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目的鉴别日本脑炎疫苗株ML-17的两个大斑变异株(ML-17L2和ML-17L4)的存在,并通过全基因组序列测定分析引起斑形和毒力改变的基因突变。方法首先对疫苗株ML-17在传代过程中出现的多斑ML-17sh的两个大斑变异株进行纯化和鉴定,再对两个大斑变异株进行全基因组序列测定和全编码氨基酸序列推导,并与小斑疫苗株ML-17及其大斑亲代野毒株JaOH0566的全核苷酸序列和全编码氨基酸序列进行比较,分析引起斑形改变的基因突变,并应用小白鼠试验检查ML-17变异前后的神经侵袭力和毒力变化。结果全基因组序列分析显示两个大斑变异株的核苷酸总数均为10 977个,与ML-17和JaOH0566相比较,分别有33和34个核苷酸变异散在分布于ML-17L2和ML-17L4的全基因组中,其中又分别有12和13个核苷酸导致了氨基酸的改变。在ML-17L2和ML-17L4的衣壳蛋白区没有氨基酸变异,但在包膜蛋白区各有一个氨基酸变异;在5’端非编码区没有核苷酸变异,但在3’端非编码区有3~4个核苷酸变异,且各增加了一个额外的10699核苷酸。ML-17和ML-17sh的小鼠LD50分别为大于1×106FFU和481FFU,变异后毒力明显增强。结论发生在两个大斑变异株的第5非结构蛋白区和其它区的氨基酸返祖变异(变回与JaOH0566相同)和新变异,可能引起了斑形的改变;而全部5个氨基酸返祖变异和2~3个氨基酸新变异可能与其毒力改变有关。本次研究为ML-17L2和ML-17L4的斑形和毒力改变提供了分子遗传学基础。
Objective To identify the presence of two large spot mutants (ML-17L2 and ML-17L4) of the Japanese encephalitis vaccine strain ML-17 and to analyze gene mutations that cause altered plaque and virulence by genome-wide sequence analysis. Methods Two major mutant strains of ML-17sh were identified during the passage of the ML-17 vaccine strain. Whole genome sequence analysis and full-coding amino acid sequence deduction , And compared with the full nucleotide sequence and full coding amino acid sequence of the spot blot vaccine strain ML-17 and its large wild-type parental strain JaOH0566 to analyze the gene mutation causing the speckle change. The mouse experiment was used to examine the effect of ML- Changes of neural invasion and virulence before and after mutation. Results Genome-wide sequence analysis showed that the total number of nucleotides in two major mutant strains was 10 977. Compared with ML-17 and JaOH0566, 33 and 34 nucleotide variations were scattered in ML-17L2 and ML The complete genome of -17L4, of which 12 and 13 nucleotides, respectively, led to amino acid changes. There is no amino acid variation in the capsid protein region of ML-17L2 and ML-17L4, but one amino acid variation in the envelope protein region; no nucleotide variation in the 5 ’non-coding region but no nucleotide variation in the 3’ non-coding region There are 3 to 4 nucleotide variations, each with an additional 10699 nucleotides added. The mouse LD50 of ML-17 and ML-17sh were greater than 1 × 106 FFU and 481 FFU, respectively, and the virulence was significantly enhanced after mutation. Conclusions The amino acid anaphoric variation (the same back to JaOH0566) and the new mutation that occurred in the fifth non-structural protein region and the other regions of the two large mutant strains may cause the change of the plaque. However, all the five amino acids returned to the ancestral Mutations and 2 to 3 new amino acid variations may be related to their virulence. This study provides a molecular genetic basis for the speckle and virulence changes of ML-17L2 and ML-17L4.