Sequence Analysis of ORF2 Gene of Type 2 Porcine Circovirus and Construction of Eukaryotic Expressing Vector of ORF2

I U; CHEN De; HUA Xiu; HUANG Wei; TAN Guo; CHEN Pu

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February 2002

I U, CHEN De, HUA Xiu, HUANG Wei, TAN Guo and CHEN Pu. Sequence Analysis of ORF2 Gene of Type 2 Porcine Circovirus and Construction of Eukaryotic Expressing Vector of ORF2[J]. Virologica Sinica, 2002, 17(1): 87-91.

Citation: I U, CHEN De, HUA Xiu, HUANG Wei, TAN Guo, CHEN Pu. Sequence Analysis of ORF2 Gene of Type 2 Porcine Circovirus and Construction of Eukaryotic Expressing Vector of ORF2 .VIROLOGICA SINICA, 2002, 17(1) : 87-91.

猪圆环病毒2型ORF2基因序列分析及真核表达载体的构建

1.
I．南京农业大学动物医学院．南京210095
2.
上海交通大学农学院，上海200030

摘要

摘要：根据GertSank中猪圆环病毒2型(PCV-2)ORF2基因序列，设计一对弓I物，应用PCR从疑患断奶仔猪多系统消耗综合症(PMWS)的死亡仔猪组筝i病料中扩增出ORF2全基因(702hp)。将此片段克隆入pGEM．T easy载体，筛选获得重组质粒pTORF2，井对此质粒中的插入序列进行了测序分析，结果表明本试验克隆的ORF2与美国PCV-2 分离株AF264039的核苷酸及氮基酸序列同源性均达到100％，与其他PCV 2毒株同源性分别为92．3％～98．6％和92．3％～％．6％。重组质粒pTORF2经BarnHl、EcoR V双酶切，回收ORFE基因，转移入真核表达载体pSec— Ta ／HygmB的相应酶切位点之间，构建成重组质粒pSecTagORF2。此重组表达载悼的构建成功为进一步研究 ORF2编码蛋白的生物学活性及建立PCV诊断试剂盘打下基础。
- 猪圆环病毒
- , ORFE
- , 真核表达载体
- , 构建

Sequence Analysis of ORF2 Gene of Type 2 Porcine Circovirus and Construction of Eukaryotic Expressing Vector of ORF2

1.
1，College of VeterinaryMedicine．NanjingAgricultural University，Nanfing 210095，China
2.
Cortege ofShanghai jiao Tong Univer~ity，Shanghai 200030，Ch ina

Abstract

Abstract：According to the published ORF2 gene sequence of PCV一2，two primers were designed．and ORF2 gene was amplified from suspected PMW S sample using PCR．Then the PCR product was cloned into pGEM ．T easy vector，and recombinant plasmid nam ed pTORF2 was obtained The cloned ORF2 gene in pTORF2 was sequenced and compared with other PCV isolates in GenBank，the results demonstrated that the ORF2 gene is closely related with a USA isolate AF264039．the homology of their nucleotide and amino acid sequence both were 100％． In addition． there existed 92．3％ to 98．6％ nucleotide sequence identity and 92．3％ to 96 6％ amino acid sequence identity with other PCV-2 isolates．Recombinant plasmid pTORF2 was digested with Bam H I／EcoR V．ORF2 gene Was purified．and introduced into eukaryotic expressing veCtor pSecTag2／HygroB between BarnH I and E∞R V enzyme site．then pSecTagORF2 recom binant plasmid was constructed ．Further research is underway to study the biological activity of ORF2 protein and establish the diagnostic kit of PCV．
- Porcine circovirus
- , ORF2：Eukasyotic expresing vector
- , Construction

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Sequence Analysis of ORF2 Gene of Type 2 Porcine Circovirus and Construction of Eukaryotic Expressing Vector of ORF2

1. 1，College of VeterinaryMedicine．NanjingAgricultural University，Nanfing 210095，China
2. Cortege ofShanghai jiao Tong Univer~ity，Shanghai 200030，Ch ina

Abstract: Abstract：According to the published ORF2 gene sequence of PCV一2，two primers were designed．and ORF2 gene was amplified from suspected PMW S sample using PCR．Then the PCR product was cloned into pGEM ．T easy vector，and recombinant plasmid nam ed pTORF2 was obtained The cloned ORF2 gene in pTORF2 was sequenced and compared with other PCV isolates in GenBank，the results demonstrated that the ORF2 gene is closely related with a USA isolate AF264039．the homology of their nucleotide and amino acid sequence both were 100％． In addition． there existed 92．3％ to 98．6％ nucleotide sequence identity and 92．3％ to 96 6％ amino acid sequence identity with other PCV-2 isolates．Recombinant plasmid pTORF2 was digested with Bam H I／EcoR V．ORF2 gene Was purified．and introduced into eukaryotic expressing veCtor pSecTag2／HygroB between BarnH I and E∞R V enzyme site．then pSecTagORF2 recom binant plasmid was constructed ．Further research is underway to study the biological activity of ORF2 protein and establish the diagnostic kit of PCV．