论文部分内容阅读
目的研究现行麻疹病毒流行株连续传代后,对非洲绿猴肾细胞(Vero细胞)感染能力的改变,并从基因层面分析引起麻疹病毒与细胞受体结合位点改变的可能原因。方法将现行麻疹病毒流行株Ningbo(宁波)05-2在Vero细胞上连续传代,观察并记录致细胞病变效应(Cytopathic Effect,CPE);采用荧光定量逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)对原始株Ningbo05-2与第18代传代株Ningbo05-2/P18进行病毒鉴定后,通过血细胞凝集(Hemagglutination,HA)试验测定Ningbo05-2/P18的HA活性,并利用RT-PCR扩增其血凝素(Hemagglutinin,H)和核蛋白(Nucleoprotein,N)基因全序列。结果现行麻疹病毒流行株Ningbo05-2连续传代17次(P18),至第13代开始观察到在Vero细胞上融合样CPE;荧光定量RT-PCR鉴定传代株为麻疹病毒,但仍无对猴红细胞的凝集活性。与Ningbo05-2比较,传代株Ningbo05-2/P18在H蛋白上存在三个位点(312aa、314aa、546aa)的氨基酸突变,导致二级结构上311位的β折叠,以及312～316位的β转角转变为α螺旋。在H基因进化树上,传代株与原始株位于同一分支,仍属于H1b基因亚型。而在N蛋白核苷酸和氨基酸水平,传代株与原始株序列完全一致。结论现行麻疹病毒流行株在Vero细胞上连续传代后,H蛋白上个别氨基酸的突变可能导致与细胞结合位点的改变,从而影响病毒对细胞的感染能力。
Objective To study the changes of infection ability of Vero cells in the continuous transmission of the current measles virus in China and to analyze the possible causes of the changes in the binding sites of measles virus and cell receptor from the gene level. Methods The current measles virus strain Ningbo (Ningbo) 05-2 was serially passaged on Vero cells, and the cytopathic effect (CPE) was observed and recorded. Reverse transcription-polymerase chain reaction (Reverse Transcription-Polymerase After the viruses of Ningbo05-2 and 18th generation, Ningbo05-2 / P18 were identified by PCR and RT-PCR, the HA activity of Ningbo05-2 / P18 was determined by Hemagglutination (HA) The full-length Hemagglutinin (H) and Nucleoprotein (N) genes were amplified by RT-PCR. Results The current measles virus epidemic strain Ningbo05-2 was continuously passaged 17 times (P18), and the CPE fusion was observed on Vero cells starting from the 13th passage. The passaged strain was identified as measles virus by fluorescence quantitative RT-PCR, Agglutination activity. Compared with Ningbo05-2, the passage strain Ningbo05-2 / P18 has three amino acid mutations (312aa, 314aa, 546aa) on the H protein, resulting in a 311-fold β-sheet on the secondary structure and a 312- β turn into α-helix. On the H gene phylogenetic tree, the passage strains are on the same branch as the original strain and still belong to the H1b gene subtype. In the N protein nucleotide and amino acid levels, the passage of strains and the original plant sequence exactly the same. CONCLUSIONS: After the continuous passage of the current measles virus strain on Vero cells, the mutation of individual amino acids in H protein may lead to the change of the binding sites with the cells, thereby affecting the ability of the virus to infect cells.