猪囊尾蚴TSOL18核酸疫苗表达载体构建、免疫原性及安全性研究
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摘要
囊虫病(Cysticercosis)是由猪带绦虫(Taenia solium)的幼虫猪囊尾蚴寄生于人或猪等中间宿主而引起的人畜共患寄生虫病,在中国大部分地区广泛流行,严重威胁着当地居民的身体健康,是一个重要的公共卫生问题。不仅如此,该病也是制约养猪业发展的重要因素,还给畜牧业生产造成重大经济损失,囊虫病的广泛存在还造成畜牧业的重大经济损失,极大影响了我国畜产品在国际市场上的竞争力,因而是公认的社会经济病之一。
1.本实验将猪带绦虫六钩蚴阶段TSOL18基因定向克隆于真核表达质粒pVAX1,经筛选、鉴定及DNA序列分析正确后,将重组质粒pVAX1/TSOL18转染BHK-21细胞,通过SDS-PAGE、Western blotting、荧光抗体染色,检测细胞中表达的TSOL18抗原。结果表明,重组真核质粒pVAX1/TSOL18可在BHK-21细胞中表达,表达的TSOL18目的蛋白能被猪囊尾蚴病阳性血清所识别。动物免疫试验表明,真核表达载体能有效地诱导机体产生细胞免疫和体液免疫应答,这为猪囊尾蚴病核酸疫苗的研究奠定了良好的基础。
2.探讨pVAX1/TSOL18重组表达质粒作为DNA疫苗在小鼠体内的组织分布和生物安全性。将pVAX1/TSOL18重组质粒经肌肉注射免疫BALB/c小鼠,免疫后不同时间剖杀小鼠,分别采集各种组织样品,抽提gDNA,利用PCR技术分析pVAX1/TSOL18在组织内的分布及残留时间;同时,以PCR技术检测免疫动物粪便,分析pVAX1/TSOL18重组表达质粒在外界环境中的释放情况。结果显示,免疫pVAX1/TSOL18重组表达质粒1d和5d后,在小鼠组织gDNA均扩增出目的片段,但不同个体的小鼠,扩增出的目的片段的组织也不完全相同,但在血液中均扩增出了目的片段,第5d较第1d扩增出的目的片段明显变暗;免疫后15d,仅在一只小鼠的血液中检测到TSOL18基因;免疫后30d在所有动物组织中均未检测到重组表达质粒;同时,免疫1d、5d小鼠的粪便中均未检测到TSOL18基因。pVAX1/TSOL18重组表达质粒作为疫苗对动物和环境是安全的。
Cysticercosis is an important zoonosis caused by Taenia solium larval cysticercus cellulosae that can infect intermediate hosts such as pigs or human, which is a major public health problem in most areas of China, and a great potential threat to human security. This disease, whose prevention is always an obstacle for veterinarians mainly because of limitation of protective antigens, is known as an economic disease for it attributes not only to great economic loses but also to decrease of international competition on meat products.
1.In this study, recombinant expression plasmid pVAX1/TSOL18 was constructed and then transfected into BHK-21 cell line with liposome 2000. The results of SDS-PAGE, Western blotting and fluorescence antibody assay showed that TSOL18 protein was successfully expressed in BHK-21 cell line and could be recognized by positive serum against cysticercosis.Immunization in mice indicated that the expression vectors could efficiently induce the specific cellular and humoral immunoresponse, which laid a solid foundation for the further development of new vaccines of cysticercosis.
2.The biosafety and tissue distribution of recombinant expression plasmid was discussed as a DNA vaccine in mouse. Which is one of the key questions which should be solved before it is to be put into practice in the clinical trail. In this test, we investigated the recombinant TSOL18 plasmid in order to evaluate its distribution and two main aspects of biosafety in mice. Recombinant pVAX1/TSOL18 plasmid was injected through the intramuscular introduction, then the mice were slaughtered to extract the gDNA of all kinds of tissues at the different time. The distribution and the possibility of integration of recombinant pVAX1/TSOL18 plasmids in the mice genome were analyzed by PCR technology. One day and five days after immuning the recombinants . the interesting gene of plasmid was amplified from the different gDNA of mice tissues. But different individuals provided the end fragment with different tissue. In the blood, we could amplify the end fragment all the time. Contrast to the first day, the concentration of fragment are darker in fifth day. Fifteen days later, only in one mice we could amplify the fragment of plasmid TSOL18. Thirty days later, we could detect no signals of recombinant plasmid in all the mouse. And we never detected the TSOL18 plasmid at first and fifth day in the feces. Recombinant plasmid pVAX1/TSOL18 is safe to both mice and the surrounding environment as a vaccine.
1.本实验将猪带绦虫六钩蚴阶段TSOL18基因定向克隆于真核表达质粒pVAX1,经筛选、鉴定及DNA序列分析正确后,将重组质粒pVAX1/TSOL18转染BHK-21细胞,通过SDS-PAGE、Western blotting、荧光抗体染色,检测细胞中表达的TSOL18抗原。结果表明,重组真核质粒pVAX1/TSOL18可在BHK-21细胞中表达,表达的TSOL18目的蛋白能被猪囊尾蚴病阳性血清所识别。动物免疫试验表明,真核表达载体能有效地诱导机体产生细胞免疫和体液免疫应答,这为猪囊尾蚴病核酸疫苗的研究奠定了良好的基础。
2.探讨pVAX1/TSOL18重组表达质粒作为DNA疫苗在小鼠体内的组织分布和生物安全性。将pVAX1/TSOL18重组质粒经肌肉注射免疫BALB/c小鼠,免疫后不同时间剖杀小鼠,分别采集各种组织样品,抽提gDNA,利用PCR技术分析pVAX1/TSOL18在组织内的分布及残留时间;同时,以PCR技术检测免疫动物粪便,分析pVAX1/TSOL18重组表达质粒在外界环境中的释放情况。结果显示,免疫pVAX1/TSOL18重组表达质粒1d和5d后,在小鼠组织gDNA均扩增出目的片段,但不同个体的小鼠,扩增出的目的片段的组织也不完全相同,但在血液中均扩增出了目的片段,第5d较第1d扩增出的目的片段明显变暗;免疫后15d,仅在一只小鼠的血液中检测到TSOL18基因;免疫后30d在所有动物组织中均未检测到重组表达质粒;同时,免疫1d、5d小鼠的粪便中均未检测到TSOL18基因。pVAX1/TSOL18重组表达质粒作为疫苗对动物和环境是安全的。
Cysticercosis is an important zoonosis caused by Taenia solium larval cysticercus cellulosae that can infect intermediate hosts such as pigs or human, which is a major public health problem in most areas of China, and a great potential threat to human security. This disease, whose prevention is always an obstacle for veterinarians mainly because of limitation of protective antigens, is known as an economic disease for it attributes not only to great economic loses but also to decrease of international competition on meat products.
1.In this study, recombinant expression plasmid pVAX1/TSOL18 was constructed and then transfected into BHK-21 cell line with liposome 2000. The results of SDS-PAGE, Western blotting and fluorescence antibody assay showed that TSOL18 protein was successfully expressed in BHK-21 cell line and could be recognized by positive serum against cysticercosis.Immunization in mice indicated that the expression vectors could efficiently induce the specific cellular and humoral immunoresponse, which laid a solid foundation for the further development of new vaccines of cysticercosis.
2.The biosafety and tissue distribution of recombinant expression plasmid was discussed as a DNA vaccine in mouse. Which is one of the key questions which should be solved before it is to be put into practice in the clinical trail. In this test, we investigated the recombinant TSOL18 plasmid in order to evaluate its distribution and two main aspects of biosafety in mice. Recombinant pVAX1/TSOL18 plasmid was injected through the intramuscular introduction, then the mice were slaughtered to extract the gDNA of all kinds of tissues at the different time. The distribution and the possibility of integration of recombinant pVAX1/TSOL18 plasmids in the mice genome were analyzed by PCR technology. One day and five days after immuning the recombinants . the interesting gene of plasmid was amplified from the different gDNA of mice tissues. But different individuals provided the end fragment with different tissue. In the blood, we could amplify the end fragment all the time. Contrast to the first day, the concentration of fragment are darker in fifth day. Fifteen days later, only in one mice we could amplify the fragment of plasmid TSOL18. Thirty days later, we could detect no signals of recombinant plasmid in all the mouse. And we never detected the TSOL18 plasmid at first and fifth day in the feces. Recombinant plasmid pVAX1/TSOL18 is safe to both mice and the surrounding environment as a vaccine.
引文
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