【作者】 林颖博；

【导师】 战文斌；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2009， 博士

【摘要】 本论文在研制的抗中国对虾(Fenneropenaeus chinensis)血细胞单克隆抗体的基础上,通过间接免疫荧光技术首次检测到中国对虾血细胞最早出现于肢芽期,大颗粒细胞最早出现于膜内无节幼体期;通过流式细胞技术研究发现,中国对虾血细胞对荧光微球的吞噬率在注射后迅速升高并在6h后达到最高值,被吞噬的荧光微球在淋巴器官中积累,血细胞吞噬率在白斑综合征病毒(WSSV)感染后虽在短时间内升高但随后迅速降低;论文建立了差速离心法提取的中国对虾血细胞膜与地高辛标记WSSV的Dot-Blot和ELISA体外结合体系作为筛选体系,从制备的中国对虾血细胞膜单克隆抗体中筛选到一株对WSSV具有阻断作用的单克隆抗体R2H1,为进一步研究WSSV与宿主的相互作用机理提供了基础。(1)利用杂交瘤技术,论文制备了特异性识别中国对虾所有三类血细胞的单抗2A3和特异性识别中国对虾大颗粒血细胞中细胞质颗粒的单抗1H11。通过对包括囊胚期、原肠胚期、肢芽期、膜内无节幼体期和无节幼体Ⅰ期在内的中国对虾胚胎的间接免疫荧光检测,最早在肢芽期胚胎中检测到2A3阳性信号;在膜内无节幼体期,2A3阳性血细胞平均直径约7μm,聚集成簇;在无节幼体Ⅰ期,血细胞形态更加完整清晰,平均直径已与成体对虾血细胞相似。1H11阳性大颗粒细胞最早在膜内无节幼体期胚胎中出现,这些细胞同样成簇存在,在其细胞质外围可见点状荧光。在无节幼体Ⅰ期样品中,血细胞总数和大颗粒细胞数量较胚胎期都显著增加,反映了对虾免疫系统随着胚胎发育的进行而加强。(2)流式细胞术分析显示,健康中国对虾血细胞对注射入体内的荧光微球的吞噬在注射后迅速升高并在6h后达到最高点12.58%;随后该比率逐渐下降,至注射后16h实验结束时,血细胞吞噬率已下降到4.02%,反映了血细胞对血淋巴中异物的清除过程。显微镜观察注射微球6h后中国对虾的外周血细胞和淋巴器官,发现主要是半颗粒细胞执行对微球的吞噬,吞噬后的荧光微球在淋巴器官中积累。WSSV感染后第一天,血细胞吞噬率(14.42%)由于应激作用而略高于对照值;随后,该比率迅速降低并维持在对照值的50%左右(6.12%);在感染后第六天,血细胞吞噬率再次上升至11.23%,随后感染对虾大量死亡。(3)论文通过差速离心法提取海捕中国对虾血细胞膜,分别利用Dot-Blot和ELISA技术,使地高辛(DIG)标记的WSSV与之4°C结合4h并以抗DIG抗体作为探针检测,实验结果皆为阳性,证明体外环境下WSSV能稳定地与血细胞膜结合。以抗中国对虾血细胞膜多克隆抗体37°C孵育血细胞膜1h,能够阻断WSSV-DIG与血细胞膜的结合,证明该体外结合体系可以应用于WSSV阻断剂的筛选。应用ELISA体外结合技术,从制备的中国对虾血细胞膜单克隆抗体中筛选具有阻断效果的阳性孔,克隆获得一株单抗R2H1可以显著(P<0.05)削弱WSSV与对虾血细胞膜的体外结合。以Dot-Blot验证,R2H1阻断组发色明显浅于未阻断组。可以推断,R2H1与中国对虾血细胞膜上的受体蛋白结合,从而抑制了WSSV与血细胞膜的体外结合。更多还原

【Abstract】 In this thesis, monoclonal antibodies apecifically against haemocytes of Chinese shrimp (Fenneropenaeus chinensis) were generated. Employing these Mabs as probes, the appearance of Chinese shrimp haemocytes were detected to be at the limb bud stage and the appearance of granular haemocytes were at the embryonic-nauplius stage. Utilizing flow cytometry technique, the phagocytic percentage of Chinese shrimp haemocytes against injected fluorescence microbeads were observed to rise rapidly after injection and peak at 12.58% in 6 hours. The phagocyted microbeads accumulated in the lymph organ of shrimp. The White Spot Syndrome Virus (WSSV) infection stimulated the phagocytosis at the early stage but impaired it soon after. Furthermore, an in vitro combination system of Digoxigenin (DIG) labeled WSSV and isolated haemocytes plasma membrane (HPM) was established as a selecting system basing on the ELISA and Dot-blot. Using this system, monoclonal antibody R2H1 was screened and identified to impair the in vitro combination of WSSV-DIG and HPM.Ⅰ. Mab 2A3 against all haemocytes and Mab 1H11 specific to cytoplasmic granules in granular haemocytes were generated and employed to study the ontogenesis of haemocytes by IFAT in Chinese shrimp embryos of blastula, gastrula, limb bud stage, embryonic-nauplius and nauplius I stages. With Mab 2A3, the first positive cells were detected at the limb bud stage of the embryos. Distinguishable positive haemocytes with a diameter of about 7μm appeared in embryonic-nauplius. At the following nauplius I stage, configuration of haemocytes became more integrated and clear. Their diameters were almost similar to those of haemocytes from adult shrimp. With Mab 1H11, the first positive granular haemocytes were identified at the stage of embryonic-nauplius which existed in clusters with positive punctate fluorescence of granules distributed around the periphery of granular haemocytes. Increased number of both total and granular haemocytes at nauplius I stage impressed a possible enhancement of the immune system as the embryogenesis progressed.Ⅱ. Flow cytometry technology was employed to investigate the phagocytosis of Chinese shrimp haemocytes against injected fluorescence microbeads. The phagocytic percentage of normal Chinese shrimp haemocytes increased rapidly after injection and reached the peak of 12.58% in 6 hours. After that, the percentage decrease slowly. Microscopy observation of peripheral haemocytes and lymph organ showed that the main phagocytic haemocytes subgroup was semi-granular haemocytes and the phagocyted microbeads accumulated in lymph organ. As a response to WSSV infection, the phagocytic percentage at 1 day post infection (14.42%) was slightly higher than control value. However, the percentage then decreased and maintained at about 50% of the control value after that until the percentage increased again to 11.23% at 6 days post infection just before most of the shrimp died.Ⅲ. Chinese shrimp HPM isolated by differential centrifugation were incubated with Digoxingenin labeled WSSV (WSSV-DIG) at 4°C for 4h and detected by antibodies against DIG. Both ELISA and Dot-Blot assay showed positive results of the WSSV-DIG and HPM in vitro combination. When polyclonal antibody against HPM of Chinese shrimp was allowed to bind to the HPM at 37°C for 1h before the incubation with WSSV-DIG, the in vitro combination was blocked in both Dot-Blot and ELISA assay. This result proved that the ELISA and Dot-Blot in vitro combination was a practicable screening system for WSSV receptor antibodies. Using the ELISA system, Mab R2H1 was identified to significantly impair the combination of WSSV and HPM. When confirmed by Dot-blot assay, the R2H1 blocked group showed a significantly lighter color comparing with the unblocked group. It can be deduced that Mab R2H1 recognizes one of the WSSV receptors on HPM, the decrease of WSSV combination was due to the occupation of WSSV binding site by Mab R2H1.更多还原