旋毛虫新生幼虫DNA结合相关蛋白基因的筛选与分析

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。

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

Objective To screen the gene encoding DNA binding-related protein from newborn larvae of Trichinella 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 Trichinella spiralis was 2 × 105 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 × 1012 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 Trichinella spiralis,putative nudix hydrolyase of Trichineall psudospiralis /SJCHGC05997 protein of Schistosoma japonicum,hypothetical protein CBG23797 of Caenorhabditis 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 cDNA library of newborn larvae of Trichinella spiralis was successfully constructed,and 4 candidate genes used for study on mechanisms of cyst formation and signal transduction of Trichinella spiralis were screened with murine muscle chromosomal DNA.