| 摘要: |
| [目的]构建猪圆环病毒2型(PCV2)ORF2基因疫苗,并对其免疫效果和安全性进行研究。[方法] 将PCV2 ORF2基因从pMD-ORF2*质粒(包含PCV2 SCH A株ORF2基因)酶切回收,插入到真核表达载体pcDNA-3。1(+)中,构建真核表达质粒pcDNA-ORF2,对该质粒进行酶切和PCR鉴定。将pcDNA-ORF2转染IBRS-2细胞,进行Western blotting检测。将构建好的pcDNA-ORF2制成核酸疫苗,按100 μg/只腿部肌肉注射免疫Balb/c小白鼠,同时设pcDNA-3。1(+),PCV2全毒和PBS为对照,共免疫2次,间隔2周。分别于首免后第14天和56天采血,用ELISA法检测抗体；于首免后第56天,采用淋巴细胞增殖试验(MTT法)和流式细胞仪(FACS)对小鼠的细胞免疫进行检测。取首免后56 d小鼠的心肝、脾、肺、肾和脑等实质器官,采用PCR方法检测pcDNA-ORF2核酸疫苗的安全性。[结果] Western blotting结果显示,pcDNA-ORF2构建成功,并在哺乳动物细胞中获得表达。pcDNA-ORF2核酸疫苗能诱导小鼠产生较强的细胞免疫和体液免疫应答。PCV2 ORF2基因未整合到小鼠染色体上。[结论] pcDNA-ORF2可诱导小鼠产生免疫反应,其对小鼠是安全的。 |
| 关键词: 猪圆环病毒2型 ORF2基因 核酸疫苗 |
| DOI： |
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| 基金项目:教育部长江学者和创新团队发展计划资助项目(IRTO555) |
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| Development of ORF2 gene vaccine against PCV2 and immunity test in mice |
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| Abstract: |
| 【Objective】 The study is to develop a new generation vaccine of PCV2 furtherly. 【Method】 The ORF2 gene of PCV2 was inserted into the eukaryotic expression vector pcDNA-3.1(+) to construct nucleotide vaccine plasmid pcDNA-ORF2.The nucleotide vaccine plasmid identified by PCR and digestion was transfected into IBRS-2 cells with lipofectamine for the analysis of western blotting.The constructed pcDNA-ORF2 was developed into nucleotide vaccine to intramuscular inject into BALB/c mice with 100ug at two weeks interval.On the 14th and the 56th day after the first vaccination,serums were collected to test by ELISA.And on the 56th day after the first vaccination,cell mediated immunity was detected by MTT method and Fluorescence Activated Cell Sorter (FACS).To confirm the safety of pcDNA-ORF2 gene vaccines,the genomes of vaccinated mice were extracted for PCR amlification.【Result】 The result indicated that the nucleotide vaccine plasmid pcDNA-ORF2 was constructed successfully and the ORF2 gene was expressed in IBRS-2 cells,and pcDNA-ORF2 gene vaccines successfully induced humoral and cellular immune response in mice.The safety test of pcDNA-ORF2 gene vaccine showed that ORF2 genes could not be integrated into the mice chromosome and it was safe. 【Conclusion】 The pcDNA-ORF2 gene vaccine successfully induced immune response in mice and was confirmed to be safe. |
| Key words: porcine circovirus type2 ORF2 gene nucleotide vaccine |
