森林脑炎病毒2BS细胞适应株全基因序列分析

Analysis of complete genome sequence of 2BS cell-adapted tick-borne encephalitis virus strain

目的分析“森张”株森林脑炎病毒(tick-borne encephalitis virus,TBEV)适应2BS细胞传代后的全基因序列。方法将“森张”株TBEV感染2BS细胞,经蚀斑克隆纯化,并连续传代培养。提取适应2BS细胞培养后的TBEV RNA,反转录为cDNA,以其为模板进行PCR扩增,扩增产物进行测序鉴定,并应用DNAMAN和MEGA软件对所获得序列进行分析。结果获得2BS细胞适应株TBEV全基因序列,共10782个碱基,编码3414个氨基酸;与鼠脑适应株TBEV全基因序列比对,2BS细胞适应株TBEV的5'和3'端非编码区(UTR)分别有1个碱基突变;结构蛋白区域有5个碱基突变,导致3个氨基酸变异;非结构蛋白区域有31个碱基突变,导致9个氨基酸变异。与主代鼠脑适应株核苷酸序列同源性为99.63%,氨基酸同源性为99.56%。结论“森张”株TBEV适应2BS细胞传代后,在分子水平方面证明相对于鼠脑适应株TBEV基因稳定,为2BS细胞森林脑炎疫苗的研发奠定了基础。

Objective To analyze the complete genome sequence of Senzhang strain of tick-borne encephalitis virus(TBEV)adapted to 2BS cells.Methods The 2BS cells were infected with Senzhang strain of TBEV,of which the plaque clones were purified and subcultured continuously.The RNA of TBEV adapted to 2BS cells were extracted and reversely transcribed to cDNA and used as a template for amplification by PCR.The PCR product was identified by sequencing,and the obtained sequences were analyzed by using DNAMAN and MEGA software.Results The obtained complete gene sequence of 2BS cells-adapted strain of TBEV consisted of 10782 bases encoding 3414 amino acids.Compared with the complete gene sequence of mouse brain-adapted TBEV strain,there were one base mutations at the 5'and 3'untranslated regions(UTRs)of 2BS cells-adapted strain respectively;there were 5 base mutations in structural protein region resulting in the variations of 3 amino acids and 31 base mutations in the non-structural protein region resulting in the variations of 9 amino acids.The homologies of nucleotide and amino acid sequences of 2BS cells-adapted strain were 99.63%and 99.56%respectively to those of primary mouse brain-adapted strain.Conclusion The genetic stability of Senzhang strain of TBEV was confirmed at molecular level after adaption to 2BS cells,which laid a foundation of development of 2BS cells-based TBEV vaccine.