摘要
构建1株能够表达凋亡素的重组减毒单增李斯特菌(Listeria monocytogenes,LM),并探究其对B16F10小鼠黑色素瘤细胞增殖活力的影响。利用同源重组技术将凋亡素编码序列定点整合至单增李斯特菌hly基因信号肽下游,用RT-PCR及Western-blot方法验证重组菌株LMΔhly::apoptin中凋亡素的转录与表达,最后测定重组菌株的生物学特性,并通过CCK-8及荧光显微镜探究了重组菌株对B16F10细胞增殖活力及凋亡水平的影响。RT-PCR及Western-blot结果表明,该重组菌株能够转录并分泌表达具有免疫反应性的凋亡素。重组菌株对B16F10细胞的侵袭率为5.11%±0.56%,显著低于野生株对B16F10细胞的侵袭率(19.17%±4.83%)(P<0.05),但具有较好的胞内增殖能力,且在B16F10细胞内能检测到凋亡素的表达。CCK-8结果表明重组菌株能够显著抑制B16F10细胞的增殖,经荧光染色观察到重组菌株可诱导B16F10肿瘤细胞凋亡。本研究成功构建了表达凋亡素的重组减毒单增李斯特菌,其能够侵袭B16F10细胞并在细胞内增殖,且能在B16F10细胞中有效表达凋亡素。该重组菌株通过促进B16F10细胞凋亡而抑制B16F10细胞的增殖。以上研究为开发新型单增李斯特菌活载体肿瘤疫苗奠定了基础。
This study aimed to construct recombinant attenuated Listeria monocytogenes which can express apoptin protein and explore its effect on the viability of B16F10 mouse melanoma cells.The apoptin sequence was integrated into the frame with the L.monocytogenes hly promoter-signal sequence by homologous recombination technology,and introduced into the chromosome of L.monocytogenes.Recom-binant strain LMΔhly::apoptin expressing the apoptin-LLO fusion protein was obtained.The biological characteristics of the recombinant strain and its effect on the proliferation and apoptosis of B16F10 cells were investigated.RT-PCR and Western-blot results showed that the recombinant strain LMΔhly::apoptin could transcribe and secrete the apoptin protein with immunological activity.The invasion rate of the recombinant strain to B16F10 cells was 5.11%±0.56%,which was significantly lower than that of the wild strain to B16F10 cells(19.17%±4.83%)(P<0.05).However,the recombinant strain had good intracellular proliferation ability,and the expression of apoptin protein could be detected in B16F10 cells.CCK-8 results showed that the recombinant strain could significantly inhibit the proliferation of B16F10 cells,and the recombinant strain could induce the apoptosis of B16F10 cells by fluorescence staining.In conclusion,we successfully constructed a recombinant attenuated L.monocytogenes strain expressing apoptin protein,which can invade B16F10 cells and proliferate in B16F10 cells,and can ef-fectively present the expression of apoptin protein in B16F10 cells.Furthermore,the recombinant strain inhibits the proliferation of B16F10 cells by promoting cell apoptosis.The above research pro-vides a theoretical basis for the development of new tumor vaccine based on L.monocytogenes vector.
作者
张昊恺
孙奇娟
许凤茹
张春杰
余祖华
廖成水
李静
丁轲
王少辉
贾艳艳
ZHANG Hao-kai;SUN Qi-juan;XU Feng-ru;ZHANG Chun-jie;YU Zu-hua;LIAO Cheng-shui;LI Jing;DING Ke;WANG Shao-hui;JIA Yan-yan(Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control,Luoyang 471003,China;Laboratory of Functional Microbiology and Animal Health,Henan University of Science and Technology,Luoyang 471003,China;Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2023年第7期911-918,共8页
Chinese Veterinary Science
基金
国家自然科学基金项目(31702219)
河南科技大学青年骨干教师培养计划项目(13450011)。
关键词
凋亡素
单增李斯特菌
同源重组
凋亡
抗肿瘤
apoptin protein
Listeria monocytogenes
homologous recombination
apoptosis
antitumor
