牛肺炎链球菌重组PsaA蛋白对牛胚气管上皮细胞黏附抑制作用的研究

Inhibitory effect of recombinant PsaA protein of Streptococcus pneumoniae on adhesion of bovine embryonic trachea epithelial cells

为研究牛肺炎链球菌的黏附蛋白—肺炎链球菌表面黏附素A (PsaA)对牛肺炎链球菌黏附牛胚气管上皮细胞的影响,本研究利用PCR扩增牛肺炎链球菌psaA基因,构建重组质粒pET32a-PsaA,经双酶切和测序鉴定正确后,将阳性重组质粒转化BL21(DE3)感受态细胞后进行IPTG诱导表达,采用SDS-PAGE和western blot鉴定重组PsaA蛋白(rPsaA)的表达情况及反应原性,结果显示,rPsaA在上清和沉淀中均获得了表达,且具有较好的反应原性。将牛胚气管上皮细胞与1.3×10^(8)cfu/mL的牛肺炎链球菌稀释液于37℃共孵育后,利用间接免疫荧光试验(IFA)检测牛肺炎链球菌对牛胚气管上皮细胞的黏附性,结果显示,可观察到黏附在牛胚气管上皮细胞的牛肺炎链球菌的绿色荧光,即牛肺炎链球菌可以黏附牛胚气管上皮细胞。利用牛肺炎链球菌稀释液孵育经不同浓度rPsaA预处理的牛胚气管上皮细胞后,对黏附的细菌计数,结果显示,rPsaA可以极显著减少牛肺炎链球菌对牛胚气管上皮细胞的黏附菌数(P<0.01)。利用本研究制备的rPsaA多克隆抗体和肺炎链球菌阳性血清分别与牛肺炎链球菌作用后,与牛胚气管上皮细胞孵育,对黏附的细菌计数,结果显示,rPsaA多克隆抗体与肺炎链球菌阳性血清均能有效减少牛肺炎链球菌对牛胚气管上皮细胞的黏附数。本研究经原核系统表达了牛肺炎链球菌rPsaA,并首次证明rPsaA具有影响牛肺炎链球菌黏附牛胚气管上皮细胞的作用,该结果为探究抑制肺炎链球菌黏附呼吸道上皮细胞的机理以及肺炎链球菌亚单位疫苗的研究奠定了基础。

In order to study the inhibitory effect of the adhesion protein of Streptococcus pneumoniae,pneumococcal surface adhesin A(PsaA),and anti-rPsaA serum on the adhesion to bovine embryonic tracheal epithelial cells,this study amplified psaA gene of S.pneumoniae by PCR.The recombinant plasmid pET32a-PsaA was constructed for prokaryotic expression.The recombinant plasmid identified as positive by double digestion was transformed into BL21(DE3) competent cells and expressed by IPTG induction.SDS-PAGE and western blot determined the solubility and antigenicity of the recombinant protein.The results showed that the recombinant PsaA protein(rPsaA) was expressed in soluble form and showed good antigenicity.The adhesion of S.pneumoniae to bovine duct epithelial cells was detected by indirect immunofluorescence assay after bovine respiratory tract epithelial cells were incubated with 1.3×10^(8)cfu/m L S.pneumoniae dilution.The results showed that S.pneumoniae could adhere to bovine embryonic trachea epithelial cells.Bovine embryonic tracheal epithelial cells treated with different concentrations of rPsaA proteins were incubated with S.pneumoniae diluted solution,and the number of adhesion bacteria was statistically analyzed.The results showed that the rPsaA protein could significantly inhibit the adhesion of S.pneumoniae to bovine embryonic tracheal epithelial cells.The bovine embryonic tracheal epithelial cells were infected with S.pneumoniae that pre-incubated with the anti-rPsaA polyclonal antibodies or anti-S.pneumoniae serum,respectively.Results showed that anti-rPsaA serum and antiS.pneumoniae serum could effectively inhibit the adhesion of S.pneumoniae to bovine embryonic tracheal epithelial cells.In this study,the rPsaA protein of S.pneumoniae bovine was successfully expressed.For the first time,the rPsaA protein of S.pneumoniae and anti-rPsaA protein serum were confirmed to inhibit the adhesion of S.pneumoniae to the trachea epithelial cells from the bovine embryo.This study laid foundation for exploring the mechanism of inhibiting the adhesion of S.pneumoniae to respiratory epithelial cells and the clinical application of S.pneumoniae subunit vaccine.