马传染性贫血病毒膜蛋白基因的改造及其在真核细胞中的表达

Modifications of the EIAV Envelope Glycoprotein and Expression in Hela Cell Line

以马传染性贫血病毒(equine infectious anemia virus,EIAV)天然膜蛋白基因(gp120)为基础设计的DNA疫苗在马体内的表达量很低,这与密码子使用偏嗜性、RNA剪切位点多和导致RNA不稳定性的腺苷丰富区的大量存在有关。为解决这些问题,合成了EIAVgp120基因(Syn-env),使其密码子偏嗜性符合高水平表达的哺乳动物基因。EIAV弱毒疫苗的制备过程中,出现了几个重要而稳定的N-糖基突变位点,以Syn-env为基础,运用重叠延伸PCR定点突变策略获得了6个具有不同N-糖基缺失组合的gp120基因。将突变后的基因克隆到真核表达载体pVRCSV1.0,然后转染Hela细胞,发现这些重组质粒均获得了表达。进而为比较6种质粒诱导机体免疫保护效果,阐明EIAV弱毒疫苗减毒机理奠定了基础。

In the context of DNA vaccines the native equine infectious anemia virus（EIAV）envelope gene（gp120） has proven to be an extremely weak immunogen in horses probably because the RNA transcripts are poorly expressed owing to an unusual codon-usage bias, the possession of multiple RNA splice sites and potential adenosine-rich RNA instability elements. To overcome these problems a synthetic version of sequences encoding the EIAV（gp120） envelope glycoprotein is produced（Syn-env）in which the codon-usage bias is modified to conform to that of highly expressed horse and human genes. Critical consensus N-glycosylation sites mutations occurr during the viral passages in vitro and in vivo for vaccine＇s preparation. Based on the Syn-env and according to mutations displayed during viral attenuation, the authors successfully constructe six gene mutants in which gp120 gene is point-mutated by overlap PCR mutagenesis strategy. Then insert into the pVRCSV1.0 mammalian expression vector, and transfect Hela cell cultures. All the plasmids can be expressed in Hela cell. The authors can compare the protective efficacy of the immune responses elicited by the six recombinant mammalian expression vectors, which may help to elucidate the attenuated mechanism of EIAV and provide instructive theoretical basis for development of potential HIV vaccine.