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pDVWS501为含有登革2型病毒全长cDNA的质粒,可利用感染性转录体技术恢复为有活力的病毒MON501.将MON501注射乳鼠脑内可引发脑炎症状,其E蛋白的62,203位分别为Glu,Asn.采用OL-PCR方法把pDVWS501 E62位氨基酸突变为Lys,得到质粒TB62;E203位氨基酸突变为Asp,得到质粒TB203.将pDVWS501,TB62和TB203酶切后体外转录得到全长登革2型病毒转录体,应用电穿孔技术转染BHK-21细胞,7天后收毒.RT-PCR证实有登革2型病毒存在,接种C6/36细胞,3～5 d可使其产生典型病变.测定突变区域的序列,结果表明得到了恢复病毒MON501和E62,E203位点突变的重组病毒HFT62,HFT203.3株病毒均在其基因组5’端加“G”,3’端则与登革2型病毒野生株相同.分别将3株病毒稀释至105～102TCID50,经脑内途径注射1日龄乳鼠,发现与MON501相比,HFT62,HFT203在同一稀释度发病乳鼠的个数减少,发病时间延长且差异显著,表明E62,E203可能是登革2型病毒乳鼠神经毒力相关位点.
pDVWS501 is a plasmid containing the full-length cDNA of dengue 2 virus, which can be recovered to the viable virus MON501 by using infectious transcript technology.The encephalitogenic condition can be induced in the brain of neonatal MON501-injected mice, and the 62203 E protein is Glu and Asn, the amino acid at position E62 of pDVWS501 was mutated to Lys by OL-PCR to obtain the plasmid TB62, and the amino acid at position E203 was mutated to Asp to obtain the plasmid TB203.The pDVWS501, TB62 and TB203 were digested to obtain the full length dengue 2 BHK-21 cells were transfected into BHK-21 cells by electroporation, and the cells were harvested after 7 days.RT-PCR confirmed the presence of dengue 2 virus, and C6 / 36 cells were inoculated 3 to 5 days to induce typical pathological changes. The results showed that the recombinant viruses HFT62 and HFT203.3 with the mutation sites of MON501 and E62 and E203 were obtained, all of which had “G” at the 5 ’end of their genome. 3 strains of virus were diluted to 105 ~ 102TCID50, respectively, by intracerebral injection of 1-day-old suckling mice and found that compared with the MON501, HFT62, HFT203 in the same dilution the incidence of reduced number of suckling mice, the incidence The results showed that E62 and E203 may be dengue type 2 virus Mouse neurovirulence related sites.