摘要
为建立利用植物细胞瞬时表达反刍动物寄生线虫捻转血矛线虫优势抗原的方法,选取了氨基肽酶基因Hc-ap-10和优选抗原分子基因Hc23作为目标基因,将经过密码子优化的Hc-ap-10(AP10^(op))和Hc23分别插入pH7lic-EGFP表达载体并在N、C两端均添加核定位信号(nuclear localization sequence,NLS),使用电转法将pH7dNLSAP10^(op)和pH7dNLSHc23分别转入GV3101根癌农杆菌中,随后将根癌农杆菌悬液侵染本氏烟草,最后通过荧光显微镜观察目标蛋白定位情况,并提取侵染烟草叶片总蛋白,使用Western blot检测蛋白的表达量。结果显示,成功构建了植物双元表达载体pH7dNLSAP10^(op)和pH7dNLSHc23,荧光显微镜观察发现在目的基因N、C两端添加了核定位信号后的质粒转化根癌农杆菌。该方法外源表达的膜蛋白pH7dNLSAP10^(op)和pH7dNLSHc23都成功表达在本氏烟草叶片的细胞核上,Western blot也成功检测到Hc23和AP10^(op)蛋白,且Hc23蛋白的表达水平相对较高。结果表明,添加了核定位信号能成功将目的蛋白表达在植物细胞核上;密码子优化有利于大分子蛋白的高效表达,但对于一些相对分子质量相对较小的蛋白可能是非必要的。该研究成功将2种抗原分子表达在植物细胞中,为后续疫苗的研究奠定了基础。
The purpose of this study was to establish a method of using plant cells to transiently express vaccine candidate antigens of Haemonchus contortus.The aminopeptidase gene Hc-ap-10 and antigen molecule gene Hc23 were selected as the target genes.Then,codon optimized Hc-ap-10(AP10^(op))and Hc23 were inserted into the pH7 lic-EGFP expression carrier and nuclear localization sequence(NLS)were added at both ends of N and C.Using Agrobacterium mediated transformation,pH7 dNLSAP 10^(op)and pH7 dNLSHc23 were transferred to GV3101 Agrobacterium tumefacien s,and then Nicotiana benthamiana was infested with agrobacterium turmefaciens.Finally,the target protein positioning was observed by the fluorescence microscope,and the total protein of Nicotiana benthamiana leaf was extracted,and the expression of the protein was detected using Western blot.The results showed that the plant binary expression vector pH7 dNLSAP 10^(op)and pH7 dNLSHc23 were successfully constructed,fluorescence microscope found that the plasmid was transformed into Agrobacterium tumefaciens after the target gene was added with nuclear localization sequence.The two proteins expressed were successfully expressed in the nucleus of Nicotiana benthamiana leaves.Western blot also successfully detected Hc23 and AP10^(op)proteins,and Hc23 had relatively high expression levels.This study indicated that the addition of nuclear positioning sequence could successfully express the target protein on the nucleus of plant cells.Codon optimization was beneficial for the efficient expression of macromolecular proteins,but might not be necessary for some proteins with relatively small molecular weight.In summary,the study successfully expressed two antigen molecules in plant cells,laying the foundation for follow-up vaccine research.
作者
叶莉莎
张挺
廖聪
李婷婷
田振东
胡敏
YE Lisha;ZHANG Ting;LIAO Cong;LI Tingting;TIAN Zhendong;HU Min(State Key Laboratory of Agricultural Microbiology,College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;Ministry of Agriculture and Rural Affairs Key Laboratory of Potato Biology and Biotechnology,Huazhong Agricultural University,Wuhan 430070,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2022年第12期2420-2426,共7页
Chinese Journal of Veterinary Science
关键词
瞬时表达
捻转血矛线虫
氨基肽酶
Hc23基因
核定位信号
根癌农杆菌
本氏烟草
transient expression
Haemonchus contortus
aminopeptidase
Hc23 gene
nuclear localization sequence
Agrobacterium tumefaciens
Nicotiana benthamiana
