的存在可以提高病毒的效价。经连续DAC处理,在第六代细胞中生产的AcMNPV多角体感染甜菜夜蛾后,幼虫死亡率明显高于对照组。这些结果表明,DAC对杆状病毒DNA复制和基因表达具有一定作用。     

Construction and function of recombinant AcMNPV with double copies of v-cath gene

Two recombinant baculoviruses, dciAcMNPV and dcdAcMNPV in which another copy of the v-cath gene controlled by ie1 promoter and polh promoter was inserted, were respectively constructed by the Bac-to-Bac system. The expression of the v-cath gene of the recombinant baculoviruses in Sf9 cells at different phases was investigated by SDS- PAGE and Western blot. The results showed that only recombinant virus dciAcMNPV containing late gene v-cath driven by early gene promoter could express V-CATH protein, cathepsin encoded by virus genome, 12 h post-infection and dcdAcMNPV containing late gene v-cath driven by late and very late gene promoters could express more V-CATH protein. Negative control ncAcMNPV, a mutant deleted v- cath gene, could not express V-CATH protein at all. The Spodopera exigua larvae were infected with viruses respectively and the results showed that the toxicity was as follows: dcdAcMNPV>dciAcMNPV>wtAcMNPV>ncAcMNPV. The toxicity of recombinant viruses and the characters of dead larvae showed that the v-cath gene was relative to viral toxicity and host liquefaction. Recombinant baculovirus dcdAcMNPV might be used as a new kind of safe viral-pes- ticide, because of its high toxicity obtained by adding another gene copy and changing the expression level of its own gene relative to virulence.     

定点突变内皮抑素Zn2+的结合位点及突变基因的克隆表达

从人胚肝组织中提取总RNA, 以逆转录聚合酶链式反应（RT-PCR）法获得人内皮抑素编码序列, 采用定点突变技术将His2和His4双突变为Leu2和Val4. 将突变基因cDNA插入含有T7启动子的质粒pET-28b中构建表达质粒pMendo, 转化大肠杆菌BL21(DE3), 筛选表达菌株BL21-Mute, 表达菌株经IPTG诱导后以包涵体方式产生大量内皮抑素突变蛋白. SDS-PAGE分析表明, 表达的重组蛋白占菌株可溶性蛋白质的30%. 复性、 纯化的内皮抑素突变蛋白纯度达到98%, 失去抑制人脐静脉内皮细胞增殖的活性.     

耐高温巴氏杜氏藻突变株的诱变和鉴定

采用紫外线辐射的方法对巴氏杜氏藻(Dunaliella bardawil)进行诱变.经过夏日高温(42~52℃)筛选,得到一耐高温的突变株,命名为Dunaliella bardawilvar.H-42 Huang&Liu(简称H-42).研究结果表明,在每天42℃处理6 h的条件下,H-42达到生长平衡期时藻细胞密度为初始密度的4.5倍,而对照在第5天时细胞密度几乎降为零.通过显微镜观察并测量,证明突变株的细胞体积为(0.65~1.27)×10-15m3,是原出发株的3.0~3.4倍.蛋白质SDS-PAGE电泳表明,H-42在大约24 kD和30 kD处分别比对照多出一条新的蛋白带,同时在大约26 kD处缺失一条蛋白带.RAPD分析表明两者的遗传相似系数为0.726.由此我们认为H-42确为一耐高温的巴氏杜氏藻突变株.     

Point Mutation Identification Using On-Chip Ligase Detection Reaction

An efficient method was developed to detect point mutations using oligonucleotide microarrays and the ligase detection reaction (LDR). Allele-specific LDR primers were immobilized on polylysine-coated glass slides to perform LDR on a chip. The spotting concentration and detection limit were analyzed using a synthesized oligonucleotide as a template. The optimal primer spotting concentration was 20 μmol/L and the lowest detectable template concentration was 0.33 nmol/L. The method was successfully employed to identify malignant mutations of hypertrophic cardiomyopathy. Asymmetric polymerase chain reaction was employed to prepare single stranded DNA as LDR templates from cloned plasmids. The discrimination ratios for AC, TC, GT, TT, GA, and AA mismatches are 32.82, 44.24, 17.75, 18.34, 11.66, and 8.91, respectively. This method may allow construction of multiple mutation detection systems.     

c-Jun氨基末端激酶3结构域突变质粒的构建、鉴定与转染

为构建c-Jun氨基末端激酶3 (c-Jun N-terminal kinase3,JNK3)的p VP16-eGFP-Myc-JNK3活化环(activation-loop,A-loop)结构域突变型重组质粒,通过A-loop突变型JNK3的c DNA序列以及p VP16-eGFP-Myc的酶切位点设计引物,PCR扩增目的基因,使用限制性内切酶Mlu I与Xba I将基因克隆到p VP16-eGFP-Myc真核表达载体中,构建p VP16-eGFP-Myc-JNK3(A-loop)结构域突变型重组质粒。转化后,通过双酶切鉴定与DNA测序判断是否成功构建重组质粒,使用Trans IntroTMEL Transfection Reagent转染人胚肾(human emborynic kidney,HEK) 293T细胞,通过荧光显微镜下观察融合蛋白表达情况。结果表明:构建p VP16-eGFP-Myc-JNK3 (A-loop)结构域突变型重组质粒,双酶切鉴定和测序结果显示构建成功; p VP16-eGFP-MycJNK3 (A-loop)成功转染HEK293T细胞且表达。鼠源JNK3结构域突变型质粒构建成功,对进一步研究JNK3结构域在相关疾病的作用和机制提供实验工具。     

Construction and function of recombinant AcMNPV with double copies of v-cath gene

Two recombinant baculoviruses, dciAcMNPV and dcdAcMNPV in which another copy of the v-cath gene controlled by ie1 promoter and polh promoter was inserted, were respectively constructed by the Bac-to-Bac system. The expression of the v-cath gene of the recombinant baculoviruses in Sf9 cells at different phases was investigated by SDSPAGE and Western blot. The results showed that only recombinant virus dciAcMNPV containing late gene v-cath driven by early gene promoter could express V-CATH protein, cathepsin encoded by virus genome, 12 h post-infection and dcdAcMNPV containing late gene v-cath driven by late and very late gene promoters could express more V-CATH protein. Negative control ncAcMNPV, a mutant deleted vcath gene, could not express V-CATH protein at all. The Spodopera exigua larvae were infected with viruses respectively and the results showed that the toxicity was as follows: dcdAcMNPV>dciAcMNPV>wtAcMNPV>ncAcMNPV. The toxicity of recombinant viruses and the characters of dead larvae showed that the v -cath gene was relative to viral toxicity and host liquefaction. Recombinant baculovirus dcdAcMNPV might be used as a new kind of safe viral-pesticide, because of its high toxicity obtained by adding another gene copy and changing the expression level of its own gene relative to virulence.