【作者】 范文韬；

【导师】 刘德立；

【作者基本信息】 华中师范大学 ， 生物化学与分子生物学， 2001， 硕士

【摘要】 1．SeMNPV vp39基因的克隆和表达 因为重组病毒杀虫剂的发展以及在构建高效表达载体方面的巨大潜力，昆虫杆状病毒（Insect Baculovirus，IBV）成为现代分子生物学研究中的重要领域。核型多角体病毒（Nuclear Polyhedrosis Virus，NPV）是昆虫杆状病毒中最重要的一个属。其模型种苜蓿银纹夜蛾多粒包埋核型多角体病毒（Autographacalifornica multiple nuclear polyhedrosis virus）和家蚕单粒包埋核型多角体病毒（Bombyx mori single nuclear polyhedrosis virus）已得到了详尽的研究。核衣壳蛋白基因（Nuclear capsid protein gene，vp39）是昆虫杆状病毒的一个重要的必需结构基因。vp39基因与病毒侵染、核衣壳迁移和子代病毒的装配有着密切的关系。本文运用聚合酶链式反应（Polymerase Chain Reaction，PCR）等分子生物学技术，将甜菜夜蛾多粒包埋核型多角体病毒（Spodoptera exigua multiplenuclear polyhedrosis virus，SeMNPV）的vp39基因克隆，并将其插入到高效表达载体pET-28a，构建了重组原核高效表达质粒pET-Se39。质粒转化入大肠杆菌BL21中，SeMNPV vp39基因在IPTG的诱导下高效表达。SDS-PAGE证实表达蛋白的分子量约为39kD。诱导4小时，蛋白表达量达到最高。利用极早期基因ie1启动子构建了真核重组表达质粒pGEM-IE1-Se39。这项研究不仅有助于对vp39基因结构和功能的进一步研究，还将有助于病毒和宿主关系的深入探讨。 卜自霎霎。1历丰兰付个寸 tZ竟目／刁 叫上丁V儿人 丫、、。y VuHRS 工HESIS LSPR生物传感器的研究和应用 生物体内的任何生命活动都离不开生物大分子相互作用。无论是基因的 复制、转录和表达还是信号传递等过程的实质都是生物大分子相互作用。表 面等离子激元共振（sufe plasmen resonance，SPR）生物传感器是一种全新的 生物大分子相互作用检测仪。它操作简便快捷，检测灵敏，而且无须酶标记， 兔疫荧光标记和放射性同位素标记。不仅简化了操作、避免了对实验样品的 影响，还减少了环境的污染。本文利用乙肝表面抗原、抗体；破伤风类毒素、 抗毒素等生物制品，对表面等离子激元共振生物传感器在免疫学检测上的特 征进行了探讨。更多还原

【Abstract】 1. Cloning and expression of the vp39 gene of SeMNPV Because of its huge potential value in the fields of the construction of high efficiency expression vector and the development of reconbinant virus pestcide ,insect baculovirus(IBV) becomes one of the hotspots of molecular biology research. Nuclear polyhedrosis virus(NPV) is the most important genus of IBV with nearly 447 species. Nuclear capsid protein gene(vp39) was proved to be closely related to the infection process.A fragment of SeMNPV (Spodoptera exigua multiplenucleocapsid nuclear polyhedrosis virus) about 1 .3kb had been amplified by employing PCR technique and by designing a pair of primers based on the sequence of capsid protein gene vp39 of AcM7NPV(Autographa caizfornica multiplenucleocapsid nuclear polyhedrosis virus). The recombinant expression plasmid pET-Se39 has been constructed by subcloning the 1.3kb fragment into the procaryotic expression vector pET-28. After transformed into E. coil BL2 1, SeMNPV vp39 gene was highly expressed by the inducement of IPTG. SDS-PAGE analysis showed that the molecular weight of the expressed protein was about 39kD. The amount of the expressed product reached to the highest level when it had been induced with IPTG for 4 hours. The encaryotic expression plasmid pGEM-IE1-Se39 containing an iel promotor has been constructed. This research would redound to the deep study of the structure and the function of the vp39 gene ,and furthermore, would be helpful to the research of the relation between virus and hosts. 2. Research and applying of surface plasmon resonance biosensor Biomacromolecule interaction is so important a mechanism that nearly every proceeding in the body of creature, the copy of gene,the transcription of gene, the expression of gene, the enzymatic reaction,the transmission of signal, etc., related to interaction of bioniacromolecule. Surface plasmon resonance(SPR) biosensor is a novel biomacromolecule interaction detection instrument. Compared with other methods, surface plasmon resonance biosensor has many unique advantages. Not only is it convenient and sensitive, but also it is unnessisary to mark samples with enzyme, fluorescence marker or radioactive tracer. With several sorts of biological products e.g. hepatitis B surface antigen/antibody and tetanus toxsoid/antitoxm,, the speciality of surface plasmon resonance biosensor has been discussed in the paper.更多还原