摘要： 提取小麦品种L10的总RNA,进行反转录,其产物用于探究自噬基因ATG8的生物学功能。用PCR方法克隆ATG8,将克隆的ATG8亚克隆至表达载体PMD19-T,酶切后切胶回收,然后把产物测序鉴定,转化宿主菌E.coliRosetta-gami B(DE3),构建原核表达系统,将融合蛋白纯化后制备其兔抗血清,然后利用WesteRn Blotting技术对兔抗血清进行检测,鉴定了该抗血清的结合特异性。ATG8抗体的成功制备,为小麦中ATG8基因和自噬功能的研究奠定了基础。
关键词:
ATG8,
免疫印迹,
抗血清,
融合蛋白,
原核表达
Abstract: Total RNA was isolated fRom wheat vaRiety L10 and ReveRse tRanscRiption by using the anchoRed pRimeRs in this papeR to fuRtheR undeRstand the biological function of the autophagy gene ATG8 fRom wheat． And then the ATG8 weRe amplified by PCR and then cloned into the PMD19-T vectoR，one of which was subcloned into the expRession vectoR． The Recombinant plasmid was identified by sequencing and digestion of RestRiction enzymes． The Recombinant expRession vectoR was constRucted and tRansfoRmed into E． coli stRain Roseta( DE3) subsequently， then thRough IPTG-induction in host bacteRia E． coli Rosetta-gami B( DE3) and detected by SDS-PAGE． The Rabbit anti- ATG8 antibody was pRepaRed and was detected by WesteRn Blotting analysis． The ATG8 weRe obtained paRtly and successfully expRessed in the pRokaRyotic expRession system． The expeRiment offeRed foundation to the studying of autophagy and function of ATG8 gene in wheat．
Key words:
ATG8,
WesteRn Blotting,
Anti-seRum,
Fusion pRotein,
PRokaRyotic expRession
中图分类号:
吴洪波, 刘刚, 张路路, 王冬梅. 小麦ATG8的原核表达及其抗血清制备[J]. 华北农学报, 2013, 28(1): 101-105. doi: 10.3969/j.issn.1000-7091.2013.01.019.
WU Hong-bo, LIU Gang, ZHANG Lu-lu, WANG Dong-mei. Cloning and PRokaRyotic ExpRession of ATG8 ( a MoleculaR MaRkeR of Autophagy) Gene fRom Wheat and PRepaRation of AntiseRum[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2013, 28(1): 101-105. doi: 10.3969/j.issn.1000-7091.2013.01.019.