铜绿假单胞菌重组Ef-LasR疫苗的构建、鉴定及表达  

Construction,identification,and expression of a recombinant Ef-LasR vaccine against Pseudomonas aeruginosa

作　　者：欧兴坤 李文桂[1] OU Xing-kun;LI Wen-gui(Institute of Infections and Parasitic Diseases,the First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,CAina)

机构地区：[1]重庆医科大学附属第一医院传染病寄生虫病研究所,重庆400016

出　　处：《中国病原生物学杂志》2021年第6期630-633,638,共5页Journal of Pathogen Biology

基　　金：重庆市科委地方病重大专项基金项目(No.2008AB5055,2008AB5008,2008AB5054)。

摘　　要：目的构建以粪肠球菌为载体的铜绿假单胞菌重组Ef-LasR疫苗,并研究其表达效率。方法采用PCR扩增铜绿假单胞菌标准株PA01的LasR基因,然后克隆至pGEX-1λT载体,构建重组质粒pGEX-LasR。将重组质粒电穿孔转化感受态粪肠球菌构建重组Ef-LasR疫苗,抽提质粒进行PCR和双酶切鉴定。重组疫苗用IPTG诱导表达,采用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果PCR扩增出717 bp的LasR基因;双酶切和PCR证实重组疫苗(rEf-LasR)构建正确;SDS-PAGE显示rEf-LasR能够高效表达分子质量单位为53 ku的目的蛋白,Western blot显示该蛋白能被铜绿假单胞菌感染的鼠血清识别。结论成功构建了重组Ef-LasR疫苗,其表达产物具有反应原性,为铜绿假单胞菌疫苗的进一步研究奠定了基础。Objective To construct a recombinant vaccine against Pseudomonas aeruginosa based on an Enterococcus faecalis vector and to observe its expression efficiency.Methods The LasR gene of P.aeruginosa PA01 was amplified with PCR and cloned into pGEX-1λT to obtain the recombinant plasmid pGEX-LasR.The recombinant plasmid was electroporated into E.faecalis to construct a recombinant vaccine,which was designated rEf-LasR,and then the plasmid was extracted for PCR and enzyme digestion.Expression of rEf-LasR was induced with 1 mM IPTG when the culture reached an OD600 of 0.6-0.8.The recombinant protein was verified using SDS-PAGE and Western blotting.Results The expected full-length lasR fragment(717 bp)was amplified with PCR,and the recombinant pGEX-LasR was successfully constructed and transformed into E.faecalis.After digestion and PCR,pGEX-LasR contained a pGEX-1λT vector fragment 4,947 bp in length and the lasR gene 717 bp in length.SDS-PAGE and Western blotting indicated that the recombinant protein with a molecular weight of about 53 ku was effectively expressed and recognized by the sera of mice infected with P.aeruginosa.The amount of recombinant protein produced was higher 5 h after expression was induced with IPTG,and the rate of expression was about 21%.Conclusion A rEf-LasR vaccine against P.aeruginosa was successfully constructed,and the fusion protein was immunoreactive.

关 键 词：铜绿假单胞菌 Ef-LasR 疫苗 

分 类 号：R378.991[医药卫生—病原生物学]