旋毛虫新生幼虫DNA结合相关蛋白基因的筛选与分析  

Biopanning and Analysis of DNA Binding-Related Protein Genes from New-born Larvae of Trichinella spiralis

作　　者：吴秀萍[1] 邓洪宽[1,2] 刘相叶[1] 于录[1] 王学林[1] Boireau Pascal 刘明远[1] 

机构地区：[1]吉林大学人兽共患病研究所人兽共患病教育部重点实验室,长春130062 [2]法国食品卫生安全局,巴黎94703

出　　处：《中国生物制品学杂志》2009年第8期737-742,共6页Chinese Journal of Biologicals

基　　金：国家杰出青年基金(NSFC30825033);"十一五"863计划生物和医药技术领域专题(2006AA02Z451);国家自然科学基金(NSFC30771885)

摘　　要：目的筛选旋毛虫新生幼虫表达的可与宿主肌细胞染色体DNA结合的相关蛋白基因,寻找旋毛虫侵入及寄生过程中与宿主肌细胞发生结合的相关蛋白,为研究寄生虫与宿主间的信号转导、侵袭及长期寄生机制奠定基础。方法利用噬菌体展示技术构建旋毛虫新生幼虫T7噬菌体展示cDNA文库,用小鼠肌肉染色体DNA对展示文库进行筛选,随机挑选30个阳性克隆,进行PCR鉴定、测序分析和编码蛋白二级结构及翻译后修饰预测。结果旋毛虫新生幼虫T7噬菌体展示cDNA文库的原始文库库容量为2×105pfu,重组率为98%,95%的插入片段长度在250～2000bp之间,扩增后文库滴度为3×1012pfu/ml。对经4轮筛选后的克隆进行PCR鉴定及测序,获得13个基因序列,其中有4个(DN14、DN24、DN9及DN23)为旋毛虫新生幼虫DNA结合相关蛋白基因,分别与旋毛虫未知蛋白及假定ORF11.30、旋毛虫nudix水解酶及血吸虫SJCHGC05997蛋白、秀丽新杆线虫未知蛋白和旋毛虫TGF-β配基具有同源性。经编码蛋白质二级结构及翻译后修饰预测,这4种蛋白可能与旋毛虫包囊形成和寄生虫与宿主间信号转导有关。结论已成功构建了旋毛虫新生幼虫T7噬菌体展示cDNA文库,并利用小鼠肌肉染色体DNA筛选出4个可用于研究旋毛虫包囊形成及与宿主间信号转导机制的候选基因。Objective To screen the gene encoding DNA binding-related protein from newborn larvae of TrichineUa spiralis, find the protein related to binding to host muscle cells of Trichinella spiralis during invasion and parasitism and lay a foundation of study on the mechanisms of signal transduction, invasion and long-term parasitism of Trichinella spiralis. Methods The T7 phage display cDNA library of newborn larvae of Trichinella spiralis was constructed by phage display technique, from which the DNA binding-related protein genes were screened with murine muscle chromosomal DNA. Thirty positive clones were selected randomly for PCR identification, sequencing as well as prediction of secondary structure and post-translational modification of proteins encoded. Results The primary capacity of constructed T7 phage display cDNA library of newborn larvae of TrichineUa spiralis was 2 × 10^5 pfu, and the recombination rate was 98%. The lengths of 95% of inserts were 250 - 2 000 bp. The titer of the constructed library reached 3 × 10^12 pfu / ml after amplification. Thirteen gene fragments were obtained by PCR identification and sequencing of clones after 4 cycles of biopanning, of which 4 （DN14, DN24, DN9 and DN23） encoded DNA binding-related proteins of newborn larvae of Trichinella spiralis, homologous to unknown protein / hypothetical ORF 11. 30 of TrichineUa spiralis, putative nudix hydrolyase of Trichineall psudospirdis / SJCHGC05997 protein of Schistosoma japonicum, hypothetical protein CBG23797 of Caenorhobditis briggsae and TGF-beta-like ligand precursor of T. spiralis respectively. The prediction of secondary structure and post-translational modification of proteins encoded indicated that the 4 kinds of proteins might be related to cyst formation and signal transduction of Trichinella spiralis. Conclusion The T7 phage display eDNA library of newborn larvae of Trichinella spiralis was successfully constructed, and 4 candidate genes used for study on mechanisms of cyst formation and signal transduetion of Trichine

关 键 词：旋毛虫 新生幼虫 DNA结合相关蛋白 噬菌体展示cDNA文库 

分 类 号：R383.15[医药卫生—医学寄生虫学] S852.7[医药卫生—基础医学]