L929细胞无血清悬浮驯化及其在流产衣原体灭活疫苗中的应用研究

Research on Serum-free Suspension Domestication of L929 Cells and its Application in Chlamydia abortus Inactivated Vaccine

为通过悬浮细胞培养方法规模化生产流产衣原体抗原,采用逐步降血清悬浮驯化法使贴壁L929细胞逐步适应悬浮培养环境,最终获得了一株可无血清悬浮培养的L929细胞株。将流产衣原体接种于L929悬浮细胞上,研究不同的促感染试剂以提高收菌量,并选择出最佳的培养方法制备流产衣原体灭活疫苗。以25μL、50μL、100μL的剂量免疫Balb/c小鼠,攻毒后检测其免疫保护效果。结果显示,当该L929悬浮细胞初始接种密度为5×10^(5)/mL时,培养72 h后细胞浓度可达到约7×10^(6)/mL,细胞活力在95%以上;在细胞密度为6×10^(6)/mL,接菌量为5.5×10^(4) IFU/mL,加入脂质体2000的量为1.7μL/mL、放线菌酮浓度为0.4μg/mL时,菌量增殖倍数最大,48 h内约扩增了52倍。经流产衣原体悬浮培养细胞灭活疫苗免疫后,小鼠产生的抗体水平随免疫剂量的增加而提升;接种50μL、100μL疫苗组小鼠的脾淋巴细胞增殖水平、IFN-γ、IL-2的含量远高于接种25μL疫苗和注射PBS组的小鼠;对免疫后的小鼠进行攻毒,接种疫苗组小鼠的脾脏、十二指肠、子宫中的流产衣原体基因组含量远低于未接种疫苗组小鼠。注射PBS组及接种25μL疫苗组的小鼠子宫出现了不同程度的坏死及炎症,而其余两组小鼠的子宫无异常变化。结果表明,本试验成功驯化出1株L929悬浮培养细胞株,并且通过悬浮培养工艺制备的流产衣原体灭活疫苗免疫效果良好,接种50μL、100μL该灭活疫苗可以有效抑制流产衣原体的感染,该L929细胞全悬浮培养株的成功驯化为疫苗规模化生产提供了技术支持。

In order to provide a suspension cell culture method for large-scale production of Chlamydia abortus,this study decreasing serum concentrations,adherent L929 cells adapted to suspension culture gradually,and became completed suspended,finally we get a L929 cell lines can cultured in serum-free suspension.Chlamydia abortus was inoculated on L929 suspension cells,and the best culture method was selected by adding different infection-promoting agents to increase the amount of Chlamydia abortus.Chlamydia abortus inactivated vaccine was prepared,and Balb/c mice were immunized with the vaccine at doses of 25μL,50μL and 100μL,and the immunoprotective effect was detected after challenge.The results showed that when the inoculation density of the suspension cells was 5×10^(5) cells/mL,after 72 hours of culture,the cell concentration reached about 7×10^(6) cells/mL,and the cell viability was more than 95%.When the cell density was 6×10^(6) cells/mL,the infection amount of pathogens was 5.5×10^(4) IFU/mL,the addition of lipofectamine 2000 was 1.7μL/mL and the concentration of actinomycetone was 0.4μg/mL,the amount of pathogens was amplified by 52 times.After immunized with the suspended cell Chlamydia abortus inactivated vaccine,the antibody level in the mice was increased with the increase of immunization dose,and the level of splenic lymphocyte proliferation and the content of IFN-γand IL-2 in 50μL and 100μL vaccine groups were much higher than those in 25μL vaccine group and PBS group.The immunized mice were challenged,the genome content of Chlamydia abortus in spleen,duodenum and uterus of mice inoculated with vaccine was much lower than that of mice without vaccination.The uterus of mice injected with PBS and 25μL of vaccine showed different degrees of necrosis and inflammation,while the uterus of the remaining two groups of mice had no abnormal changes.The results showed that a L929 suspension culture cell line was successfully acclimated in this experiment,and the inactivated Chlamydia abortus vaccine prepared from L929 suspension culture had strong immune effect.Inoculating 50μL and 100μL of the inactivated vaccine can have a protective effect on mice to a certain extent.The successful domestication of the L929 cell suspension culture strain provides a scientific basis for the large-scale preparation of vaccines.