摘要
根据GenBank中猪圆环病毒2型(PCV-2)ORF2基因序列,设计了1对引物,应用PCR从广西PCV-2的DNA扩增出ORF2基因,将ORF2基因克隆至真核表达载体pcDNA3.1(+)载体上,获得重组质粒,经PCR酶切以及序列分析鉴定,表明插入的片段为ORF2目的基因,插入的位置、大小和阅码框均正确,成功构建了真核表达载体重组质粒pcDNA-PCV-ORF2。对阳性真核表达质粒pcDNA-PCV-ORF2大量抽提后,以每只小鼠0.2 mg免疫5周龄左右的雌性小鼠,然后用ELISA检测免疫小鼠血清中抗体的产生情况,同时进行pcDNA-PCV-ORF2的体内分布和安全性检测。结果表明,pcDNA-PCV-ORF2在小鼠体内广泛存在,产生了抗猪圆环病毒的特异性抗体,从第7天开始产生抗体,第28天抗体水平达到最高,抗体可维持7周以上,并具有良好的安全性。本研究通过对小鼠的免疫试验,产生了猪圆环病毒的特异性抗体,表明成功构建了能够在活体细胞中高效表达的真核表达载体,为进一步研究猪圆环病毒DNA疫苗奠定了基础。
According to the published ORF2 gene sequence of porcine circovirus type 2 （PCV-2） in GenBank, a pair of primers were designed to amplify the ORF2 gene from PCV-2 Guangxi isolate. The PCR product was purified and inserted into pcDNA3.1 （＋） vector. The recombinant plasmid was identified by restriction endonuclease analysis and PCR. It was proved by DNA sequencing that the acquired recombinant plasmid contained complete ORF2 gene and the recombinant DNA vaccine plasmid of pcDNA-PCVORF2 was constructed. After quantities of the 5 recombinant plasmids were produced and purified, 5- week-old female mice were immunized with the plasmids, and levels of the antibody against PCV-2 in sera from the immunized mice were detected by ELISA. The distribution and the security of pcDNA-PCV- ORF2 were detected by PCR. The antibody emerged in 7th day, and reached maximum at the 28th day, then declined slowly, lasted for over 7 weeks. The distribution of pcDNA-PCV-ORF2 in the mice was wide and the pcDNA-PCV-ORF2 was safe in the mice. The results showed that pcDNA-PCV-ORF2 can induced the specific antibody.
出处
《动物医学进展》
CSCD
2008年第4期18-21,共4页
Progress In Veterinary Medicine
基金
广西水产畜牧局项目(桂渔牧科061909)
国家百千万人才工程入选专项资金项目(945200603)
