摘要
为构建新城疫病毒(NDV)V蛋白C端酵母双杂交诱饵质粒,用RNA试剂盒提取NDV F48E9株的RNA,通过反转录试剂盒将抽提的RNA反转录,以反转录合成的c DNA为模板,通过PCR扩增,获得V蛋白C端基因(VCTD)。通过酶切连接的方法将V蛋白C端基因克隆至p GBKT7载体上,命名为p GBKT7-VCTD。将p GBKT7-VCTD转化酵母Y2H Gold酵母感受态细胞,转化产物涂布营养缺陷型平板,观察平板上菌落生长情况,判定诱饵质粒有无自激活能力;同时挑取平板上的生长的菌落接种液体培养基培养,绘制生长曲线,判定p GBKT7-VCTD诱饵质粒有无毒性。酶切和测序结果表明,成功构建p GBKT7-VCTD诱饵质粒,且该诱饵质粒在酵母细胞中无毒性和自激活能力。本研究成功构建了p GBKT7-VCTD诱饵质粒,为筛选NDV V蛋白C端纳米抗体奠定基础。
To construct a Newcastle disease virus(NDV)V protein C terminal region yeast-two hybrid bait plasmid,total RNA from NDV F48 E9 strain was extracted with an RNA kit.The reverse transcription was then performed to synthesis cDNA.The gene encoding C terminal domain of V protein(VCTD gene)was amplified through polymerase chain reaction(PCR)with the synthesised cDNA as template.The acquired VCTD gene was subsequently cloned into p GBKT7 vector through restriction enzyme digestion and ligation and was designed pGBKT7-VCTD.The pGBKT7-VCTD was transformed into Y2 H Gold competent cells and the transformed product was spread on nutrient deficient plates to test for autoactivation.Meanwhile,a single colony appeared on the above plates was pick and inoculated into liquid nutrient deficient medium to plot the growth curve for toxicity detection.The enzyme digestion and sequencing results suggested that pGBKT7-VCTD bait plasmid was successfully constructed.And the bait had no autoactivation and toxicity in yeast.In conclusion,our study successfully constructed NDV V protein C terminal domain yeast-two hybrid bait plasmid pGBKT7-VCTD,which laid the foundation for screening of nanobody against C terminal domain of NDV V protein.
作者
高小龙
仝丽娜
郭承意
周雷
杨峻鹏
张诚
李增魁
文英
李英
GAO Xiaolong;TONG Lina;GUO Chengyi;ZHOU Lei;YANG Junpeng;ZHANG Cheng;LI Zengkui;WEN Ying;LI Ying(College of Agriculture and Animal Husbandry,Qinghai University,Xining 810000)
出处
《安徽农业大学学报》
CAS
CSCD
2021年第2期241-244,共4页
Journal of Anhui Agricultural University
基金
青海省科技厅自然科学基金青年项目(2018-ZJ-952Q)资助。
关键词
新城疫病毒
V蛋白
诱饵质粒
Newcastle disease virus
V protein
bait plasmid
