摘要
为了进一步研究锦鲤疱疹病毒主要囊膜蛋白(KHV-MEP)的功能及锦鲤疱疹病毒(KHV)的感染机制,根据KHV-MEP基因序列设计并合成1对引物,从自然感染KHV发病的锦鲤(Cyprinus carpio Koi)肝组织总DNA中扩增获得特异性基因片段。将所得基因片段克隆到pMD18-T Simple Vector载体中,获得重组质粒T-KMEP;酶切鉴定后进行序列测定,并采用氨基酸亲水性分析软件TMpred对其编码氨基酸序列进行分析;在对该片段所编码氨基酸可能抗原位点分析的基础上,进行PCR改造构建原核表达载体,获得重组表达载体pBV-KMEP1和pBV-KMEP2。所获得的基因片段大小为771bp,该基因片段与GenBank中已登录的KHV-MEP基因(AB178537)的同源性为100%,是一个完整的开放阅读框,所编码的蛋白由256个氨基酸组成,分子量为28.2kD,等电点(PI)为8.65。该序列含有4个跨膜区,可构成主要抗原决定簇。结果显示所获得的目的基因片段就是锦鲤主要囊膜蛋白全基因。
In the present study, the total DNA of Koi Herpesvirus （KHV） was extracted from the liver of Koi carp （Cyprinus carpio Kio）. To study the function of the major envelop protein （KHV-MEP） and the infection mechanism of KHV. KHV-MEP gene was amplified using the primers which were designed based on the KHV- MEP gene sequence in GenBank （AB178537, AB178334）. The PCR product was then ligated to pMD18-T simple vector and transformed into E.coli DH5α. The sequencing result of recombinant plasmid indicates that the major envelop protein gene of KHV is 771 bp long. It is an integrate exoteric Open Reading Frame （ORF）, encoding a protein composing 256 amino acids whose molecular mass is 28.2 kD and isoelectric point （PI） is 8.65. It has the highest homology of 100% with the major envelop protein gene of KHV （AB178537）in GenBank. Amino acid hydrophilic analysis indicates that there are 4 transmembrane regions, which are the main antigenic determinant of the major envelop protein. The results show that the target fragment is the major envelop protein gene of KHV.
出处
《中国水产科学》
CAS
CSCD
北大核心
2008年第5期880-884,共5页
Journal of Fishery Sciences of China
基金
广东省重大科技兴海(兴渔)项目(070103)
珠江水产研究所所长基金项目(2006G3)
广州市番禺区科技计划项目(2007-Z-46-1).
关键词
锦鲤疱疹病毒
主要囊膜蛋白基因
序列分析
原核表达载体
Koi Herpesvirus （KHV）
major envelop protein gene
sequence analysis
prokaryotic expressionvector
