Ⅱ型登革病毒D01090株在人胚肺二倍体细胞KMB17上适应株选育及初步鉴定

Adaptability of Dengue-ⅡVirus D01090 Strain in KMB17 Cells and Its Preliminary Identification

目的:选育能在人胚肺二倍体细胞KMB17上稳定增殖的Ⅱ型登革病毒适应株,为研发以人源性细胞为基质的登革疫苗候选株奠定基础。方法:将Ⅱ型登革病毒中国株D01090提取病毒基因组,通过RT-PCR法进行登革病毒型别鉴定后,在C6/36和Vero细胞上进行毒种扩增和滴度测定;将D01090毒株以4.0MOI接种KMB17细胞并反复传代至病毒完全适应在细胞内扩增,并连续传代10代,选育出良好的KMB17细胞适应株;病毒培养液经蔗糖梯度离心和超速离心后获得高浓度病毒液,接种KMB17细胞后通过透射电镜超薄切片检测细胞的病理变化;然后经过三轮蚀斑纯化筛选出纯化病毒株,免疫荧光法检测病毒纯化株的抗原性。结果:以Ⅱ型登革病毒中国株D01090基因组为模板,能扩增出511bp的登革病毒特异基因和119bp的Ⅱ型登革病毒型特异性基因。病毒经C6/36细胞扩增后滴度达4.5CCID50/ml,感染KMB17细胞至第三代可产生明显的细胞病变(cytopathic effect,CPE),连续传10代细胞病变速度逐渐增快,至第10代达到增殖高峰,病毒滴度达5.0CCID50/ml;将病毒感染6天后病变达+++的KMB17细胞进行超薄切片后经透射电镜观察细胞的病理变化,镜下可观察到内质网中新组装成的病毒颗粒,细胞周围产生很多分裂的小碎片,伴有游离出胞的病毒;三轮蚀斑纯化后筛选出纯化克隆,免疫荧光法检测病毒的抗原性呈阳性。结论选育出了能稳定传代且病毒扩增量高的Ⅱ型登革病毒KMB17细胞适应株,经蚀斑纯化后仍保持较好的抗原性。

Objective ： To select the adaptive strain of Dengue- Ⅱvirus D01090 strain in KMB17 cells, which lay the foundation of the exploration of dengue vaccine using human cells as host. Methods： After extraction of dengue- Ⅱ D01090 strain genome and identification of the serotype of dengue virus through RT-PCR, the virus was replicated an detected for the titer;The Dengue- Ⅱ D01090 strain was subcultured in KMB17 cells with 4.0 MOI till the virus completely adapted to multiply in cells, then continued subculturing in KMB17 cells for 10 passages. The adapted strain was screened out, Virus culture fluid was purified by sucrose gradient centrifugation and uhracentrifugation, KMB17 cells was uhrathinsected and observed the pathology under transmission electron microscope after infected with virus; Adapted strain was purified through plaque assay, and the antigenicity was detected by IFA. Results： After dengue-Ⅱ virus DO1090 strain RNA was extracted as templete, a typical 511 bp gene segment of dengue virus and a specific 119bp gene segment of dengue-Ⅱ virus were amplified by RT-PCR. After replication in C6/36 cells, the virus titer could reach 4. 5 CCIDso/ml, The CPE of KMB17 cells was appeared earlier after continuous subculture, their titer increased with the increasing passages and get the highest 5.0 CCIDs0/ml on passage 10. KMB17 cells was ultrathinsected and observed the pathology under transmissionelectron microscope after 6 days infected with virus and CPE get ＋ ＋ ＋, the new packaging virus particles were observed in the endoplasmic reticulum, many small fragment were generated around the cell, with the virus drifting out of the cell. Purified virus strain was screened through three cycles of plaque purification, while antigenicity of purified strain was positive detecting by IFA. Conclusion： Dengue-ll virus D01090 （China） adapted strain was screened out which could stably proliferated in KMB17 ceils and keep a high virulence, also maintained good antigenicity through plaque purification.