摘要目的 构建嗜热吸水链霉菌cDNA文库并进行表达序列标签(EST)测序,从中筛选具有毒力作用的基因进行体外克隆并诱导表达.方法 利用RNA转录5’末端转换技术构建致农民肺嗜热吸水链霉菌的cDNA文库,将平板内克隆进行编号,取前1020个克隆提取质粒,进行EST测序.用生物信息学方法分析EST测序结果并预测基因功能,从中筛选出可能引起人体免疫反应的毒力因子.将这些目的基因亚克隆入pET-28a载体,将重组子转化大肠杆菌BL21,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导相应蛋白的表达.结果 成功构建了较高质量的嗜热吸水链霉菌cDNA文库,得到有义序列978个,获得单一基因347个,其中2段基因分别与胸膜肺炎放线菌外膜蛋白、转铁蛋白B的同源性为51％和42％,其开放阅读框长度分别为1554 bp和726 bp,分别编码517和241个氨基酸.将2段基因亚克隆入原核表达载体中,IPTG诱导表达产物的相对分子质量分别为63 000和30 000.结论 所构建的嗜热吸水链霉菌cDNA文库符合构建基因文库的质量要求,文库中的EST数据将有助于农民肺特异性毒力分子的进一步筛选.

abstractsObjective To construct a cDNA library from Streptomyces thermohydroscopicus and screen genes with virulence,obtain the recombinant fusion virulence proteins by prokaryotic expression system. Methods The Streptomyces thermohydroscopicus cDNA library was constructed by switching mechanism at 5'end of RNA transcript approach.A total of 1020 clones randomly selected from the cDNA library were sequenced and these expressed sequence tags (EST) were further analyzed for the screen of antigen-specific genes. The two candidate genes were subcloned into expression vector pET-28a. The recombinants were transformed into BL2 and proteins were expressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG).Results A high-quality cDNA library from Streptomyces thermohydroscopicus was constructed and a set of 978 valid sequences were obtained.Clustering and assembly of these cDNA sequences resulted in 347 unique genes,among which 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein (51％) and transferring-binding protein B (42％) from Actinobacillus pleuropneumoniae serotype (APP).The open reading frame (ORF) of the two candidate genes are 1554 bp and 726 bp,which coded two peptides with 517 and 241 amino acids,respectively.The molecular weights of the recombinant fusion proteins were 63 000 and 30 000.Conclusion The cDNA library of Streptomyces thermohydroscopicus reached the quality requirement of gene library.EST database in the library would greatly facilitate further screening of virulence genes.