万方数据
万方数据
第1期李新华等:鱼类口服免疫的研究进展
鳟和无胃的鲤鱼,3周后取血和肠黏膜ELISPOT
法检验发现,抗体效价远比对照组未包裹抗原免疫
的要高,同时取黏膜和鳃切片特异性染色,结果浆
细胞大量增加。李新华等[3叩用海藻酸钠包裹嗜水
气单胞菌(APrD7,zD撇s.Ilydro夕^i如)疫苗口服免疫
银鲫(&r口s““s口“m£“sgi抛ziD),结果发现,微胶
囊疫苗组鱼血清抗体效价较高,峰值为1:80,且维
持时间较长;各肠段的黏膜抗体中,后肠黏膜抗体效价最高,微胶囊疫苗组峰值达1:320,前肠的肠黏膜抗体效价也有一定的水平为1:40;肠道免疫组化试验也得到相似结果:在不同时间对前、中、后肠切片进行免疫组化染色,从光镜下观察到肠黏膜外层和黏膜下层均有阳性着色,以包裹全菌苗为最多,未包裹菌苗较少。由此得出结论:口服海藻酸钠包裹后的抗原不仅能激发鱼体全身免疫,还能诱发局部黏膜免疫,口服免疫将是一种行之有效的免疫方法。
(2)聚交酯醣酯聚合物(Polylactide—co—glycol—ide,PLG)目前,用得最为广泛的是聚交酯醣酯聚合物类。它安全、降解性好(活体中2~3个月降解)[3卜33。。PLG必须先用有机溶剂溶解。乙酸乙酯(EA)毒性小,口味好,但对PLG的溶解性和沸点不如二氯甲烷(DCM)[3引。PLG的包裹过程包括PLG的溶解,乳剂的形成,乳剂的稳定,有机溶剂的清除以及颗粒性状的检验:先将PLG溶解在有机溶剂(DCM)内与抗原的水溶液相混合,搅拌形成油包水的乳剂,然后加稳定剂聚乙烯醇(PVA)之后通过蒸发(一般采用真空旋转蒸发仪清除残留的有机溶剂)或者萃取将有机溶剂分离,干燥成1~10肛m的抗原微粒,PLG的操作流程见图1[2。,定量检查包裹效果和显微观察微粒性状见图2。然后拌入饲料投喂。
l抗原溶液II聚合物溶液I
I(水相)II(有机相)l
图1PLG包裹疫苗的操作流程
圈Z扫描电锈下的PL(j包翼颗粒
(3)脂质体磷脂分散在水中自然形成多层囊泡,每层均为脂质的双分子层;囊泡中央和各层之间被水相隔开,双分子层厚度约为4nm。这种具有类似生物膜结构的双分子小囊称为脂质体。脂质体是纳米级材料,使用时先用氯仿溶解,真空旋转蒸发干燥后与菌悬液混合,搅拌形成乳剂,喷雾干燥即得抗原微囊颗粒,与饲料拌匀即可。脂质体包裹的疫苗不仅可以延缓被消化酶破坏降解的时间,同时也是一种很好的疫苗佐剂,能增强口服疫苗的免疫原性,刺激机体产生细胞免疫和体液免疫。
此外,用来包裹的材料还有肠衣、明胶和淀粉等等。但是,Park等[353用肠衣包裹创伤弧菌(y.口“Z”i∥c“s)制成弧菌微囊疫苗免疫牙鲆(P口raZi—cJIl胁ysDzi抛fP甜s),发现免疫效果反不如不包裹直接口服,原因是肠衣的保护影响了抗原的释放。因此不同的抗原应采取不同的包裹材料和包裹方式。同时,要不断开发新的包裹材料,如PLEA和PLEG等。张小江等[363采用可生物降解的合成高分子聚DL-乳酸一聚乙二醇共聚物(DL_polylactide-co-polyethyleneglycol,PELA),包裹哈维氏弧菌(V.|}l口r口e∥)重组外膜蛋白OmpK后,制备成PELA—OmpK微球。斜带石斑鱼口灌四甲基异硫氰酸罗达明(TRITC)荧光素标记的0mpK—PELA微球后,第1d在其后肠观察到大量红色荧光微球,第3d荧光微球数量明显减少,第7d荧光微球基本消失。免疫组化研究结果表明,口服PELA-OmpK组在斜带石斑鱼后肠组织黏膜层可见较强的棕黄色阳性着色。口服PELA—OmpK组血清抗体效价显著高于口服OmpK蛋白溶液组与对照组;口服PELA-OmpK组的粘液抗体效价显著高于口
服OmpK蛋白组。
万方数据
万方数据
万方数据
万方数据
鱼类口服免疫的研究进展
作者:李新华, 沈锦玉, 潘晓艺, 郝贵杰, LI Xin-hua, SHEN Jin-yu, PAN Xiao-yi,HAO Gui-jie
作者单位:浙江省淡水水产研究所,浙江,湖州,313001
刊名:
水产科学
英文刊名:FISHERIES SCIENCE
年,卷(期):2010,29(1)
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