摘要
目的探讨重组Ⅱ型单纯疱疹病毒(OH2)基因组修饰的稳定性。方法型别鉴定实验根据Ⅰ型(HSV-1)及Ⅱ型(HSV-2)单纯疱疹病毒g D糖蛋白基因的保守序列设计引物进行PCR扩增,HSV-1 PCR片段上有一个Msp I酶切位点,经酶切及电泳实验鉴别OH2病毒的型别;基因修饰实验根据被敲除或插入的序列位点上下游病毒核酸序列设计引物进行PCR扩增及电泳,并用电泳图分析OH2病毒基因组中是否剔除ICP34.5基因、ICP47基因及插入hGM-CSF基因;ELISA检测OH2上清中hGM-CSF含量验证插入的hGM-CSF基因能否正常表达。结果型别鉴定实验中HSV-1经酶切实验出现130bp和70bp两条条带,而HSV-2和OH2病毒PCR条带和酶切条带均只有一条,且为200bp。基因修饰实验中OH2病毒在四对引物下扩增,能扩增出对应大小的条带。ELISA实验中hGM-CSF浓度在31.25~500pg/m L范围内线性关系良好,且OH2病毒中hGM-CSF表达量均大于2ng/mL。结论 OH2病毒中被修饰的基因稳定地存在,同时插入的hGM-CSF基因能够正常表达。
Objective To evaluate the genome stability of a recombinant oncolytic HSV-2 （ 0H2） modi-fied with deletions for neural virulence gene ICP34.5 and immune suppressor gene ICP4 7 ,and insertion of the expression cassette for granulocyte macrophage colony stimulating factor （ hGM-CSF） at the ICP34. 5 site. Methods The HSV-1 and HSV-2 are distinguished by restriction fragments after PCR amplification. This experi-ment is designed based on gD glycoprotein conserved sequence in which HSV-1 has a MspI sequence. The 0H2 modifications can be identified by PCR/agrose gel analysis with the primers designed from the sequences for de-letion, insertion and down/up-stream flanking regions. hGM-CSF expression in OH2 culture supernatant can be validated by ELISA. Results The HSV1 has two fragments in 130bp and 70bp by enzyme digestion in identifica-tion experiment, while HSV-2 and OH2 have only one fragment in 200bp. OH2 virus can amplify the corre-sponding size of the strips under the four pairs of primers amplification in genetic modification experiments. The liner of hGM-CSF in range 31.25 500pg/mL is good,and the expression of hGM-CSF is more than 2ng/mL in OH2 virus. Conclusion The modified genes in OH2 virus genome are stable,and the hGM-CSF expression can be detected.
出处
《湖北科技学院学报（医学版）》
2017年第2期97-99,107,185,共5页
Journal of Hubei University of Science and Technology(Medical Sciences)
基金
湖北省自然科学基金面上项目(2014CFB186)
