抗恙虫病东方体噬菌体抗体库的构建和初步筛选

Construction and Preliminary Screening of Phage-Displayed Antibody Library against Orientia tsutsugamushi

目的构建抗恙虫病东方体噬菌体抗体库并进行初步筛选,获得与恙虫病东方体膜抗原特异性结合的抗体克隆,为研制恙虫病的快速诊断试剂盒奠定基础。方法从感染恙虫病东方体小鼠的脾细胞中提取RNA,经逆转录、PCR扩增出抗体轻链和重链基因,将轻链基因和表达载体pComb3进行酶切、连接,转化大肠杆菌XL1-blue,构建轻链库;再对轻链重组质粒和重链Fd基因进行酶切、连接,转化大肠杆菌XL1-blue,构建组合文库,以辅助噬菌体VCSM13进行超感染。用恙虫病东方体56kDa型特异性抗原作为筛选抗原,进行4轮富集筛选后用ELISA法进行阳性克隆的鉴定。结果构建了库容为3.46×106的鼠源性抗恙虫病东方体的噬菌体Fab片段抗体库。经过4轮富集筛选,抗体库中的目的抗体得以富集约160倍,并用ELISA法对其进行鉴定,得到了7个阳性克隆。结论构建的抗恙虫病东方体噬菌体抗体库,库容为3.46×106,滴度为2.5×1012cfu/ml,基本上达到了建库要求,能够满足多样性的需求,并成功的筛选出抗恙虫病东方体56kDa蛋白的抗体,用过氧化物酶标记的抗M13抗体进行了初步鉴定,认为具有一定的特异性。

Objective For screening effective antibody to diagnosis Orientia tsutsugamushi infection,we constructed the phage-displayed antibody library against O. tsutsugamushi. Methods Total RNA extracted from spleen cell of BALB/c mice infected with O. tsutsugamushi karp strain was reverse transcribed, and immunoglobulin heavy chain Fd genes and light chain were amplified with polymerase chain reaction by the designed primers. After digestion with Xho I ＋ Spe I and Sac I ＋ Xba I, the amplified Fd and light chain fragments were cloned into phagemid pComb3 and transformed into E. coli XLl-blue. The phage antibodies were displayed on the surface of recombinant phage after rescue by helper phage VCSM 13. Results Fab phage library containing 2.5×10^12 clone against Orientia tsutsugamushi was constructed by using phage surface display technique. After four times of combining-eluting-amplifying, the phage antibodies were screened and enriched. The positive phage antibodies were identified by ELISA. Conclusion A phage display library against Orientia tsutsugamushi was successfully constructed.