致农民肺嗜热吸水链霉菌cDNA文库的构建及体外表达

Construction of cDNA library and antigen expression of Streptomyces Thermohydroscopicus in vitro

目的构建致农民肺嗜热吸水链霉菌cDNA文库并进行EST测序,通过生物信息学软件分析EST序列同源性及生物学功能,筛选重组阳性基因克隆进行体外表达。方法利用SMART方法构建致农民肺嗜热吸水链霉菌的cDNA文库,随机挑选重组阳性克隆进行EST测序。挑选有关目的基因连接入pET-28a载体并转化大肠埃希菌BL2(DE3),IPTG诱导体外表达。结果该文库的重组率为96.3%,是一个较高质量的文库。从文库中随即挑选的1 020个重组阳性的克隆,获得978个有义序列,平均长度为495bp,含有347个单一基因,同源性比较发现部分序列与猪胸膜肺炎放线菌外膜蛋白(omla),转铁蛋白B(TbpB)具有较高的同源性,分别为51%和42%。将重组阳性的基因克隆至载体中,IPTG诱导得到表达产物的相对分子质量分别为63×103和30×103。结论所构建的致农民肺嗜热吸水链霉菌cDNA文库包含全部EST序列,可能包含嗜热吸水链霉菌相关疾病巨大部分毒力分子,这些数据的积累将有助于农民肺特异性毒力分子的进一步筛选,同时为研究农民肺的发病机制奠定基础,如果能准确筛选出相关毒力基因,将为农民肺血清学诊断以及特异性疫苗的研发奠定基础。

Objective To construct the cDNA library of Streptomyces Thermohydroscopicus and to express some of the specific antigens in vitro. Methods The cDNA library of Streptomyces Thermohydroscopicus was constructed by switching mechanism at 5＇end of RNA transcript approach. The recombinant positive clones were selected randomly for EST sequencing and two candidate genes were sub-cloned into expression vector pET-28a. The recombinants were then transformed into BL2 （DE3） and proteins were expressed by the induction of IPTG. Results A high-quality cDNA library of Streptomyces Thermohydroscopicus was constructed with the recombination rate of 96.3%. A set of 978 valid sequences were obtained out of total 1020 positive clones with the average length of 495 bp which contained 347 unique genes. The homology analysis revealed that 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein （51%） and transferring-bind- ing protein 13 （42 %） from Actinobacillus pleuropneumoniae serotype （APP） . The molecular weights of the two expression products were 63kDa and 30kDa. Conclusions The cDNA library of Streptomyces Thermohydroscopicus harbors all EST sequences and most likely contains the majority of the virulence genes. It not only provides the basis for elucidating the etiology of farm＇s lung disease of serological diagnosis and vaccine for farm＇s lung disease.