牙鲆鳃细胞与中国对虾淋巴细胞杂交细胞的体外培养

In vitro culture of hybrid cells derived from flounder gill cells and shrimp lymphocytes

利用PEG介导的细胞融合方法 ,将已成系的牙鲆鳃细胞与中国对虾淋巴细胞进行细胞杂交 ,对所获得的杂交细胞用MEM∶MPS( 1∶1)混合培养液进行体外培养。培养 5天后发现 ,杂交细胞长成了单层 ,传代培养后仍可继续生长和分裂。通过逐渐提高混合培养液中MPS的所占比例所筛选出的杂交细胞 ,目前已传 12代 ,生长和分裂势头仍然十分旺盛。通过接种对虾杆状病毒发现 ,10天后杂交细胞出现了明显的病变特征 (细胞收缩变圆、脱落、死亡 ) ,至 14天细胞便几乎完全脱壁死亡。取病变细胞的培养上清 0 .3mL再加入到一瓶已长成单层的杂交细胞中 ,结果 5天后杂交细胞也出现了明显的病变特征 ,而未接种病毒的对照组杂交细胞生长正常。可初步认为我们所得到的杂交细胞确为对虾 (杆状 )

The hybrid cells produced by PEG mediated cell fusion method with gill cells from flounder, Paralichthys olivaceus, and lymphocytes from shrimp, Penaeus chinensis, were cultured in mixed medium of MEM∶MPS (1∶1). After 5 days culture and observation, it was found that the hybrid cells cultured in the mixed medium of MEM∶MPS(1∶1) grew into monolayer and with good growing and proliferating appearance. As the concentration of MPS in the mixed medium was raised gradually, ideal hybrid cells had been screened out. By now, the obtained hybrid cells have been subcultured for 12 generations and still had very good growing and dividing appearance. Hybridoma cells acquired obvious disease appearance such as,the cell contracted to round, disattachment and death, after inoculation with white spot syndrome virus (WSSV) of shrimp for 10 days, and died almost completely at the 14th day after inoculation. After 0.3 mL culture supernatants of the cells with obvious disease appearance added into a new monolayer of normal hybrid cells, the hybrid cells acquired obvious disease appearance 5 days later, while the control cells still with normal appearance. It can be concluded that the hybrid cells obtained in this study are propably the target cells of shrimp viruses.