中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2012年
3期
193-197
,共5页
许蕾%王玲玲%刘朔%凌媛%马列%王群%张丽娇%何晓雨%赵明静%王笑歌
許蕾%王玲玲%劉朔%凌媛%馬列%王群%張麗嬌%何曉雨%趙明靜%王笑歌
허뢰%왕령령%류삭%릉원%마렬%왕군%장려교%하효우%조명정%왕소가
农民肺%基因文库%抗原%嗜热吸水链霉菌
農民肺%基因文庫%抗原%嗜熱吸水鏈黴菌
농민폐%기인문고%항원%기열흡수련매균
Farmer's lung%Gene library%Antigens%Streptomyces thermohydroscopicus
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.
目的 構建嗜熱吸水鏈黴菌cDNA文庫併進行錶達序列標籤(EST)測序,從中篩選具有毒力作用的基因進行體外剋隆併誘導錶達.方法 利用RNA轉錄5’末耑轉換技術構建緻農民肺嗜熱吸水鏈黴菌的cDNA文庫,將平闆內剋隆進行編號,取前1020箇剋隆提取質粒,進行EST測序.用生物信息學方法分析EST測序結果併預測基因功能,從中篩選齣可能引起人體免疫反應的毒力因子.將這些目的基因亞剋隆入pET-28a載體,將重組子轉化大腸桿菌BL21,用異丙基-β-D-硫代半乳糖苷(IPTG)誘導相應蛋白的錶達.結果 成功構建瞭較高質量的嗜熱吸水鏈黴菌cDNA文庫,得到有義序列978箇,穫得單一基因347箇,其中2段基因分彆與胸膜肺炎放線菌外膜蛋白、轉鐵蛋白B的同源性為51％和42％,其開放閱讀框長度分彆為1554 bp和726 bp,分彆編碼517和241箇氨基痠.將2段基因亞剋隆入原覈錶達載體中,IPTG誘導錶達產物的相對分子質量分彆為63 000和30 000.結論 所構建的嗜熱吸水鏈黴菌cDNA文庫符閤構建基因文庫的質量要求,文庫中的EST數據將有助于農民肺特異性毒力分子的進一步篩選.
목적 구건기열흡수련매균cDNA문고병진행표체서렬표첨(EST)측서,종중사선구유독력작용적기인진행체외극륭병유도표체.방법 이용RNA전록5’말단전환기술구건치농민폐기열흡수련매균적cDNA문고,장평판내극륭진행편호,취전1020개극륭제취질립,진행EST측서.용생물신식학방법분석EST측서결과병예측기인공능,종중사선출가능인기인체면역반응적독력인자.장저사목적기인아극륭입pET-28a재체,장중조자전화대장간균BL21,용이병기-β-D-류대반유당감(IPTG)유도상응단백적표체.결과 성공구건료교고질량적기열흡수련매균cDNA문고,득도유의서렬978개,획득단일기인347개,기중2단기인분별여흉막폐염방선균외막단백、전철단백B적동원성위51％화42％,기개방열독광장도분별위1554 bp화726 bp,분별편마517화241개안기산.장2단기인아극륭입원핵표체재체중,IPTG유도표체산물적상대분자질량분별위63 000화30 000.결론 소구건적기열흡수련매균cDNA문고부합구건기인문고적질량요구,문고중적EST수거장유조우농민폐특이성독력분자적진일보사선.
Objective 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.
