精氨酸-甘氨酸-天冬氨酸序列修饰对流感病毒溶瘤能力的影响  

Effect of RGD modification on oncolytic potential of influenza virus

作　　者：杨英 龙琼[1] 杨颖 李多[1,3] 杨姝[1] 马雁冰[1] YANG Ying;LONG Qiong;YANG Ying;LI Duo;YANG Shu;MA Yan-bing(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming,650000,Yunnan Province,China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650000 [2]昆明医科大学,云南昆明650000 [3]云南省疾病预防控制中心,云南昆明650000

出　　处：《中国生物制品学杂志》2022年第5期551-556,561,共7页Chinese Journal of Biologicals

基　　金：云南省创新疫苗技术与产业转化研发平台(106001016-14)。

摘　　要：目的探讨精氨酸-甘氨酸-天冬氨酸(RGD)序列修饰对流感病毒溶瘤能力的影响。方法采用流感病毒反向遗传操作体系,将含PB2、PB1、PA、HA、NP、NA、M和NS的质粒PHW200(八质粒)及经RGD修饰的八质粒分别共转染293T细胞,拯救出野生型和重组RGD流感病毒。采用胶体金法试纸条进行鉴定,血凝试验检测病毒效价,PCR法检测病毒传代稳定性,噬斑试验检测病毒滴度。用拯救的两种流感病毒感染不同细胞(B16-F10、A549、TC-1、CT26、4T1和Vero细胞),检测细胞活力和细胞中乳酸脱氢酶(lactate dehydrogenase,LDH)释放率。用B16-F10细胞注射小鼠,建立小鼠黑色素瘤模型后,分别注射两种流感病毒及PBS,隔1 d给药1次,共5次,肿瘤细胞接种后19 d,测量肿瘤大小;肿瘤细胞接种后25 d,统计小鼠存活数量。结果野生型和重组RGD流感病毒经胶体金法试纸条鉴定均为阳性,血凝效价分别为1∶2^(8)和1∶2^(9),传代稳定性良好,病毒滴度分别为3×10^(7)和1.8×10^(7) PFU/mL。两种流感病毒感染后B16-F10和A549细胞活力均大幅降低,其他细胞活力变化较小;与相应野生型流感病毒组比较,重组RGD流感病毒组B16-F10细胞中LDH释放率明显升高(P<0.05),A549细胞略升高,但差异无统计学意义(P>0.05),其他细胞均较低。与PBS组比较,两种流感病毒组小鼠肿瘤大小明显减小(P<0.001),小鼠存活率明显升高(P<0.05)。结论RGD修饰的流感病毒对B16-F10具有较好的亲嗜性,且溶瘤能力增强。本研究为溶瘤流感病毒在肿瘤治疗中的应用奠定了基础。Objective To investigate the effect of RGD modification on oncolytic potential of influenza virus.Methods The 293T cells were co-transfected with plasmid PHW200(octave plasmid)containing PB2,PB1,PA,HA,NP,NA,M and NS genes and RGD-modified octave plasmid by reverse genetic manipulation to rescue wild and recombinant RGD influenza viruses respectively.The rescued viruses were identified with colloidal gold test strips,determined for titer by hemagglutination(HA)test and plaque assay,and for stability during subculture by PCR.Various tumor cells(B16-F10,A549,TC-1,CT26,4T1 and Vero cells)were infected with the rescued viruses,and determined for viability and lactate dehydrogenase(LDH)release rate.Mice were injected with B16-F10 cells to establish the model of melanoma.The model mice were treated with the rescued viruses and PBS respectively,every other day for 5 times.The tumors were measured19 d,while the surviving mice were counted 25 d,after the inoculation with B16-F10 cells.Results Both the wild and recombinant RGD influenza viruses were positive in colloidal gold strip test and stable in subculture,of which the HA titers were 1∶2^(8)and 1∶2^(9)and 3×10^(7) and 1.8×10^(7)PFU/mL in plaque assay,respectively.The viabilities of B16-F10and A549 cells decreased significantly after infection with the viruses,while those of other cells showed no significant changes.Compared with those infected with wild virus,the LDH release rate in the B16-F10 cells infected with recombinant RGD influenza virus increased significantly(P<0.05),while that in the A549 cells increased insignificantly(P>0.05),and those in other cells decreased.Compared with those treated with PBS,the tumors of mice treated with both the influenza viruses decreased significantly in size(P<0.001),while the survival rate increased significantly(P<0.05).Conclusion The RGD-modified influenza virus showed high affinity to B16-F10 cells,while the oncolytic potential was enhanced,which laid a foundation of study on the application of oncolytic influenza viru

关 键 词：流感病毒 溶瘤能力 精氨酸-甘氨酸-天冬氨酸序列 黑色素瘤 

分 类 号：R392-33[医药卫生—免疫学]