论文部分内容阅读
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 order to further study the function of KHV-MEP and the mechanism of KHV infection, a pair of primers were designed and synthesized according to the KHV-MEP gene sequence. The carp (Cyprinus carpio Koi) liver tissue total DNA amplified specific gene fragments. The resulting gene fragment was cloned into the pMD18-T Simple Vector vector to obtain the recombinant plasmid T-KMEP; enzyme digestion identified sequence analysis and amino acid hydrophilicity analysis software TMpred its encoded amino acid sequence analysis; in the fragment Based on the possible analysis of the amino acid positions of the encoded amino acids, a prokaryotic expression vector was constructed by PCR and the recombinant expression vectors pBV-KMEP1 and pBV-KMEP2 were obtained. The obtained gene fragment was 771bp in size. The gene fragment was 100% identical to the KHV-MEP gene (AB178537) registered in GenBank. It was a complete open reading frame, and the encoded protein consisted of 256 amino acids , Molecular weight of 28.2kD, isoelectric point (PI) of 8.65. This sequence contains four transmembrane regions that make up the major epitopes. The results showed that the target gene fragment obtained was the main gene of the major envelope protein of Koi carp.