抗铜绿假单胞菌PcrV蛋白单克隆抗体的筛选和功能验证

摘要

Objective:To obtain a high specificity and high affinity anti-PcrV protein monoclonal antibody which can be used for the treatment of Pseudomonas aeruginosa infected.Methods: The PcrV gene was amplified by PCR using P.aeruginosa PAO1 genome DNA as the template.The expression vector(pET-28a-PcrV) was constructed and transformed into E.coli BL21(DE3).The re-combinant PcrV protein was expressed by IPTG induction and purified by Ni2+affinity chromatography.The specific binders of PcrV were screened by phage display.The genes encoding VH and VL were amplified respectively by PCR using the plasmid of positive clone as the template.Then the recombinant expression vectors were constructed and transfected into 293E cells.Monoclonal antibody were purified by the Protein A affinity resin from the culture supernatants.The affinity of antibody was detected by ELISA and the function of YG5 was verified in murine pneumonia model caused by P.aeruginosa.Results: Recombinant PcrV protein was expressed and purified.A full human monoclonal antibody(named as YG5) against PcrV was obtained by phage display.The results of ELISA showed that YG5 had a high affinity with EC50=61 ng/ml.Furthermore,it was found that YG5 could protect mice from infection caused by P.aeruginosa.Conclusion:Our findings present a novel human monoclonal antibody YG5 against PcrV,which inhibits the infection casued by P.aeruginosa and may be a potential drug for treatment of P.aeruginosa infection.%目的:获得能应用于治疗铜绿假单胞菌(Pseudomonas aeruginosa)感染的抗PcrV蛋白全人源单克隆抗体.方法:以铜绿假单胞菌PAO1基因组为模板,PCR 扩增 PcrV 基因并构建重组表达载体 pET-28a-PcrV,转化至大肠杆菌 BL21 (DE3),利用IPTG诱导表达,并通过镍离子螯合树脂亲和纯化PcrV蛋白.利用噬菌体抗体库筛选PcrV结合的噬菌体克隆并进行序列测定,以测序正确的阳性克隆质粒为模板,PCR扩增该抗体的重链可变区基因和轻链可变区基因并构建重组表达载体转染至293E细胞,收取培养细胞上清,并利用Protein A树脂亲和纯化抗体.利用ELISA实验检测抗体亲和力,并通过小鼠铜绿假单胞菌感染肺炎模型验证抗体活性.结果:获得重组蛋白PcrV,通过噬菌体展示技术筛选并制备了一株全人源抗PcrV单克隆抗体YG5.ELISA实验结果表明YG5抗体具有较高的亲和力(EC50=61 ng/ml),进一步的实验结果表明,YG5能有效抵抗铜绿假单胞菌感染,降低小鼠的死亡率.结论:靶向铜绿假单胞菌PcrV蛋白的全人源单克隆抗体YG5能够有效地抑制铜绿假单胞菌致病性,降低其对机体感染,有可能成为抗铜绿假单胞菌感染的治疗性抗体.