pH值对巴西橡胶树胶乳β-1,3-葡聚糖酶结合黄色体膜的影响

基于黄色体内含物中的多种可溶性蛋白质以及黄色体膜在胶乳凝固中所起到的重要作用,已提出了多种阐明胶乳凝固机制的假说,但有关β-1,3-葡聚糖酶在胶乳凝固中的作用尚无报导.通过聚丙烯酰胺凝胶电泳(Tricine-SDS-PAGE)和免疫印迹技术(western bloning),对不同pH值条件下破裂收集的黄色体内含物和膜组分中的蛋白质进行研究.结果表明,在pH5.5左右的酸性环境中,β-1,3-葡聚糖酶主要表现为可溶性蛋白质,很少结合黄色体膜;但在pH6.9左右的中性及偏碱性的环境下,大量β-1,3-葡聚糖酶结合到黄色体膜上,可溶性蛋白所占的比例很低.首次发现了β-1,3-葡聚糖酶可以结合黄色体膜,二者结合的程度明显受环境pH值的影响.该结果对于认识β-1,3-葡聚糖酶的生物功能,完善黄色体在胶乳凝固中的作用及阐明乳管堵塞机制具有重要意义.     

3型鲤疱疹病毒白介素10单克隆抗体制备与应用

白介素（Interleukin，IL）10是 脊椎动物Th2细胞（T helper 2 cell）和先天淋巴细胞（Innate lymphoid cell）分泌的一种多效应细胞因子。3型鲤疱疹病毒（Cyprinid herpesvirus 3, CyHV3）ORF134编码IL10样蛋白，本研究构建了ORF134原核表达质粒，在大肠杆菌DE3感受态细胞中表达CyHV3-IL10重组蛋白。SDS-PAGE分析结果表明，重组CyHV3-IL10蛋白约为18 ku，主要以包涵体形式表达。将CyHV3-IL10包涵体进行变性和复性，经分子筛凝胶柱层析纯化获得了纯度较高的重组CyHV3-IL10蛋白，利用CyHV3-IL10重组蛋白免疫小鼠制备了单克隆抗体，通过酶联免疫吸附测定法（Enzyme linked immunosorbent assay，ELISA）筛选，获得了2株亲和力强的抗体，并利用激光共聚焦技术和Western blotting对抗体进行了鉴定。结果表明，FITC标记的单克隆抗体能够特异性识别HEK293细胞中表达的CyHV3-IL10重组蛋白和感染CyHV3的镜鲤脾和鳃组织中表达的CyHV3-IL10蛋白。CyHV3-IL10单克隆抗体的制备为后续研究CyHV3-IL10蛋白功能和建立CyHV3诊断方法奠定了基础，为CyHV3病毒检测试剂盒的研发提供了新思路。     

Wnt3a在不同毛色羊驼皮肤中的表达和定位

本研究旨在探索Wnt3a在羊驼皮肤中的表达与定位.以不同毛色的成年羊驼为研究对象,应用荧光定量PCR技术分析不同毛色羊驼皮肤Wnt3a基因的相对表达量,并运用Western blotting及免疫组织化学法对Wnt3a蛋白在不同毛色羊驼皮肤中进行表达和定位研究.结果:荧光定量PCR结果显示棕色羊驼中Wnt3a mRNA相对表达量是白色羊驼的2.9702倍;Western blotting结果表明,羊驼皮肤组织组蛋白提取物中存在相对分子质量约39 ku的产物,棕色羊驼皮肤平均蛋白表达量显著高于白色羊驼;免疫组织化学结果显示Wnt3a在羊驼皮肤毛囊的根鞘和毛球部呈阳性表达,根据光密度值分析得出Wnt3a在棕色和白色羊驼毛囊中的表达差异显著(P＜0.05).通过以上研究显示Wnt3a可能与羊驼毛色形成具有相关性.     

转基因稻谷外源蛋白在肉仔鸡体内的安全评价

为了研究肉仔鸡体内器官中是否存在转基因稻谷所表达的外源蛋白,为转基因水稻品系进一步的饲用或食用安全性研究奠定基础,试验将200只1日龄的AA肉仔鸡随机分成2组,试验组、对照组,分别饲喂转基因稻谷及其亲本配制的基础日粮,采用Western blotting方法对饲喂转基因稻谷及其亲本稻谷种子、饲料及其对应粪便,各组肉仔鸡的胸肌、腿肌、肝脏、肾脏、血液、腺胃、肌胃、十二指肠肠壁、空肠肠壁、回肠肠壁的转基因外源蛋白在肉仔鸡体内的表达情况进行了检测。结果表明:采用Western blotting技术未在肉仔鸡体内各器官中检测到转基因稻谷所转录表达的外源蛋白,转基因蛋白成分能够被机体完全消化,肉仔鸡粪便中未检测到所转录的外源蛋白,转基因蛋白不会通过粪便在自然界传播。     

Serological characterization of fluorescent Pseudomonas strains cross-reacting with antibodies against Erwinia chrysanthemi

Sixteen bacterial strains, cross-reacting with antibodies againstErwinia chrysanthemi (Ech), were isolated from potato peel extracts, ditch water, and the rhizosphere of wheat, onion, sugar beet and chicory using the immunofluorescence colony-staining procedure. Based on fatty acid profiles, isolates were classified as belonging to thePseudomonas fluorescens group.These strains, together with two previously isolated cross-reactingP. fluorescens strains, crossreacted with polyclonal antibodies against Ech in immunofluorescence cell-staining, Ouchterlony double diffusion, and ELISA. Seventeen strains also reacted strongly with monoclonal antibodies against the lipopolysaccharides (LPS) of Ech in ELISA.Cell envelopes (CE) and proteinase-K-treated CE (mainly LPS) of cross-reacting bacteria were further characterized with SDS-PAGE and Western blotting. Based on CE protein and LPS patterns, the cross-reacting bacteria were classified into two groups, each existing of two subgroups. Both CE and proteinase-K-resistant antigens strongly cross-reacted on immunoblots with antisera against a wild type strain of Ech. With an antiserum against a LPS O-chain lacking mutant of Ech only protein bands but no proteinase-K-resistant antigens were detected on immunoblots. These data suggest that in all cases the highly antigenic LPS O-chain is responsible for the cross-reactions.     

病毒外壳蛋白基因在马铃薯基因组中的整合与转录表达

以表达马铃薯卷叶病毒（ＰＬＲＶ）外壳蛋白（ＣＰ）基因的转基因马铃薯植株为供试材料,在接种病毒后续发感染条件下提取转基因植株ＤＮＡ和ＲＮＡ,用克隆的病毒ＣＰ基因ｃＤＮＡ为探针进行Ｓｏｕｔｈｅｒｎ和Ｎｏｒｔｈｅｒｎ杂交分析,结果表明,外源ＣＰ基因稳定整合于马铃薯基因组内,并在转录水平上高效表达．     

南美白对虾人工感染细菌后肝胰脏中主要变化蛋白的研究

以采自汕头的南美白对虾为研究对象,运用病原菌人工感染、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、基质辅助激光解吸电离飞行时间质谱分析(MALDI-TOF/MS)、生物信息学和免疫印迹(Western-blot ting)等现代分子生物学方法,探索和鉴定对虾感染后肝胰脏中的主要变化蛋白。结果表明,南美白对虾人工感染溶藻酸弧菌6h之后,实验组和对照组肝胰脏的SDS-PAGE图谱出现12条明显差异条带,其中分子量分别为75kDa(命名为p75)和73kDa(命名为p73)的两种蛋白变化最为明显,进一步研究显示,p75和p73分别为血蓝蛋白的两个不同的亚基。提示,肝胰脏中的血蓝蛋白可能在对虾抗感染免疫中发挥重要的作用。     

Enhanced expression of stress protein 70 in the brains of goldfish, Carassius auratus, reared with bluegills, Lepomis macrochirus

Induction of stress protein (HSP70) was studied in goldfish (Carassius auratus) reared with bluegills (Lepomis macrochirus), a predator of small fish. HSP70 was identified by Western blotting and quantified by optical density after SDS-PAGE. The expression of HSP70 was significantly enhanced in the brains of goldfish reared with bluegills for 6 and 12 h in a single tank. The hepatopancreas and the kidney were not affected by the treatment. When goldfish were separated from bluegills with a partition net (1×1 cm mesh size), this protein also increased in the brains after 6 h but then returned to the control level after 12 h. When the goldfish and bluegills were kept in separate tanks and were not able to see each other but were connected by circulating water, HSP70 levels in the goldfish were unaffected. Immunohistochemical observations indicated that an anti-HSP70 antibody was found to react predominantly with the optic and vagal lobes of the brains. These results suggest that visual perception plays a primary role in enhancement of HSP70 expression in the goldfish reared with bluegills.     

白斑综合征病毒(WSSV)基因VP28原核表达与检测

VP28是对虾白斑综合征病毒(White Spot Syndrome Virus,WSSV)的囊膜蛋白.将VP28基因序列密码子优化后合成,经限制性内切酶NdeⅠ、Xho Ⅰ酶切后,按正确的阅读框顺序插入到pET-28a(+)表达载体上.重组质粒转化大肠杆菌BL21(DE3),经质粒双酶切、测序鉴定后成功地构建了VP28基因原核表达载体pET-28a-VP28.转化菌经IPTG诱导,SDS-PAGE显示含有与预期大小一致的约30 ku蛋白带,主要以包涵体形式表达.采用Ni-IDA-Sepharose CL-6B亲和层析柱对重组蛋白进行纯化,获得了纯度为95％的大肠杆菌重组蛋白VP28.该纯化蛋白作为绝对定量Western的标准品,梯度稀释建立标准曲线,以定量检测转基因鱼腥藻7120中的VP28绝对表达量.实验结果表明,大肠杆菌BL21(DE3)表达的重组蛋白VP28与鱼腥藻7120中表达的VP28分子量基本相同,纯化后的大肠杆菌重组蛋白梯度稀释作为绝对定量Western标准曲线,计算出转基因鱼腥藻7120在培养第17天时,VP28的表达量最大,为5.14 μg/mL,占转基因鱼腥藻7120总蛋白浓度的1.45％.这不仅对转基因鱼腥藻的高效培养具有重要意义,也为日后确定投喂对虾口服疫苗有效剂量防治白斑综合征奠定了基础.     

羊附红细胞体抗原的分析及初步应用

将提取的羊附红细胞体抗原进行SDS-PAGE电泳及免疫印迹分析,并以此抗原为诊断抗原,用ELISA方法对64份羊的血清样本进行了初步检测。结果表明,56kDa和26kDa的羊附红细胞体抗原具有较好的免疫性,而且不与羊附红细胞体阴性血清和羊支原体病阳性血清发生交叉反应,具有较好的特异性;ELISA检测的64份血清样本的阳性率为66.7%,比涂片镜检高22.9个百分点。