基于测序技术探究卡介菌生物制剂核酸物质基础

Exploration of Nucleic Acid Material Basis of BCG Biological Preparation Based on Sequencing Technology

目的明确免疫调节剂卡介菌多糖核酸注射液中起免疫增强作用的核酸物质基础。方法(1)核酸提取:试剂盒法分离提取卡介菌及卡介菌多糖核酸注射液的核酸部分;(2)测序:采用第三代测序技术和第二代宏测序技术分别对卡介菌基因组和注射液核酸片段序列进行测序,获得核酸序列;(3)数据分析:统计分析核酸序列中免疫功能的含胞嘧啶-磷酸-鸟嘌呤(cytosine-phosphoric acid-guanine,CpG)的典型CpG基序数量及分布情况;(4)免疫活性分析:采用酶联免疫法分析提取核酸刺激细胞释放免疫因子TNF-α效果。结果注射液核酸片段中平均每拷贝含有4000个典型CpG基序,占总基因组的0.5%;富含CpG基序核酸片段刺激细胞释放TNF-α浓度显著强于无CpG基序核酸片段(P<0.01),显示了较强的免疫调节作用。结论卡介菌多糖核酸注射液核酸物质中富含CpG基序而表现较强的免疫活性,为进一步开发新型免疫调节制剂提供理论基础。

Objective To clarify the nucleic acid material basis of immunomodulator BCG polysaccharide nucleic acid injection for immune enhancement.Methods(1)Nucleic acid extraction:the nucleic acid part of BCG and BCG polysaccharide nucleic acid injection were isolated and extractedby kit method;(2)Sequencing:the third-generation sequencing technology and second-generation macro-sequencing technology were used respectively to sequence the BCG genome and the injection nucleic acid fragment sequences to obtain the nucleic acid sequence;(3)Data analysis:statistical analysis was performed for the typical cytosine-phosphoric acid-guanine(CpG)motif quantity and distribution of CpG containing immune function in nucleic acid sequence;(4)Immunoactivity analysis:Enzyme linked immunoassay was used to analyze the effect of extracted nucleic acid to stimulate cells to release immune factor TNF-α.Results There were about 4000 typical CpG motifs in each copy on average in injection nucleic acid fragments,accounting for 0.5%of the total gene group.Nucleic acid fragments rich in CpG motifs stimulate cells to release TNF-αat a concentration that is significantly stronger than nucleic acid fragments without CpG motifs,showing a stronger immunomodulatory effect.Conclusion The nucleic acid fractions of BCG polysaccharide nucleic acid injection are rich in CpG motif and show strong immune activity,which provides theoretical basis for further development of new immunomodulatory agents.