摘要
为了筛选柔嫩艾美耳球虫子孢子入侵相关的蛋白,利用酵母双杂交体系构建了柔嫩艾美耳球虫子孢子酵母双杂交cDNA文库。提取纯化的子孢子总RNA,经MMLV-RT反转录合成cDNA第一链后,利用LD-PCR扩增合成双链cDNA（ds cDNA）,dscDNA经纯化柱CHROMA SPINT＋TE-400 Columns纯化剔除小于200 bp的片段。将纯化后dscDNA、pGADT7-Rec及Carrier DNA共转化到已经制备好的酵母感受态细胞Y187中,dscDNA与pGADT7-Rec以同源重组的方式在细胞内进行连接,经过缺陷性培养基（SD/-Leu）的筛选得到酵母双杂交cDNA文库。结果显示,构建的柔嫩艾美耳球虫子孢子酵母双杂交cDNA文库的转化率为4.33×10^5/μg pGADT7-Rec,文库滴度为3.62×10^7 cfu/m L。随机挑取32个单克隆进行PCR检测,插入片段大小为200~2000bp,重组率为93.75%,并以该文库作为模板,用柔嫩艾美耳球虫5对特异性引物进行PCR扩增,获得了这5个基因片段。结果表明成功构建了柔嫩艾美耳球虫子孢子酵母双杂交cDNA文库,为进一步筛选和研究球虫入侵互作蛋白奠定了基础。
In order to screen the proteins related to the invasion of Eimeria tenella sporozoites, the yeast two-hybrid cDNA library of E. tenella sporozoites was constructed in the present study. The total RNA was isolated from the sporozoites and used as the template to synthesize the first-strand cDNAs. The dscDNAs were acquired by long-distance polymerase chain reaction （LD-PCR） using the first- strand cDNAs as the template and purified with Chroma Spin TE-400 Column to remove less than 200 bp fragments. The dscDNA,pGADT7-Rec and CarrierDNA were co-transformed into Y187 yeast competent cells. The dscDNA were then connected with pGADT7- Rec by homologous recombination reaction to construct the yeast two-hybrid cDNA library orE. tenella sporozoites. The results showed that the conversion ratio and titer of the eDNA library were 4.33 ×1^05/μg pGADT7-Rec and 3.62 ×10^7cfu/mL, respectively, As demonstrated in PCR amplification, the cDNA library contained approximately 93.75 % recombinant clones and the inserted eDNA fragments were between 200 bp and 2000 bp. Furthermore, 5 gene fragments were obtained by PCR amplication using the eDNA library as template and 5 specific pairs of primers of E. tenella. Therefore, it concluded that a yeast two-hybrid eDNA library of the sporozoites was constructed for screening invasion-related interaction proteins of the sporozoites of E. tenella.
作者
徐帅兵
黄兵
赵其平
董辉
朱顺海
崔晓霞
谢雨翔
唐敏
杨志远
韩红玉
XU Shuai-bing HUANG Bing ZHAO Qi-ping DONG Hui ZHU Shun-hail CUI Xiao-xia XIE Yu-xiang TANG Min YANG Zhi-yuan HAN Hong-yu(Key Laboratory of Animal Parasitolgy of Ministry of Agriculture, Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China)
出处
《中国动物传染病学报》
CAS
北大核心
2016年第6期36-42,共7页
Chinese Journal of Animal Infectious Diseases
基金
国家自然科学基金(31272557,31572266)
农业部中央级公益性科研院所项目(2015JB10)
