抗铜绿假单胞菌PcrV的scFv-Fc抗体的制备及体外活性研究

Preparation and in vitro functions study of scFv-Fc antibodies targeting Pseudomonas aeruginosa PcrV

目的制备靶向铜绿假单胞菌(Pseudomonas aeruginosa)III型分泌系统组件蛋白PcrV的scFv-Fc形式的单克隆抗体MFa,并研究其体外对铜绿假单胞菌生物膜形成及细胞黏附和侵入的影响。方法将编码MFa的DNA序列合成后,插入到真核表达载体中,获得重组质粒,再采用聚乙烯亚胺(PEI)将质粒转染HEK293E细胞并获得瞬时表达;表达的产物通过HiTrap MabSelect SuRe柱进行纯化,所得到的抗体采用SDS-PAGE分析纯度,采用BCA法测定蛋白浓度。为了检测MFa抗体与PcrV抗原的亲和力,对PcrV抗原进行表达纯化,方法是将PcrV目的序列插入到pET28a表达载体中,转化E.coli BL21(DE3)获得宿主菌,在0.5mmol/L IPTG 15℃的条件下诱导过夜,并通过His-Tag标签进行亲和纯化。对于纯化的MFa抗体与PcrV抗原通过Western blot、ELISA、SPR(表面等离子共振)研究抗体与抗原的亲和力,并研究抗体体外对生物膜形成的影响,对铜绿假单胞菌黏附与侵入HeLa细胞的影响。结果MFa抗体在HEK293瞬转系统成功获得表达与纯化,且对PcrV蛋白具有较好的亲和活性,KD值为492.2nmol/L。功能实验证实,MFa抗体能够降低铜绿假单胞菌生物膜的形成,降低铜绿假单胞菌对HeLa细胞黏附与侵入作用。结论靶向PcrV的scFv-Fc抗体MFa具有对铜绿假单胞菌引起的感染的潜在效用,具有进一步开发为抗铜绿假单胞菌治疗药物的价值。

Objective To prepare monoclonal antibody MFa against PcrV antigen and to study the in vitro activities of MFa against Pseudomonas aeruginosa via inhibition of biofilm formation,cell adhesion and invasion.Methods The DNA fragment encoding MFa was synthesized and inserted into the eukaryotic expression vector to obtain recombinant plasmids,which were then transfected into HEK293E cells by PEI to express MFa antibody.The expressed product was purified by HiTrap MabSelect SuRe column.The purity of the purified MFa was analyzed by SDS-PAGE,and the protein concentration was determined by the BCA method.In order to detect the affinity between MFa antibody and PcrV antigen,PcrV antigen was expressed and purified by inserting the target sequence of PcrV into pET28a expression vector and transforming E.coli BL21(DE3)to obtain the host bacteria.The host bacteria was induced overnight by 0.5mmol/L IPTG at 15℃,followed by purification with Ni-NTA column.Western blot,ELISA and SPR(surface plasma resonance)were used to study the affinity between the purified MFa antibody and PcrV antigen,and to study the effect of the antibody on the formation of biofilm in vitro,as well as on Pseudomonas aeruginosa adhesion and invasion of HeLa cells.Results MFa antibody was successfully expressed and purified in HEK293 transient expression system.MFa antibody showed good affinity to PcrV with a KD value of 492.2nmol/L.It could significantly reduce the formation of Pseudomonas aeruginosa biofilm formation,the adhesion and invasion of HeLa cells.Conclusion The PcrV targeting scFv-Fc antibody MFa has potential effects on the infections caused by Pseudomonas aeruginosa,and is promising to be developed as a novel therapeutic drug against Pseudomonas aeruginosa.