马铃薯环腐病棒杆菌单克隆抗体的制备

PRODUCTION OF MONOCLONAL ANTIBODIES TO CLAVIBACTER MICHIGANENSE SUBSP.SEPEDONICUM

用马铃薯环腐病棒杆菌的细胞壁蛋白粗提物作为抗原,应用杂交瘤技术,成功地建立了六株分泌抗马铃薯环腐病棒杆菌单克隆抗体的杂交瘤细胞株CH1、CH2、CH3、CH4、CH5和cH6。对这些杂交瘤细胞株所分泌的相应六种单抗McAb1、McAb2、McAb3、McAb4、McAb5和McAb6进行专化性测定的结果表明,McAb4对环腐病菌具有亚种特异性,其它5种单抗除与环腐病菌呈阳性反应外,还与其它亚种的棒杆菌有不同程度的交叉反应。细胞株CH4经体外培养传代30余代和液氮冻存5个月,其分泌抗体的滴度无明显改变。McAb4属于IgG2a亚类,其诱发的小鼠腹水抗体效价达1:2×105,CH4的染色体数为95—102条。应用ELISA夹心法,McAb4所能检测环腐病菌的最低浓度为103个细菌/毫升。检测感染环染环腐病的马铃薯汁液,稀释1000倍仍呈阳性反应。

The cell wall protein extract from strain HECL of Clavibacter michigan-ense subsp.sepedonicum(Spiech and Koith) Davis et al was prepared by LiCltreatments.This protein extractwas used as antigen for immunizing Balb/cmice.The spleen cells from immunized mice were then fused with myelomacell line Sp2/o-Ag14.Six hybridoma cell lines CH1,CH2,CH3,CH4,CH5,CH6 secreting monoclonal antibodies McAb1,McAb2,McAb3,McAb4,McAb5,and McAb6 were established respectively.By testing their specificities toother phytopathogenic bacteria,McAb4 was specific to Cl.m.subsp.sepedoni-cum,While the other 5 lines reacted not only with Cl.m.subsp.sepedonicum,but also with other 8 species of coryneform pathogenic bacteria There was noobvious reduction in the ability of secreting antibody and its titer after thehybridoma cell line CH4 was reproduced for thirty generations or keeped inthe liquid nitrogen for five months. The McAb4 belonged to IgG2a.A high titer(1:10~5) of ascitic fluid wasproduced by injecting CH4 cell in to Balb/c mice.The number of chromosomeof CH4 cell ranged from 96-102.Electroblot immunoassay showed the molec-ular weight of the protein antigen specific to McAb4 was 5900kd,The sensiti-vities of double antibody sandwich (DAS-ELISA) and indirect IF test were7.8*10~3 cells/ ml and 10~3 cells/ml,respectively,A positive reaction occuredin DAS-ELISA for the tissue juice from ring rot-infected tubers by diluted1000 times.