CAJ | 학술논문

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。
위제고외원항원단백적표체수평，근거이득도적 HA-LTB융합기인，통과첨가쌍증강자CaMV35S계동자、증강자Ω서렬、내질망체류신호KDEL서렬급가장poly（A）서렬등표체조공원건，구건 HA-LTB고효융합식물표체재체。이용농간균개도방법전화백맥근，획득전기인양성식주21주，경PCR 화RT-PCR 검측표명 HA-LTB융합기인이정합도백맥근기인조중。이용ELISA검측전기인식주，결과표명LTB면역좌제여 HA항원단백균구유면역원성，LTB최고표체량체도0．086μg/mg（단백조제액），HA최고표체량체도0．25μg/mg（단백조제액）。통과식물표체재체적설계화구건，사금류감혈응소재백맥근중적표체량득도료현저제고，위금류감가사역묘적연제전정료기출。
A safe and efficient plant expression vector,HA-LTB,which added double 35S promoter,Ωenhan-cer,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 Escherichiacoli heat-labile enterotoxin (LTB)showed that LTB could be ex-pressed 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 re-search on the H5N1 avian influenza vaccine was established by optimizing the expression vector.