禽流感血凝素HA超表达载体的构建及高表达百脉根的获得

Vector construction and over-expression of an avian influenza virus hemagglutinin (HA) in transgenic Lotus corniculatus

为提高外源抗原蛋白的表达水平,根据已得到的HA-LTB融合基因,通过添加双增强子CaMV35S启动子、增强子Ω序列、内质网滞留信号KDEL序列及加长poly(A)序列等表达调控元件,构建HA-LTB高效融合植物表达载体。利用农杆菌介导方法转化百脉根,获得转基因阳性植株21株,经PCR和RT-PCR检测表明HA-LTB融合基因已整合到百脉根基因组中。利用ELISA检测转基因植株,结果表明LTB免疫佐剂与HA抗原蛋白均具有免疫原性,LTB最高表达量达到0.086μg/mg(蛋白粗提液),HA最高表达量达到0.25μg/mg(蛋白粗提液)。通过植物表达载体的设计和构建,使禽流感血凝素在百脉根中的表达量得到了显著提高,为禽流感可饲疫苗的研制奠定了基础。

A safe and efficient plant expression vector,HA-LTB,which added double 35 Spromoter,Ωenhancer,endoplasmic reticulum retention signal KDEL sequence and extended poly（A）sequence was constructed.Twenty one transgenic Lotus corniculatus plants were obtained by the Agrobacterium-mediated method and the results of PCR and RT-PCR showed that the HA-LTB gene had been obtained from the plant genome.ELISA results of the B subunit of Escherichiacoliheat-labile enterotoxin（LTB）showed that LTB could be expressed in transgenic L.corniculatus and had immunogenicity.It was concluded that hemagglutinin（HA）which was fused with LTB had immune activity.The highest expressions of LTB and HA were 0.086μg/mg（crude protein extracts）and 0.25μg/mg（crude protein extracts）respectively which has approached advanced international standards.A high level exogenous antigen protein expression platform which was a basis for research on the H5N1 avian influenza vaccine was established by optimizing the expression vector.