淋巴囊肿病病毒主要衣壳蛋白基因片段原核载体的构建及表达

淋巴囊肿病病毒感染牙鲆鳃细胞系FG-9307，提取细胞总RNA，采用RT-PCR法成功获得了淋巴囊肿病病毒主要衣壳蛋白0．6kb基因片段。构建其原核表达载体，IPTG诱导表达。结果表明，该融合蛋白分子量约48kD，可与抗LCDV多克隆血清特异反应。为LCDV基因工程疫苗的研制奠定了实验基础。     

牙鲆(Paralichthys olivaceus)淋巴囊肿病毒的分离

通过不同的样品处理和离心方法，发现了影响淋巴囊肿病毒（LCDV）得率和纯度的因子，通过消除该因子并结合计算机软件设计程序，建立了高纯度LCDV的快速分离技术。     

牙鲆淋巴囊肿病毒主要衣壳蛋白基因片段真核表达载体的构建、表达与免疫效果评价

用淋巴囊肿病毒LCDV-cn感染牙鲆鳃细胞系FG-9307,提取细胞总RNA,用RTPCR法获得了LCDV-cn主要衣壳蛋白(MCP)0.6kb基因片段.将该LCDV-cn-MCP0.6kb片断克隆入真核表达载体pEGFP-N2,得到重组质粒pEGFP-N2-LCDV-cn-MCP 0.6.采用脂质体法将重组质粒转染入牙鲆鳃细胞系FEC,并进行瞬时表达.通过荧光显微镜观察和特异性RT-PCR检测,证实重组质粒pEGFP-N2-LCDV-cn-MCP 0.6kb已成功转染到FEC细胞,并得到了初步表达.将重组质粒肌注入牙鲆体内,检测牙鲆外周血、肠、脾脏、前肾和淋巴细胞的增殖反应、呼吸爆发活性及抗体产生水平.结果表明,构建的核酸疫苗pEGFP-N2-LCDV-cn-MCP 0.6可诱导牙鲆特异性体液免疫和细胞免疫,具有明显的免疫保护作用.     

牙鲆淋巴囊肿病的PCR诊断方法研究

以中国养殖牙鲆 (Paralichthysolivaceus)淋巴囊肿病毒 (Lymphocystisdiseasevirus,LCDVcn)主要衣壳蛋白 (majorcapsidprotein ,MCP)基因的中间保守序列为目标基因 ,设计了一对特异性引物。该引物可扩增出 172bp的病毒DNA片段 ,其最小DNA检出量为 0 0 183ng。用PCR法从人工感染淋巴囊肿病毒 3天的牙鲆血、鳃、肝、脾、肠、胃及自然发病牙鲆的肿瘤中 ,分别检测到了LCDV的存在。本实验结果证明 ,PCR法对于早期检测LCDV是十分有效的。     

牙Ping淋巴囊肿病的PCR诊断方法研究

以中国养殖牙Ping(Paraclichthys olivaceus)淋巴囊肿病毒（Lymphocystis disease virus,LCDV_cn)主要衣壳蛋白（major capsid protein,MCP)基因的中间保守序列为目标基因，设计了一对特异性引物。该引物可扩增出172bp的病毒DNA片段，其最小DNA检出量为0.0183ng。用PCR法从人工感染淋巴囊肿病毒3天的牙Ping血，鳃，肝，脾，肠，胃及自然发病牙Ping的肿瘤中，分别检测到了LCDV的存在。本实验结果证明，PCR法对于早期检测LCDV是十分有效的。     

牙鲆淋巴囊肿病的诊断技术研究

使用本实验室设计的PCR引物，在标记反应体系下，通过PCR反应合成了碱基数为172bp的牙鲆（Paralichthys olivaceus）囊肿病（Lymphocysits disease,LCD）的地高辛标记探针，在其琼脂糖凝胶电泳图172bp处有明显的亮带，由软件分析得出，合成探针的浓度为500ng/μl，此结果证明，该产物确是所需的探针，且具有很好的合成效率。通过实验证明本文采用的核酸探针-杂交诊断方法可以有效地定性和定位LCDV。实验结果还提示。LCDV是通过血液在牙鲆体内传播的，并可能同时存在垂直和水平两种传播方式。     

抗牙鲆淋巴囊肿病毒单克隆抗体的制备

用本实验室分离纯化的淋巴囊肿病毒(Lymphosystis Disease Virus China,LCDVcn)作为抗原,感染BALB／c小鼠,取被免疫小鼠的脾细胞与骨髓瘤细胞(Sp2／0融合;ELISA法检测、筛选阳性克隆,经过3次克隆化,首次成功获得18株抗牙鲆淋巴囊肿病毒的单克隆抗体。     

牙鲆抗淋巴肿病毒免疫球蛋白的纯化

采用饱和硫酸铵沉淀，DEAE－52纤维素离子交换柱层析和Sepharose 6B凝胶过滤的方法，首次得到了纯化的牙鲆抗淋巴囊肿病毒IgM，该IgM的重链与轻链分子量分别为74KD和22KD。     

螺旋藻多糖抗HSV-1作用的体外实验研究

报道从钝顶螺旋藻中提取的一种水溶性多糖类化合物，即钝顶螺旋藻多糖（PSP），在培养细胞内抗单纯疱疹病毒1型（HSV－1）作用的研究。以不同剂量的PSP作用于病毒复制周期的各个阶段，以病毒半数感染量（TCID50），细胞病变（CPE），蚀斑形成（PFU），MTT染色细胞保护率（MTT法）及核酸分子杂交作为评价指标，判断药效。结果表明：PSP对Vero细胞毒性极低；对HSV－1无直接灭活作用，可干扰病毒向宿主细胞吸附，且经PSP预处理的细胞，能明显阻滞病毒产生细胞病变；PSP可有效地抑制病毒复制，但不影响病毒的释放；PSP可明显抑制HSV－1糖蛋白gG mRNA的表达。提示PSP抗病毒靶位在于阻断病毒吸附和抑制感染细胞内病毒的复制及抑制HSV－1糖蛋白gG基因的转录。     

人类骨桥蛋白(hOPN)在细胞增殖中的功能研究

为了研究人类骨桥蛋白（hOPN)与293细胞增殖。细胞周期及与细胞周期有关基因表达的关系。并对其机制进行探讨。成功地构建了hOPN真核表达载体并获得了稳定表达hOPN的细胞系，也同时获得了稳定表达EGFP的细胞系，hOPN对293细胞具有促增殖效应，hOPN蛋白激活了293细胞细胞周期蛋白A的表达。实验结果表明：hOPN通过一定的信号通路刺激了细胞周期蛋白A的表达。加快了细胞进入通过S期，从而促进了细胞的增殖。     

胶原酶消化法纯化牙鲆淋巴囊肿病毒(Lymphosystis Disease Virus China,LCDV-cn)

先后采用各种胶原酶和疏水性制剂,消化、水解病鱼囊肿细胞中带状包涵体外围的胶原蛋白和糖蛋白。经过设计蔗糖密度梯度离心条件,成功分离出了大量的LCDV-cn粒子,并最终建立起一套较完备的、分离纯化LCDV-cn的方法。     

Effect of carbon black content on microcellular structure and physical properties of chlorinated polyethylene rubber foams

Chlorinated polyethylene rubber (CPE) foams filled with various content of carbon black were prepared by compression molding. The effect of carbon black content on the cure characteristics, foam characteristics and physical properties of the CPE compounds were investigated. The results showed that adding ZnSt as activator could lower the decomposition temperature of azodicarbonamide. The results of a moving die Rheometer suggested that the cure characteristics were influenced by the content of carbon black, and the cure rate index increased with carbon black content. The morphology and physical properties results indicated that the content of carbon black played important roles in the cell morphology and physical properties of CPE rubber foams.     

Chlorinated polyethylene nanocomposites: thermal and mechanical behavior

Chlorinated polyethylene (CPE) nanocomposites prepared with natural and organically treated montmorillonite (MMT) clays by solution intercalation method were investigated. X-ray diffraction and transmission electron microscopy techniques showed separation of organically modified clay MMT layers and indicated formation of exfoliated nanocomposites. Fourier transform infrared spectroscopy results showed interaction between the CPE matrix and the clay intercalants of Cloisite^® 30B and Cloisite^® 15A (natural MMT modified with quaternary ammonium salts). Organically treated MMT clays were found to be better dispersed in CPE in comparison to natural MMT clay. Mechanical testing showed enhanced tensile strength, Young’s modulus, and storage modulus of chlorinated-polymers/organically treated MMT clay nanocomposites. Significant improvements in the above properties were obtained with Cloisite^® 15A nanoclay. The temperature, at which maximum degradation occurred, was higher for the nanocomposite having 5 wt% Cloisite 15A than that of neat CPE. Differential scanning calorimetric results revealed that the same composition also absorbed more heat during the heating, indicating better thermal stability. CPE rubber nanocomposite could be a promising heat resistant polymeric material.     

脂质体对牙鲆淋巴囊肿病毒核酸疫苗免疫活性的影响

将淋巴囊肿病毒核酸疫苗pEGFP-N2-LCDV0.6kb用脂质体梭华-SoftastTM包裹后肌肉注射入牙鲆体内,通过测定牙鲆外周血、肠、脾脏和前肾的淋巴细胞增殖反应、呼吸爆发活性以及抗体产生水平,评价脂质体对pEGFP-N2-LCDV0.6kb免疫活性的影响.结果表明,脂质体可显著增强该核酸疫苗的免疫活性和提高保护率,可作为一种有应用前景的核酸疫苗佐剂.     

PMMA、SAN改性PVC/CPE共混体的研究

研究了刚性聚合物(PMMA、SAN)对PVC/CPE共混体力学性能、冲击断面形貌及流变性的影响。结果表明,PMMA对PVC/CPE=100/10、100/15体系,SAN对PVC/CPE=100/10体系都具有显著的增韧作用和一定的增强作用;初步的测定显示,刚性聚合物能改善共混熔体的流变性,促进PVC/CPE共混体系中CPE网络结构的形成和分散性。     

Rapid adhesion and proliferation of keratinocytes on the gold colloid/chitosan film scaffold

The gold colloid/chitosan film scaffold, which could enhance the attached ratio and accelerate proliferation of newborn mice keratinocytes, was fabricated by nanotechnology and self-assembly technology. This nanometer scaffold was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The keratinocytes were cultured and observed on three different extracellular matrices (ECM): gold colloid/chitosan film scaffold, chitosan film and cell culture plastic (control groups). 6 h, 12 h, 24 h after inoculation, the cell attached ratios were calculated respectively. In comparison to control groups, this scaffold could significantly (P < 0.01) increase the attached ratio of keratinocytes and promote their growth. Meanwhile, there were not any fusiform fibroblasts growing on this scaffold. The rapidly proliferating keratinocytes were indentified and characterized by immunohistochemistry and transmissive electron microscope (TEM), which showed the cells maintain their biological activity well. The results indicated that gold colloid/chitosan film scaffold was nontoxic to keratinocytes, and was a good candidate for wound dressing in skin tissue engineering.