牙鲆淋巴囊肿病毒一抗原蛋白的确认及定位

以牙鲆淋巴囊肿病毒（LCDV）为抗原免疫Balb/c小鼠，而后将小鼠脾细胞与P3U1骨髓瘤细胞融合，以囊肿组织冰冻切片的免疫荧光染色筛选杂交瘤细胞，阳性结果显示特异性块状荧光信号集中在囊肿细胞的细胞质边缘部分，且多个荧光信号相连呈现链圈状，有限稀释 法克隆阳性杂交瘤细胞，三次克隆后获得4株稳定产生抗LCDV抗体的单克隆杂交瘤细胞株（1A8、1D7、2B6、2D11）。应用Western-blotting法分析单抗识别蛋白的分子量，结果显示，单抗1D7 和2B6均能特异性结合一条分子量116 kD病毒多肽；应用免疫电镜技术定位单抗识别的抗原决定簇，结果发现胶体金颗粒集中吸附在病毒粒子衣壳周围，且背景清洁，无散在的金颗粒或其他污染物。实验结果说明分子量约为116 kD的蛋白多肽为LCDV病毒衣壳蛋白，且具有线性抗原决定簇。

Detecting of an antigenic protein of lymphocystis disease virus (LCDV) using monoclonal antibodies

Lymphocystis disease(LCD) is a chronic disease characterized by papilloma-like lesions typically on the skin,fins and tail.The causative agent of the lymphocystis disease,lymphocystis disease virus(LCDV),as been reported in over one hundred teleost species and it has a world-wide geographical distribution.In China LCD has resulted in a great economic loss in marine culture industry and become a factor restricting aquaculture development.In recent years,monoclonal antibody(Mab) technology has had an important impact on fish virus disease management.A large number of Mabs have been developed for fish viruses,In the present study,Mabs against LCDV were produced and characterized. In this experiment,LCDV was separated from lymphocystis nodules by cell disruption,differential centrifugation and density gradient centrifugation in sucrose.The purified virus preparation was used for mice immunizations,Western blotting and immunogold electron microscopy(IEM).Four-week old Balb/c mice were immunized 4 times within 4 weeks with purified LCDV preparations.Three days after the last immunization,spleens of the immunized mice were dissected into cells and then fused with P3-X63-Ag8U1 myeloma cell line using polyethylene glycol(PEG) as fusongen.The fused cells were cultured in HAT-GIT selecting medium for about 2 weeks.The survival cells(hybridoma cells) were cultured in GIT.Mediums of hybridoma cells were detected by indirect immunofluorescence assay test(IFAT). Many positive hybridomas were found and 4 of them were cloned because of secreting high titer antibodies.As they were cloned 3 times continuously,it could be verified that the antibodies raised by these hybridoma cell lines were monoclonal.Then the monoclonal antibodies were used in IFAT,Western blotting and IEM.In the IFAT,cryosections were prepared from nodule tissues of diseased flounder.The specific fluorescence signals were granularm and observed only at the peripheral zone of hypertrophied cells cytoplasm where was the cytoplasmic inclusion bodies location and many of them formed ribbon-shaped.Western blotting analysis identified Mabs to the LCDV proteins.The Mabs demonstrated differences in their polypeptide binding patterns.Two Mabs(1D7,2B6) react specifically with the 116 kD LCDV polypeptide.This result suggests that these Mabs target linear epitopes within the LCDV protein.However,the identity of the target antigen of two other Mabs(1A8,2D11) could not be determined by Western blotting analysis.This observation suggests that these Mabs likely target conformational epitopes that are sensitive to the conditions employed in such analyses.Transmission electrton microscopy immunogold localization results showed that the high density gold particles were located at the outermost surface of freshly purified virus particles,but not the viral nucleocapsid or outside the virions.Very little background labeling was observed.This study provided direct evidence that these four Mabs were anti-LCDV and the epitopes recognized by these Mabs were located on the surface of the virion.In conclusion,four Mabs were produced and characterized for LCDV which recognize structurally different epitopes and confirmed the 116 kD polypeptide was viral proteins.It is anticipated that these Mabs will prove useful in understanding virus host cell interactions,and in tracing the transport of virus-specific proteins through the various cell and virus-induced compartment.