摘要
为了研究猪圆环病毒2型（Porcine circovirus type 2,PCV-2）ORF2基因在Vero细胞中的表达情况,试验利用PCR方法将扩增的PCV-2 ORF2基因片段克隆到真核表达载体pcDNA3.1（＋）上,构建重组表达质粒pcDNA-ORF2,测序后对4株毒株的ORF2基因进行核苷酸序列和推导氨基酸序列同源性比较分析,并分别采用磷酸钙法和脂质体法将重组表达质粒pcDNA-ORF2导入哺乳动物Vero细胞内,通过间接免疫荧光试验验证重组表达质粒在Vero细胞中的表达情况。结果表明：PCR方法成功扩增出4株PCV-2毒株的ORF2基因;成功构建出重组表达质粒pcDNA-ORF2;4株毒株的ORF2基因核苷酸序列同源性在98.6%-99.1%之间,推导氨基酸序列的同源性在97.4%-98.7%之间;间接免疫荧光试验检测到Vero细胞内发出绿色荧光。说明重组表达质粒pcDNA-ORF2在Vero细胞中得到了正确表达。
The purpose of this study was to identify the expression of Porcine circovirus type 2（ PCV-2） ORF2 gene in Vero cells. The polymerase chain reaction（ PCR） method was used to amplify the ORF2 gene which was cloned into mammalian expression vector pcDNA3. 1（ ＋）. The recombinant expression plasmid pcDNA-ORF2 was constructed. The nucleotide sequence and the derivation of amino acid homology were analyzed for the ORF2 gene of 4 strains. The recombinant expression eukaryotic plasmid was transfected into Vero cells in vitro by using Ca3（ PO4）2 transfection and lipofectin 2000. The transient expression products were analyzed by immunofluorescence. The results showed that the ORF2 gene of four PCV-2 strains was amplified by PCR and the recombinant expression plasmid pcDNA-ORF2 was successfully constructed.The homology of the ORF2 gene nucleotide sequence of four strains was 98. 6%-99. 1%,and the homology of the amino acids was 97. 4%-98. 7%. The green fluorescence could be detected in the cells by using the indirect immunofluorescence. The results indicated that the pcDNAORF2 recombinant expression plasmid was correctly expressed in Vero cells.
作者
马晓莉
林彦星
董建国
刘燕玲
刘清神
宋长绪
徐铮
MA Xiaoli;LIN Yanxing;DONG Jianguo;LIU Yanling;LIU Qingshen;SONG Changxu;XU Zheng(Laboratory Animal Center of South China Agrieuhural University, Guangzhou 510642, China;Inspeetion and Quarantine Center for Animals and Plants, Shenzhen Entry-exit Inspeetion and Quarantine Bureau, Shenzhen 518045, China;National Engineering Researeh Center for Breeding Swine Industry and College of Animal Science of South China Azrieultural University. Guanzzhou 510642. China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2018年第11期12-15,19,250,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
“十二五”农村领域国家科技计划项目(2015BAD12B02-5)
现代农业产业共性技术创新团队项目(2016LM2150)
前沿与关键技术创新项目(2015B020230004)
广东省省级科技计划项目(2016A020212021)
广州市科技计划项目(201508020062)
