利用玉米作为生物反应器表达PEDV中和表位抗原蛋白

Expression of Core Neutralizing Epitope Gene of Porcine Epidemic Diarrhea Virus in Maize

以玉米为生物反应器,将猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)主要中和抗原表位(core neutralizing epitope,COE)基因导入玉米自交系,获得了高效表达PEDV-COE的转基因玉米稳定株系。首先,根据玉米密码子的偏好性,设计合成优化的COE编码基因,构建了植物表达载体p BAC9036。然后,利用PDS1000/He基因枪转化玉米幼胚,经过愈伤组织诱导、分化再生培养,获得71株转化再生植株,进而通过田间草甘膦抗性筛选,获得2个T1代转基因玉米稳定株系。通过PCR、RT-PCR、间接ELISA和Western blot等鉴定结果显示,COE基因已成功整合到玉米基因组并进行了转录和蛋白表达。并且种子中COE蛋白占可溶性总蛋白的0.122%和0.078%。最后,提取转基因玉米稳定株系的种子可溶性蛋白,与佐剂混合皮下注射免疫小鼠并取可溶性蛋白灌胃免疫小鼠,均可产生针对PEDV抗原的特异性抗体。获得的转基因玉米可有效表达COE蛋白,表达产物具有显著的免疫原性,为进一步研制PEDV-COE转基因玉米疫苗奠定了基础。

The core neutralizing epitope gene of porcine epidemic diarrhea virus（PEDV-COE） was expressed in maize（Zea may L.） bioreactor.The COE gene was introduced into the maize inbred line and stable lines highly expressing PEDV-COE were obtained.Firstly,the coding sequence of the COE gene was optimally modified according to the codon usage bias in maize.Then a synthetic COE gene was cloned into the plant expression vector p BAC9036.Following callus induction,plant regeneration 71 converted regenerated plants were obtained.Through field glyphosate resistance selection,2 stable T1 transgenic corn lines were obtained.The result of PCR,RT-PCR,indirect ELISA and Western blot,etc.indicated that the recombinant COE protein constituted up to 0.122% and 0.078% of the total soluble protein was transcripted in the seed of transgenic maize.Finally,the soluble proteins extracted from the seed of transformed plants were emulsified with Freund＇s adjuvant and immunized mice by subcutaneous injection route,and the extracts was fed directly to mice for oral immunization.The immunized mice developed specific serumantibody against PEDV comparing with that of the non-transgenic maize group.These results demonstrated that transgenic maize could not only express the COE polypeptides,but also induce specific antibody against PEDV in mice.Thus,it has laid a solid foundation for RD of PEDV-COE transgenic maize vaccine.