白喉菌株主要抗原基因遗传稳定性及相应抗原蛋白结构确证的研究  

Genetic Stability of Major Antigen Genes and Confirmation of Corresponding Antigen Protein Structure of Diphtheria Strains

作　　者：顾琴[1] 姬秋彦[1] 梁疆莉[1] 高娜[1] 李婧妍 蔡路奎[1] 史荔[1] 孙明波[1] 马艳[1] GU Qin;JI Qiu-yan;LIANG Jiang-li;GAO Na;LI Jing-yan;CAI Lu-kui;SHI Li;SUN Ming-bo;MA Yan(Joint Lab.of Inst.of Med.Biol.,Chinese Acad.of Med.Sci.&Peking Union Med.Coll.,Kunming 650118)

机构地区：[1]中国医学科学院,北京协和医学院医学生物学研究所联合实验室,云南昆明650118

出　　处：《微生物学杂志》2021年第6期44-49,共6页Journal of Microbiology

基　　金：中国医学科学院医学与健康科技创新工程项目(2016-I2M-1-019)。

摘　　要：对白喉棒状杆菌(Corynebacterium diphtheria)主要保护性抗原基因序列的遗传稳定性进行研究,对相应的抗原蛋白结构进行确证,并进一步验证此前一代测序的结果,为疫苗的安全性和有效性提供依据。采用二代测序技术(next generation sequencing,NGS)对白喉棒状杆菌主代代次(P3)、工作代次(P7)、疫苗代次(P12)及疫苗代次后连续传3代的第3代样品V3代次(P15)的主要抗原基因序列进行测序并分析,并用Phyre 2软件预测出白喉毒素结构与文献发表的白喉毒素结构模型进行比对分析,采用蛋白免疫印迹(Westen blot,WB)试验对蛋白结构进行确证。结果显示,白喉棒状杆菌主要抗原基因序列最高突变率位点,主代代次为A_(580) C 0.46%、A_(868) C 0.46%,工作代次为A_(580) T 0.47%,疫苗代次为T_(507) G 0.64%,V3代次为位点T_(507) G 0.49%,其中主代代次、工作代次最高突变位点在B肽链的T区(α-螺旋区)内,不是主要抗原表位区;疫苗代次和V3代次最高突变位点在A肽链的C区(催化区)内,是主要抗原表位区域,但突变率较低,小于1%。36个能影响白喉毒素进入细胞和催化活性的碱基位点中,442碱基位点的各代次均比其他位点偏高(0.41%),其他位点均小于0.45%;蛋白结构模型预测与文献一致,均包含3个结构功能区;WB试验中,在58 kDa处均出现条带,大小与预期一致,说明此蛋白结构正确。传代过程中白喉主要碱基位点突变率较低,结构正确,说明白喉棒状杆菌菌种基因遗传稳定性好。Genetic stability of the major antigen gene sequence and confirmation of corresponding antigen protein structure of Corynebacterium diphtheriae strains were studied,the results of the previous generation sequencing were further verified to provide the basis for the safety and effectiveness of the vaccine.The main antigenic gene sequences of master generation of C.diphtheriae(P3),working generation(P7),passage for vaccine production(P12)and the third passage V3(P15)after the passage for vaccine of diphtheria strains were sequenced and analyzed by second-generation sequencing technology.The predicted structure of the diphtheria toxin was compared with the structure model published in literature by Phyre 2 software.The protein structure was confirmed by western blot.The highest mutation rate of the major antigen gene sequence of master passage were A_(580) C 0.46%,A_(868) C 0.46%,and working passage A_(580) T 0.47%,vaccine passage T_(507) G 0.64%,and the site of V3 passage T_(507) G 0.49%.Among them,the highest mutation sites of the master passage and working passage were T-region(α-helix region)of B-peptide chain instead of the major epitope region.While the highest mutation sites of vaccine passage and V3 passage were at C region(catalytic region)of a peptide chain,being the main epitopes,but the mutation rate was lower than 1%.Among the 36 base sites that can affect the entry of diphtheria toxin into cells and catalytic activity,the mutation rate of the 442 base sites of all passages were higher than all the other sites(0.41%),and the other sites were all less than 0.45%;The predicted protein structure model was consistent with that in the literature,contained 3 structural functional regions;for western blot,the band all appeared at 58 kDa consistent with the expected size,indicating that the protein structure was correct.Therefore,the mutation rate of the major base site of diphtheria was relatively low and the structure was correct,which proved as good genetic stability of diphtheria strain seed.

关 键 词：二代测序 白喉 突变率 蛋白结构 遗传稳定性 

分 类 号：Q939.93[生物学—微生物学]