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目的研究鱼类传染性造血器官坏死病病毒(IHNV)糖蛋白不同区域对免疫原性的影响。方法本研究通过DNAStar 6.0软件分析糖蛋白跨膜区、疏水性及抗原性后,对其进行截短表达;纯化后的糖蛋白制备兔抗血清,ELISA检测兔抗血清的效价,用间接免疫荧光技术检测纯化后蛋白的免疫原性。结果 SDS-PAGE结果显示,截短糖蛋白相对分子质量(Mr)为40 000,纯化后的糖蛋白经高效液相色谱检测纯度达到90%。利用纯化后的蛋白制备兔抗血清,ELISA结果显示,所制备的兔抗血清与糖蛋白的反应效价为1∶80 000,与IHNV-Sn株细胞培养物的反应效价为1∶20 000;间接免疫荧光结果显示,兔抗血清与IHNV-Sn分离株及参考毒株均能发生特异性的反应。结论实验所表达的糖蛋白与天然的病毒表面糖蛋白具有几乎相同的免疫原性。
Objective To study the effect of different regions of the glycoprotein of infectious hematopoietic necrosis virus (IHNV) on immunogenicity. METHODS: The transmembrane domain, hydrophobicity and antigenicity of glycoprotein were analyzed by DNAStar 6.0 software. The purified glycoprotein was used to prepare rabbit antisera. The titer of antiserum was detected by ELISA. Fluorescence detection of purified protein immunogenicity. Results SDS-PAGE showed that the relative molecular mass of truncated glycoprotein (Mr) was 40 000 and the purity of the purified glycoprotein was 90% by high performance liquid chromatography. Rabbit antiserum was prepared by using the purified protein. The ELISA results showed that the titer of rabbit antisera and glycoprotein prepared was 1:80 000, and the reaction titer with IHNV-Sn strain cell culture was 1:20 000 ; Indirect immunofluorescence results showed that the rabbit antiserum and IHNV-Sn isolates and reference strains can react specifically. CONCLUSIONS The glycoproteins expressed in the experiment have almost the same immunogenicity as the native viral surface glycoproteins.