肺炎链球菌免疫小鼠程序的优化及杂交瘤细胞株的建立

Optimization of immune procedure of Streptococcus pneumoniae in mice and establisment of hybridoma cell strains

目的优化肺炎链球菌CSR SCS2 clone1小鼠的免疫程序,同时建立肺炎链球菌C多糖(C polysaccharides,CPs)杂交瘤细胞株。方法制备免疫抗原CSR SCS2 clonel,进行小鼠免疫程序优化:抗原量[(0. 5~2)×10~8个、(1~4)×108个、(2. 4~10)×10~8个)]、免疫间隔时间(3 d、1周、2周)、免疫途径(腹腔、腹股沟及尾静脉),同时确定抗原是否添加佐剂,间接ELISA法检测小鼠血清抗体水平。采用优化程序免疫小鼠,取血清效价最高的小鼠脾细胞,经传统单克隆抗体制备技术建立肺炎链球菌C-Ps杂交瘤细胞株,并检测细胞株的染色体数目及其分泌抗体的稳定性及特异性。结果确定小鼠最适免疫程序为:抗原量为(1~4)×108个,且需添加弗氏佐剂,免疫间隔时间为1周,免疫途径为腹股沟注射。最适程序免疫小鼠血清抗体效价达1∶12 000以上。建立了3株肺炎链球菌C-Ps杂交瘤细胞,命名为A4、G8、H10株,染色体数目分别为98、102和102,分泌的抗体具有良好的稳定性和特异性。结论成功优化了肺炎链球菌小鼠的免疫程序,并建立了肺炎链球菌C-Ps杂交瘤细胞株,为肺炎链球菌荚膜多糖中C-Ps含量检测方法的建立奠定了基础。

Objective To optimize the immune schedule of Streptococcus pneumoniae CSR SCS2 clone1 in mice,and establish pneumococcal C polysaccharide(C-Ps)hybridoma cell strains.Methods Antigen was prepared with S.pneumoniae CSR SCS2 clone1,with which mice were immunized.The immune schedule,including amount of antigen[(0.5~2)×108,(1~4)×10~8 and(2.4~10)×10~8 bacteria],time interval(3 d,1 week and 2 weeks)and immunization route(intraperitoneal,inguinal and intravenous injections),was optimized,and whether the adjuvant should be added into the antigen was determined.The serum antibody level of mice was determined by indirect ELISA.Mice were immunized by the optimized schedule,and the splenocytes of the mouse with the highest serum antibody titer were collected,with which the C-Ps hybridoma cell strain was established by routine technique and determined for the chromosome number as well as the stability and specificity of secreted antibody.Results The immune schedule was optimized as follows:mice were immunized with antigen containing Freund adjuvant at an amount of(1~4)×10~8 bacteria and a time interval of one week by inguinal injection.The serum antibody titer of mice immunized by the optimal schedule was more than 1∶12 000.Three C-Ps hybridoma cell strains were established and named as A4,G8 and H10,of which the chromosome numbers were 98,102 and 102,respectively.The secreted antibodies showed high stability and specificity.Conclusion The immune schedule of S.pneumoniae in mice was successfully optimized,and C-Ps hybridoma cell strains were established,which laid a foundation of development of a method for determination of C-Ps content in S.pneumoniae capsular polysaccharide.