组织蛋白酶B在棉铃虫个体发育过程中的表达及活性研究

运用原位水解电泳、免疫印迹和免疫组织化学等方法系统研究了组织蛋白酶B（HCB）在棉铃虫个体发育过程中的表达及活性变化规律．研究显示,HCB的表达和活性随着胚胎发育的进行逐渐下降;整个幼虫阶段都没有HCB的表达和活性;整个蛹和成虫阶段虽然都有HCB的表达,但HCB的活性只能在发育晚期的蛹和成虫组织中检测到．这些现象表明,HCB的表达和活化属于翻译后控制,同时与棉铃虫的个体发育和组织分化有着密切的关系、     

抗棉铃虫组织蛋白酶B单克隆抗体的制备及鉴定

分别以棉铃虫组织表达的组织蛋白酶B和基因重组的大肠杆菌表达的组织蛋白酶B为抗原免疫BALB／c小鼠，选择血清抗体滴度高的免疫小鼠，取其脾细胞和Sp2／0骨髓瘤细胞进行细胞融合．经ELISA法筛选阳性克隆和有限稀释法克隆细胞，共获得分泌抗组织蛋白酶B的单克隆抗体杂交瘤细胞3株，分别命名为9D5，100E2和100H6．以ELISA法和Western blotting法对3株杂交瘤细胞株分泌的单克隆抗体进行了特异性鉴定，其中9D5和100E2能够同时识别两种不同来源的组织蛋白酶B，而100H6只能识别基因重组的大肠杆菌表达的组织蛋白酶B．     

利用昆虫-杆状病毒表达系统制备HPVl6E6蛋白

目的 利用昆虫一杆状病毒表达系统高效表达HPVl6E6蛋白.方法 将HPVl6E6基因克隆入供体质粒pFastBac-HTB),然后再重组入杆状病毒基因组;用带有目的 基因的杆状病毒转染昆虫(Sf-9)细胞,27℃培养72 h,收集被转染细胞,SDS-PAGE,westem-b10t分析鉴定表达蛋白.结果 SDS-PAGE分析显示所表达蛋白分子量约为24KD,western-blot结果表明表达产物为HPV16E6蛋白.结论 利用昆虫杆状病毒表达系统获得HPV16E6蛋白的高效表达.     

neuritin基因杆状病毒表达载体的构建

为研究Neuritin蛋白的功能,将neuritinORF在杆状病毒-昆虫表达系统中表达,构建杆状病毒表达载体.以308D10为模板,利用PCR方法扩增neuritinORF,定向克隆法将其克隆至PFASTBAC-HTA转移载体中.然后利用同源重组原理将其转移至杆粒bacmid中.得到携带neuritin ORF的重组杆粒.本实验获得了重组目的基因真核表达载体并经PCR鉴定,插入片段方向、大小正确,DNA测序分析表明插入处接头和读框与预期序列相符,成功构建了Neuritin的杆状病毒表达载体.     

狂犬病病毒基质蛋白的真核表达及鉴定

本文设计并合成了狂犬病病毒PV株基质蛋白(matrixprotein,MP)基因与优化的PV株基质蛋白基因,分别克隆至转移载体pVL1393中构建穿梭质粒,再以穿梭质粒与线性化的杆状病毒BaculoGoldTM进行重组,构建表达MP的重组杆状病毒,然后感染Sf9昆虫细胞进行表达.通过SDS-PAGE电泳分析显示,表达的MP和优化后MP约分别为27kDa和26kDa.间接免疫荧光检测(IFA)显示,表达的MP和优化后MP均可与鼠抗His单克隆抗体及鼠抗RV血清特异性结合,出现明显的特异性绿色荧光.Western blot检测显示,表达的MP和优化后MP均可被鼠抗His单克隆抗体特异性识别.     

利用昆虫-杆状病毒表达系统制备HPV16E6蛋白

目的利用昆虫-杆状病毒表达系统高效表达HPV16E6蛋白。方法将HPV16E6基因克隆入供体质粒pFastBac-HTb,然后再重组入杆状病毒基因组;用带有目的基因的杆状病毒转染昆虫（Sf-9）细胞,27℃培养72 h,收集被转染细胞,SDS-PAGE,Western-blot分析鉴定表达蛋白。结果SDS-PAGE分析显示所表达蛋白分子量约为24KD,Western-blot结果表明表达产物为HPV16E6蛋白。结论利用昆虫杆状病毒表达系统获得HPV16E6蛋白的高效表达。     

组织蛋白酶B研究进展

组织蛋白酶B(cathepsinB:EC3.4.22.1)是溶酶体内半胱氨酸内切蛋白水解酶,其作用非常广泛,参与机体多种生理、病理过程.尤为重要的是,它能促进肿瘤细胞浸润转移,因此成为目前诊断、治疗恶性肿瘤的研究热点.综述了组织蛋白酶B的生物学特性、基因与蛋白结构特点、表达调控机制,以及应用方面的研究.     

丝氨酸蛋白酶抑制剂可抑制昆虫细胞表达的蛋白质降解

干细胞因子是一种多功能细胞因子,能在多级造血水平与其他细胞因子协同作用促进造血干/祖细胞及各种血细胞的存活、增殖和分化.重组人二连体干细胞因子具有比干细胞因子单体更高的比活,可以避免副反应.重组人二连体干细胞因子在Sf9细胞和大肠杆菌中表达时,发现有特异性降解.片段缺失实验证实切割位点位于重组人二连体干细胞因子的145位到165位氨基酸之间.丝氨酸蛋白酶抑制剂aprotinin和PMSF能抑制这种特异性降解.试验了不同浓度的aprotinin和PMSF对Sf9细胞存活和重组人二连体干细胞因子产量的影响,显示当aprotinin的浓度为1.0μg/mL时,重组人二连体干细胞因子的产量是没有加aprotinin时产量的2倍,而且aprotinin可以完全抑制这种特异性降解.     

棉铃虫泛素基因的克隆及序列分析

泛素介导的泛素-蛋白酶体通路(Ubiquitin-proteasome pathway,UPP)是真核细胞内依赖ATP的非溶酶体蛋白质降解途径,该途径对细胞内蛋白的选择性降解起着重要作用.设计一对简并引物,从棉铃虫Helicoverpa armigera卵巢细胞Ha831中克隆了泛素基因的编码区,GenBank登录号AY456195.序列分析表明,该编码区的长度为228bp,编码76个氨基酸、其编码蛋白的相对分子质量为8 560,等电点为6.56.同源性比较发现,棉铃虫泛素基因与其他真核生物泛素基因在氨基酸水平上具有96%以上的相似性,而与棉铃虫核多角体病毒泛素的相似性为76%,所有已知的泛素关键功能位点在该泛素蛋白中均保守存在.     

沿江棉区棉田棉铃虫卵的空间格局

对安徽沿江棉田棉铃虫卵的空间分布动态规律进行了调查研究,结果表明棉铃虫卵在棉田的聚集程度随发生代次而逐渐增强,发现不同代次虫口密度相同对理论抽样数明显不同,提出了沿江各代估值调查中的理论抽样数,并对几种空间格局模型拟合棉铃虫的空间分布情况作了比较。     

棉铃虫致病菌分离、鉴定及高活力苏云金杆菌的筛选

从山东省棉田自然死亡的棉铃虫上分离到常见致病菌至少有8种,经鉴定为:苏云金杆菌(Bacillusthuringiensis)、蜡状芽孢杆菌(Bacillus cereus)、铜绿假单胞菌(Pseudomonas aeruginosa)、球孢白僵菌(Beauveriabassiana)、卵孢白僵菌(B.tenella)、青霉菌属真菌(Penicilliumspp.)、拟青霉属真菌(Paecilimycesspp.)和蛾霉属真菌(Nomuraespp.);得到一株对棉铃虫高毒的苏云金杆菌菌株Hu,其毒力比普遍使用的Bt菌系7216高,二者的LC50分别为15.1μg/mL和26.28μg/mL。蛾霉属真菌对棉铃虫有明显的拒食作用和杀伤作用,但人工培养时生长十分缓慢。     

滞育和非滞育棉铃虫脑的组织解剖学研究

滞育和非滞育棉铃虫在不同的发育阶段，脑的形态组织学结构存在着一定的差异，主要表现在：非滞育脑的神经纤维体发达，神经分泌细胞中的核较大，且细胞质中除了有线粒体外，还有大量的粗面内质网及游离的核糖体；而滞育脑的神经纤维体相对固缩、不发达，神经分泌细胞中核较小，细胞质中含有线粒体、光滑内质网和特有的脂滴，不具粗面内质网．这些证据表明滞育棉铃虫的脑活性相对较低．     

棉铃虫滞育解除的相关基因鉴定

运用差异显示PCR(DD-PCR)方法分析棉铃虫滞育解除蛹差异表达的基因,结果得到了56个差异片段.通过RT-PCR和Real-time PCR的方法,鉴定了滞育解除过程中有3个基因表达量在下调,有4个基因表达量在上调.这7个差异表达基因中有3个和已知的基因有较高的同源性:FKBP12,esr16和NADH dehydrogenase 1 alpha subcomplex subunit 6.这些差异基因为今后研究滞育解除的分子机制提供了新的线索.     

Tissue-specific expression of glutathione S-transferases induced by 2-tridecanone or quercetin in cotton bollworms, Helicoverpa armigera (Hübner)

The tissue-specific expression of glutathione S-transferases (GSTs) in the cotton bollworm and the expression level induced by 2-tridecanone and quercetin were examined using the methods of biochemistry and the quantitative PCR. The relative expression level of GST mRNA was unanimous with the GSTs activity conjugaging with 1-chloro-2, 4-dimitro-benzene (CDNB) in fat bodies,midguts, heads and integuments of cotton bollworms. The GSTs activity in fat bodies was the highest, then midguts, heads and integuments in turn, which was in consistent with the relative expression level of GST mRNA. The specific activity of GSTs and the relative expression level of GST mRNA could be significantly induced by 2-tridecanone and quercetin, and after the induction the order of the GSTs activity and the relative expression level of GST mRNA in the above four tissues in cotton bollworms was not different from the control.The induction of GSTs by 2-tridecanone was stronger than by quercetin in all four tissues, which was in accordance with the relative expression level of GST mRNA. It suggested that the increase of GSTs activity induced by plant allelochemicals was associated with the elevated expression of GST mRNA in cotton bollworms.     

Host selection of Helicoverpa armigera and H. assulta and its inheritance

The difference in host selection between the polyphagous Helicoverpa armigera (Hübner) and the oligophagous H. assulta Quenée was examined, with cotton (Gossypium hirsutum), tomato (Lycopersicon esculentum) (hosts of H. armigera), tobacco (Nicotiana tobaccum) (host of both H. armigera and H. assulta), and bush redpepper (Capsicum frutescens) (host of H. assulta) as testing plants. A multiple-choice test was used with caged plant cuttings for adult oviposition and with leaf discs for larval feeding. A no-choice test was run for evaluating larval growth rate. The results indicated that the relationship between larval performance and adult preference of H. assulta was more conspicuous than that of H. armigera. Reciprocal hybridization between H. armigera and H. assulta followed by backcrossing of the hybrids (F1) with H. armigera was also carried out for genetic study on host selection of these two insect species. A two-choice test with cotton and bush redpepper leaf discs showed that H. armigera larvae preferred to feed on cotton, and H. assulta larvae to bush redpepper; feeding preferences of the two F1 lines were intermediate between those of their parents, but close to that of their female parent; preference indexes of backcross lines also showed that both maternal factor and chromosomal inheritance were involved in feeding selection of two Helicoverpa species.     

Synthesis dynamic and developmental profile of prothoracicotropic hormone between diapause-and nondiapause-destined individuals in Helicoverpa armigera

Biosynthesis and secretion of prothoracicotropic hormone (PTTH) of diapause-and nondiapause-destined individuals in Helicoverpa armigera were studied using whole-mount immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). The immunocytochemistry revealed that PTTH is expressed in two pairs of lateral neurosecretory cells of the brain. The presence of immunoreactivity has not significant difference between the brains of the diapause-and nondiapause-destined 6th instar larvae. However,the obvious differences of expressional pattern from day 4 pupae were observed be-tween the two types. PTTH titers in hemolymph from the 6th instar larvae to pharate adults were measured by the ELISA. Although there were similar titer changes between the two types of individuals at the larval stage,a significant difference from developmental expression was detected at the pupal stage,suggesting that the expression and secretion of PTTH does play a crucial role in regulation of pupal diapause of H. armigera.