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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 for the related proteins that can bind to the chromosomal DNA of host muscle cells expressed by the newborn larva of Trichinella spiralis and find out the related proteins that bind to the host muscle cells during the process of invasion and parasitism of Trichinella spiralis. In order to study the signal transduction between parasites and host , Invasion and long-term parasitic mechanisms. Methods The phage display technique was used to construct T7 phage display cDNA library of Trichinella spiralis larvae. The display library was screened by mouse muscle chromosomal DNA and 30 positive clones were randomly selected for PCR identification, sequencing analysis and protein secondary structure translation Modification prediction. Results The original library size of T7 phage display cDNA library of Trichinella spiralis larvae was 2 × 105pfu, the recombination rate was 98%, the length of insert between 95% was between 250 ~ 2000bp, the amplified library titer was 3 × 1012pfu / ml. After 4 rounds of screening, the clones were identified by PCR and sequenced. Thirteen gene sequences were obtained, of which 4 (DN14, DN24, DN9 and DN23) were the DNA binding related genes of neonate Trichinella spiralis, And hypotheses ORF11.30, Trichinella nudix hydrolase and SJCHGC05997 protein of Schistosoma japonicum, homologous to Trichinella spiralis TGF-beta ligand. The secondary structure of the encoded protein and posttranslational modifications predict that these four proteins may be involved in the cyst formation of Trichinella and the signal transduction between parasites and hosts. Conclusion T7 phage display cDNA library of Trichinella spiralis larvae has been successfully constructed and four candidate genes that can be used to study the cyst formation and the signal transduction mechanism between Trichinella spiralis and host cells were screened by using mouse muscle chromosomal DNA.