摘要
制备可表达斯氏艾美耳球虫天冬氨酸蛋白酶(aspartic protease,ASP)的重组腺相关病毒(rAAV9-ZsGreen1-EsASP),并探究其体外活性。通过PCR扩增EsASP基因,利用同源重组法将重组腺相关病毒载体pAAV-IRES-ZsGreen1与EsASP基因相结合,构建pAAV-ZsGreen1-EsASP表达质粒;采用脂质体转染法将pAAV-ZsGreen1-EsASP表达质粒、pHelper和pAAV-RC9质粒共转染至HEK-293T细胞中,包装并生产出能够表达EsASP蛋白的重组腺相关病毒rAAV9-ZsGreen1-EsASP;利用氯仿处理-PEG/NaCl沉淀-氯仿抽提法纯化rAAV9-ZsGreen1-EsASP,通过银染法鉴定病毒纯度,TEM观察病毒形态,qPCR法检测病毒滴度;进一步将纯化的重组病毒感染HEK-293T细胞,通过观察绿色荧光蛋白ZsGreen1和Western blot法检测EsASP的表达情况。结果显示双酶切及DNA测序证明EsASP基因已成功构建到pAAV-IRES-ZsGreen1表达质粒中,HEK-293T细胞表达绿色荧光蛋白提示共转染成功,细胞蛋白制样Western blot结果显示EsASP蛋白成功表达;纯化的重组病毒衣壳蛋白呈现3条清晰条带(VP1-3);rAAV9-ZsGreen1-EsASP大小均一,20 nm左右且滴度高于1.0×10^(12) vg/mL;重组病毒感染HEK-293T细胞后观察到绿色荧光蛋白表达,Western blot检测到EsASP的表达。结果表明成功获得了较高纯度且具有体外感染活性的rAAV9-ZsGreen1-EsASP,为研制新型斯氏艾美耳球虫病疫苗提供物质基础。
To prepare a recombinant adeno-associated virus(rAAV9-ZsGreen1-EsASP)capable of expressing the aspartic protease protein of Eimeria stiedai(E.stiedai),and explore its in vitro ac-tivity.The EsASP gene was amplified by PCR,and recombinant adeno-associated viral vector pAAV-IRES-ZsGreen1 was combined with the EsASP gene using homologous recombination to construct the pAAV-ZsGreen1-EsASP expression plasmid;pAAV-ZsGreen1-EsASP expression plasmid,pHelper,and pAAV-RC9 plasmids were cotransfected into HEK-293T cells by liposomal transfection to package and produce recombinant adeno-associated virus rAAV9-ZsGreen1-EsASP capable of expressing EsASP protein.rAAV9-ZsGreen1-EsASP was purified using chloroform treatment-PEG/NaCl precipitation-chloroform extraction method,the purity of the virus was iden-tified by silver staining,the virus morphology was observed by TEM,and virus titer was detected by qRT-PCR;the purified recombinant virus was further infected into HEK-293T cells,and EsASP expression was detected by observing green fluorescent protein ZsGreen1 and Western blot method.The results indicated that double enzyme digestion and DNA sequencing confirmed that the EsASP gene had been successfully constructed into the pAAV-IRES-ZsGreen1 expression plas-mid.The expression of green fluorescent protein in HEK-293T cells suggested that co-transfection was successful.Western blot results of cell protein preparation showed that EsASP protein was successfully expressed;The purified recombinant viral capsid proteins show three distinct bands(VP1-3).The purified rAAV9-ZsGreen1-EsASP was uniform in size,around 20 nm,and had a titer higher than 1.0×10^(12) vg/mL;Green fluorescent protein expression was observed after infection of HEK-293T cells with the recombinant virus,and EsASP expression was detected by Western blot.The results suggest that rAAV9-ZsGreen1-EsASP with in vitro infectious activity was suc-cessfully obtained,providing a material basis for the development of a novel vaccine against Eimer-ia stiedai.
作者
李亚欢
李超凡
陈梦阁
李新
王晓岑
张旭
宫鹏涛
张楠
李建华
LI Yahuan;LI Chaofan;CHEN Mengge;LI Xin;WANG Xiaocen;ZHANG Xu;GONG Pengtao;ZHANG Nan;LI Jianhua(State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases/Key Laboratory for Zoonosis Research of the Ministry of Education,College of Veterinary Medicine,Jilin University,Changchun 130062,China)
出处
《中国兽医学报》
北大核心
2025年第11期2387-2393,共7页
Chinese Journal of Veterinary Science
基金
吉林省科技发展计划项目(20220202054NC)。
