脊髓灰质炎Ⅲ型病毒Sabin株C抗原及D抗原生物学特性研究  

Biological characteristics of C and D antigens of the poliovirus typeⅢSabin strain

作　　者：刘硕 喻刚 王继麟 郝鹏亮 郭长福 陈晓琦 LIU Shuo;YU Gang;WANG Ji-lin;HAO Peng-liang;GUO Chang-fu;CHEN Xiao-qi(Institute of Biological Products of Wuhan City,Wuhan,Hubei 430207,China)

机构地区：[1]武汉生物制品研究所有限责任公司,湖北武汉430207 [2]国家联合疫苗工程技术研究中心,湖北武汉430207

出　　处：《中国病毒病杂志》2020年第3期169-174,共6页Chinese Journal of Viral Diseases

基　　金：国家“重大新药创制”科技重大专项(2018ZX09737003-005)。

摘　　要：目的对脊髓灰质炎Ⅲ型病毒Sabin株C抗原和D抗原在形态、感染特性、核酸含量和结构基因核苷酸序列、抗原不同结构蛋白质含量及2种抗原免疫原性等方面的差异进行比较观察,为脊髓灰质炎疫苗的生产评估、检定及进一步在蛋白质翻译后水平研究2种抗原的差异提供依据。方法采用二次氯化铯(CsCl)密度梯度离心的方法分离纯化脊髓灰质炎Ⅲ型病毒Sabin株C、D抗原,对分离的C、D抗原采用微量BCA(bicinchonininc acid)法检测蛋白质浓度,电镜观察病毒形态和纯度,聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)检测病毒不同结构蛋白质组成和含量,逆转录PCR(reverse transcription-PCR,RT-PCR)定量检测2种抗原单位质量携带核酸含量,并测序比较结构基因核苷酸序列差异;以中高剂量的C、D抗原分别免疫Wistar雌性大鼠,观察2种抗原体内诱生中和抗体的能力。结果脊髓灰质炎Ⅲ型病毒Sabin株单位质量D抗原的病毒滴度高于C抗原约4个对数值;单位质量D抗原的核酸含量高于C抗原近64倍,D抗原和C抗原结构基因核苷酸序列无差异;单位质量D抗原VP1蛋白质含量高于C抗原1.67倍;一次免疫后,D抗原诱导的中和抗体是C抗原的10~50倍。结论脊髓灰质炎Ⅲ型病毒Sabin株D抗原比C抗原具有较强的免疫原性,上述研究结果为脊髓灰质炎疫苗C、D抗原的检测、鉴定及进一步的疫苗效力研究和提高疫苗效力提供了实验依据。Objective To compare the C antigen and D antigen of the poliovirus typeⅢSabin strain in morphology,infection characteristics,nucleic acid sequence,different structural proteins and the immunogenicity.Methods The C and D antigens of poliovirus typeⅢSabin strain were isolated and purified by twice by cesium chloride density gradient centrifugation.The protein concentration of the separated C antigen and D antigen was detected by micro BCA assay.The morphology and purity of the virus were observed by electron microscope.The composition and content of different structural proteins of the virus were detected by polyacrylamide gel electrophoresis.The content of nucleic acid carried by unit mass of the two antigens was quantitatively detected by reverse transcription-PCR,and the structure gene was sequenced and compared.Female Wistar rats were immunized with medium and high doses of C and D antigens respectively,and the ability of the two antigens to induce neutralizing antibodies was observed.Results The virus titer of unit mass D antigen of poliovirus typeⅢSabin strain was about 4logarithms higher than that of C antigen,the nucleic acid content of unit mass D antigen was nearly 64times higher than that of C antigen.However,there was no difference in nucleotide sequence of structural gene between D antigen and C antigen.The VP1 protein content of unit mass D antigen was 1.67times higher than that of C antigen,and the neutralizing antibody induced by D antigen was 10-50times of C antigen after primary immunization.Conclusions The D antigen of poliovirus typeⅢSabin strain showed much stronger immunogenicity than C antigen.

关 键 词：脊髓灰质炎病毒 脊髓灰质炎Ⅲ型病毒Sabin株 C抗原 D抗原 生物学特性 

分 类 号：R373.22[医药卫生—病原生物学]