【作者】 康桦华；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 人工感染对虾白斑综合征病毒(WSSV),克氏原螯虾在感染的2,4,8,16,32h后测定螯虾血淋巴中的酚氧化酶活力,通过交叉吸附实验及红细胞凝集实验测定凝集素的凝集活性。结果显示,在感染的最初8h酚氧化酶活力呈上升趋势,随后的16h和32h则出现下降现象;而血淋巴上清对鸡红细胞的凝集效价没有发生阶段性变化。说明WSSV在螯虾体内的增殖对酚氧化酶活力有影响,而对其凝集素的凝集活性没有影响。 据本课题组从克氏原螯虾中新发现的丝氨酸蛋白酶抑制物的基因序列(GenBank登录号CD644775)设计一对引物,应用逆转录-聚合酶链式反应(RT-PCR)技术,从克氏原螯虾血淋巴细胞中扩增出丝氨酸蛋白酶抑制物基因PCI188,将其连入原核表达载体pET-32a,转化至大肠杆菌Rosetta菌株和BL21菌株中进行蛋白表达,结果该蛋白只在前者表达。表达产物用免疫转印检测,出现50kD的特异性条带,与已知的与螯虾免疫相关的PCI188基因编码的蛋白大小相符。 组氨酸亲和层析柱纯化丝氨酸蛋白酶抑制物融合蛋白后免疫新西兰兔,经琼脂双向扩散实验测定抗体水平合格,用免疫血清与克氏原螯虾血淋巴作用后测定酚氧化酶活力,结果显示,酚氧化酶活力有所升高,从而首次证实螯虾PCI188编码的蛋白对丝氨酸蛋白酶有抑制作用。 此外,比较了对虾白斑综合征病毒(WSSV)的泰国株(TH株)、中国株(CN株)、中国台湾株(TW株)易变区的差异,根据GenBank中这些毒株的两端外延保守序列设计一对特异性引物,分别用PCR方法扩增并克隆2004年中山株(ZHSH株)、2002年湛江株(ZHJ株)、2002年海南株(HN株)、1996年青岛株1(QD1)和传代后的青岛2株(QD2)5株毒株的易变区基因,采用DNAStar软件对序列进行比对、同源性计算和基因进化树的绘制,分析了不同毒株间易变区的特点,推测易变区与WSSV的进化有密切的关系。更多还原

【Abstract】 Hemolymph of crayfish(Procambarus clarkii) were collected from the ventral sinuse of crayfish while they were injected white spot shrimp virus(WSSV)at 2h,4h,8h,16h and 32h .Then phenoloxidase(PO) activities were measured by spectrophotometer and properties of hemagglutinins were measured by cross adsorption tests and haemagglutination assays.The results were that PO activities showed an upregulation until 8h p.i.,while at 16 and 32h p.i.the PO activities reduced,but the agglutinating titers that agglutinating activities against chicken erythrocyte had no obvious changes.The results showed while WSSV were multiplicated in the crayfish,it would effect on the PO activities but not on the agglutinating activities.The specific primers were designed referring to the reported gene sequences of GenBank accession number CD644775. Serine proteinase inhibitor gene(PCI188) was amplified by reverse transcription polymerase chain reaction (RT-PCR) method using total RNA isolated from crayfish (Procambarus clarkii )hemolymph as template. The amplified cDNA fragments were purified and inserted into pET-32a vector ,then transformed into E.coli BL21 (DE3)and Rosetta.They induced by IPTG for the expression of recombinant protein. The protein was only expressed in Rosetta other than in BL21.Expressed fusion protein was identified by SDS-PAGE and Western-blotting. The results showed that 50 kD fusion protein was identical with the known protein coded by gene PCI 188 of crayfish.Fusion protein of serine proteinase inhibitor was purified by His Bind Resin purification Kit and used to immunized to rabbit. Then phenoloxidase(PO) activities were measured by spectrophotometer after immunized serum and haemolymph of crayfish were cultivated.The results showed that immunized serum increased the PO activity of crayfish . This experiment is firstly to proved that the protein coded by gene PCI 188 of crayfish can inhibit serine proteinase.The specific primers were designed referring to the reported gene sequences of GenBank of white spot shrimp virus(WSSV)isolates from Thailand,China,Taiwan.Variable region of WSSV isolates from ZHSH,ZHJ,HN,QD1,QD2 was amplified by polymerase chain reaction (PCR) method and aloned,sequenced them.Sequence comparisons and phylogenetic analyses were performed by DNAstar software.We analysed the characteristic of variable regions and then discovered that variable region is closely related to evolution of WSSV.更多还原