论文部分内容阅读
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 compare the immunogenicity and cross-protection between four strains of Coxsackie virus groups A type 16 (CoxA16). Methods The VP1 gene of CoxA16 G20, KM / M08, MY08 and KM208 was extracted and sequenced by PCR. Thirteen CoxA16 sequences were constructed based on VP1 gene sequence using Bootstrap Test of phylogeny → Neighbor-joining in MEGA4.0 software CoxA16 phylogenetic tree was constructed and genotyped. The DNAMAN software was used to analyze the homology of the VP1 nucleotide sequences of the four strains and their amino acid sequence differences. BALB / c mice were immunized with 4 CoxA16 purified and inactivated respectively, and blood was collected on the 14th and 28th day after the primary immunization and the second immunization. Serum antibodies were detected by Western blot in mice immunized with G20 Specificity; serum neutralizing antibody titers were tested by micro-neutralization test and cross-neutralization test was carried out. Results The genotypes of G20, MY08 and KM208 belonged to B2b genotype, while the KM / M08 genotype belonged to genotype B2a. The nucleotide sequence homology of VP1 gene among the 4 strains was 91.8% -99.0% There were 3 different amino acid sites in VP1 at position 102 (N → D), position 241 (E → K) and position 248 (T → A). The antiserum of G20 strain could specifically recognize the VP1 and VP2 proteins of CoxA16 and had good immunogenicity. The neutralizing antibody titers of all the experimental groups on the 28th day after secondary immunization were higher than those of the other test groups, in which the MY08 group’s neutralization The antibody titer was significantly lower than the other three groups (P <0.01). The cross-protection ability of G20, KM / M08 and KM208 strains was better than that of MY08 strains against G20, KM / M08 and KM208 Strain cross protection, but the difference was not statistically significant (P> 0.05). CONCLUSION: All four CoxA16 inactivated induce neutralizing antibodies and cross-protection in vivo. Among them, G20, KM / M08 and KM208 strains showed better immunogenicity and cross-strain after inactivation Protection ability.