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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。
OBJECTIVE: To construct the anti-angiospray neovascularis antibody library from mouse splenocytes infected with A. cantonensis and to screen the antibodies that can be used for the early diagnosis of angiostrongylus cantonensis. Methods The RNA of splenocytes infected by C. elegans was extracted and reverse transcribed into cDNA first strand. The light and heavy chain genes were amplified by PCR. The light and heavy chain genes were ligated into the expression vector pComb3 and transformed into E. coli XLI-Blue to construct a combinatorial library. The Estrogen receptor of Escherichia coli was used as screening antigen, and the positive clones were identified by ELISA. Results The mouse anti-Anemia gondii phage antibody library was constructed successfully with a capacity of 2.32 × 106 and a titer of 1.7 × 1013 cfu / ml. A total of 5 strains of anti-Eumycotina antibodies were screened against the C. elegans, and identified by peroxidase-labeled anti-M13 antibody. Conclusion The construction of anti-Anemia gondii phage antibody library has met the requirements, and laid the foundation for the development of the GAS diagnostic kit.