抗广州管圆线虫噬菌体抗体库的构建及初步筛选

目的以广州管圆线虫感染的小鼠脾细胞为源,构建抗广州管圆线虫抗体库,并筛选可用于广州管圆线虫病早期诊断的目的抗体。方法提取感染广州管圆线虫的小鼠脾细胞RNA,并逆转录合成cDNA第一链,PCR扩增出抗体轻链和重链基因,将轻、重链基因先后连接到表达载体pComb3上,转化大肠杆菌XLI-Blue,构建组合文库。用广州管圆线虫排泄分泌抗原（EsAg）作为筛选抗原,进行富集筛选,用ELISA法鉴定阳性克隆。结果成功构建了小鼠抗广州管圆线虫噬菌体抗体文库,库容为2.32×106,滴度为1.7×1013cfu/ml。筛选到了抗广州管圆线虫排泄分泌抗原特异性抗体克隆5株,用过氧化物酶标记的抗M13抗体进行初步鉴定,具有一定的特异性。结论构建的抗广州管圆线虫噬菌体抗体库达到了要求,为研制广州管圆线虫金标诊断试剂盒奠定了基础。

Construction and preliminary screening of phage display antibody library against Angiostrongylus cantonesis

Objective To construct a phage display antibody library from the spleen cells of BALB /c mice infected with Angiostrongylus cantonesis.These phage Fab antibodies will provide very useful immunological reagents for detecting Angiostrongylus cantonesis infection.Methods Total RNA was extracted using Trizol method.Random Primers were used to reverse transcribe first-strand cDNA,and the κ light chain and the Fd regions of heavy chain products were amplified from the cDNA template by polymerase chain reaction（PCR） with specific primers.After digestion with XhoⅠ＋ SpeⅠand SacⅠ＋ XbaⅠ,the amplified Fd and light chain fragments were cloned into phagemid pComb3 and electrotransinfected into competent E.coli XL1-blue cells.Results A phage display library against Angiostrongylus cantonesis was constructed,with about a size of 2.32×10 6,titer of 1.7×10 13 cfu /ml.After four times combining-eluting-amplifying,the phage antibodies were screened and enriched.The positive phage antibodies were identified by ELISA.Conclusion A phage-displayed antibody library against Angiostrongylus cantonesis was successfully constructed.