柯萨奇病毒A组16型免疫原性及毒株间交叉保护能力的比较分析  

Immunogenicity and cross-reactivity of four Coxsackie virus group A type 16 strains

作　　者：谢振锋[1] 普晶[1] 黄泓泰 刘正玲[1] 董承红[1] 刘龙丁[1] 王丽春[1] 

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病毒免疫室云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2013年第10期1366-1370,1375,共6页Chinese Journal of Biologicals

基　　金：国家"863"计划项目(2012AA02A404);"十二五"重大新药创制国家科技重大专项(2012ZX09101319)

摘　　要：目的对柯萨奇病毒A组16型(Coxsackie virus groups A type 16,CoxA16)4个毒株进行免疫原性及毒株间交叉保护能力的比较分析。方法提取CoxA16 G20、KM/M08、MY08和KM208病毒RNA,PCR扩增VP1基因,并进行测序;利用MEGA4.0软件中Bootstrap Test of phylogeny→Neighbor-joining方法对13株CoxA16基于VP1基因全序列构建CoxA16种系进化树并进行基因分型,使用DNAMAN软件对4个毒株VP1核苷酸序列的同源性及其蛋白质氨基酸序列差异位点进行分析。分别用纯化灭活后的4株CoxA16免疫BALB/c小鼠,于初次免疫和二次免疫后的第14、28天采血,分离血清,Western blot法检测G20毒株免疫的小鼠血清中抗体的的特异性;微量中和试验法检测血清中和抗体效价并进行交叉中和试验。结果 G20、MY08和KM208毒株属于B2b基因型,而KM/M08毒株属于B2a基因型;4个毒株间VP1基因核苷酸序列的同源性为91.8%～99.0%;MY08与其他3个毒株相比,在VP1的第102位(N→D)、241位(E→K)和248位(T→A)上存在3个差异氨基酸位点。G20毒株的抗血清可特异识别CoxA16的VP1和VP2蛋白,具有良好的免疫原性;各实验组二次免疫后第28天中和抗体效价均高于其他检测时期,其中MY08组中和抗体效价明显低于其他3组,差异有统计学意义(P﹤0.01);G20、KM/M08、和KM208毒株间的交叉保护能力优于MY08毒株对G20、KM/M08和KM208毒株的交叉保护能力,但差异无统计学意义(P>0.05)。结论 4株CoxA16灭活后均能诱导机体产生相应的中和抗体和交叉保护能力,其中G20、KM/M08和KM208毒株在灭活后,显示了更好的免疫原性和毒株间交叉保护能力。Objective To analyze the immunogenicity and cross-reactivity of four strains of Coxsackie virus groups A type 16 （CoxA16）. Methods RNAs were extracted from CoxA16 G20, KM/M08, MY08 and KM208 strains, with which VP1 gene was amplified by PCR and sequenced. The phylogenetic tree of 13 strains of CoxA16 was constructed based on the Vpl sequence by Bootstrap Test of phylogeny→Neighbor-joining method in MEGA4. 0 software and subjected to genotyping. The homologies of nucleotide and amino acid sequences as well as amino acid diversity sites of VP1 of the 4 strains were analyzed by DNAMAN software. BALB / e mice were immunized with the four CoxAl6 strains after purification and inactivation, of which the serum samples were collected on days 14 and 28 after the first and the second immunization separately. The specificity of antibody in sera of mice immunized with G20 strain was analyzed by Western blot. The serum neutralizing antibody titer was determined by micro-neutralization assay, based on which cross neutralization test was performed. Results G20, KM208 and MY08 strains were clustered to genotype B2b while KM/M08to B2a. The homology of nucleotide sequences of VP1 gene of the four strains was 91. 8% N 99. 0%. There were three amino acid diversity sites, i.e. N→D at site 102, E→K at site 241 and T→A at site 248, were observed in VP1 gene of MY08 strain as compared with those of the other three strains. The antisera of mice immunized with G20 strain recognized the VP1 and VP2 of CoxA16, indicating high immunogenicity. The neutralizing antibody titers of mice in various test groups on day 28 after the second immunization were higher than those at other time points. However, the neutralizing antibody titer in MY08 group was significantly lower than those in other three groups （P 〈 0. 01 ）. The cross- reactivity among strains G20, KM208 and KM/M08 strains were insignificantly higher than that between MY08 and the three strains （P 〉 0. 05）. Conclusion The four strains of Coxl6 after inactivation i

关 键 词：柯萨奇病毒 免疫原性 交叉保护 序列分析 

分 类 号：R373.23[医药卫生—病原生物学]