铜绿假单胞菌LasR蛋白的表达及抗血清制备

The expression of LasR of Pseudomonas aeruginosa and preparation of related rabbit antiserum

目的将铜绿假单胞菌(Pseudomonas aeruginosa,PA)中的调控蛋白LasR在原核系统中进行表达,制备LasR抗血清,为PA中las调控系统的研究奠定基础。方法以PA模式菌株PAO1全基因组为模板,PCR扩增lasR基因,PCR产物用Bam HI/EcoRI双酶切连接至p GEX-4T-1载体,获得重组表达载体p GEX-4T-1-lasR;PCR产物经Nco I/Xho I双酶切连接至p ET28a载体,获得重组表达载体p ET28a-lasR。将重组表达载体分别转化至大肠杆菌感受态细胞BL21中,分别以异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)诱导表达GST-LasR和His-LasR蛋白,通过GST或Ni^(2+)亲和层析纯化重组蛋白。将纯化后的His-LasR蛋白免疫新西兰大白兔,制备LasR抗血清;纯化后的GST-LasR蛋白用于LasR抗血清ELISA效价的检测。结果 lasR基因序列大小为717 bp;成功构建p GEX-4T-1-lasR和p ET28a-lasR表达载体。构建的工程菌能够有效表达目的蛋白His-LasR和GST-LasR,蛋白纯度大于95%;LasR抗血清的效价为1∶60 000。结论在原核系统中成功表达了可溶性LasR重组蛋白,并获得高效价的LasR抗血清,为PA中las调控系统的研究奠定了基础。

Objective This study is aimed to express the protein LasR of Pseudomonas aeruginosa（ P. aeruginosa） in Escherichia coli（ E.coli） and prepare the specific LasR-rabbit antiserum,in order to provide a background in las regulation system in PA. Methods The lasR gene was amplified by PCR from P.aeruginosa PAO1 by using of it＇s whole genome as a templet. The amplified PCR fragment was then digested with Bam HI and EcoRI restriction enzymes and the restriction fragment was sub-cloned into the p GEX-4T-1 vector to form p GEX-4T-1-lasR expression vector. The amplified PCR fragment was then digested with Nco I and Xho I restriction enzymes and the restriction fragment was sub-cloned into the p ET28 a vector to form p ET28a-lasR expression vector. The obtained plasmid p GEX-4T-1-lasR and p ET28a-lasR were transformed into E. coli BL21（ DE3）,respectively. After induction with isopropyl-β-D-thiogalactoside（ IPTG）,the target proteins were purified by GST or Ni^2＋ affinity chromatography. Purified His-LasR protein was used to immunize New Zealand rabbits to prepare LasR antiserum; the purified GST-LasR protein was used in evaluation of the titer of antiserum by ELISA. Results The amplified lasR PCR fragment was 717 bp,the expression vectors p GEX-4T-1-lasR and p ET28a-lasR were successfully constructed,and fusion proteins were efficiently expressed; the purity of the fusion proteins was more than 95%.The rabbit antiserum titer reached to 1 ∶ 60 000. Conclusion The expressed soluble LasR recombinant protein in prokaryote system was successfully obtained,and the rabbit antiserum prepared as well,on which the basis can be provided in further investigation of las regulation system in P. aeruginosa.