铜绿假单胞菌黏附素蛋白的表达、纯化及多克隆抗体制备

Expression,purification and polyclonal antibody preparation of adhesin protein from Pseudomonas aeruginosa

[目的]克隆铜绿假单胞菌(Pseudomonasaeruginosa)黏附素(adhesin)基因,表达纯化黏附素蛋白和获得多克隆抗体。[方法]设计特异性PCR引物,以铜绿假单胞菌DNA为模板,PCR法扩增铜绿假单胞菌黏附素基因,并将其进行双酶切后连接到表达载体pET28a,将重组质粒pET28a-adhesin转人大肠杆菌BL21,IPTG诱导目的蛋白表达,然后通过亲和层析进行纯化。再利用各种生物信息学软件分析该蛋白的生物学特性。将adhesin蛋白免疫小鼠,并获得了高浓度的多克隆抗体。[结果]显示成功得到了重组质粒pET28a-adhesin,并通过亲和层析得到重组adhesin蛋白。Ad-hesin的a-螺旋含量为46.37%,无规卷曲的含量为35.96%,β片层的含量为17.67%。模拟得到了adhesin蛋白的三级结构。该蛋白是一个跨膜蛋白,具有两个跨膜区。Adhesin蛋白具有良好的免疫原性,获得的抗血清效价高于1:5120。[结论]纯化得到了重组adhesin及其抗血清,为研究该蛋白的功能提供了实验基础。

[Objective]To clone adhesin gene from Pseudomonas aeruginosa and express and purify adhesion molecules.[Method]A specific PCR primer was designed to amplify the adhesin gene of P.aeruginosa using the DNA of P.aeruginosa as the template.After double enzyme digestion,the adhesin gene was connected to the expression vector pET28a.After verification,pET28a-adhesin was transferred into Escherichia coli and the target protein was induced by IPTG which was purified by affini-ty chromatography.Then use various bioinformatics software to analyze the biological characteristics of the protein.The mice were immunized with adhesin protein to gain polyclonal antibody.[Result]The recombinant plasmid pET28a-adhesin was successfully obtained,and the purified recombinant adhesin protein was obtained.Adhesin has an alpha-helix content of 46.37%,a random coil content of 35.96%,and a beta sheet content of 17.67%.And the three-level structure of adhesin protein was obtained by simulation.The protein is a transmembrane protein with two transmembrane regions and has good immunogenic-ity.The titer of antiserum was higher an 1:5120.[Conclusion]In this experiment,recombinant adhesin protein and higher titer of polyclonal antibody were purified which provided experimental evidence for studying the function of the protein.