Peng Liang, Ou Jing-Yi, Pan Jia-Yun, Deng Cong, Chen Jing-Hong, Cao Hong
Department of Clinical Laboratory, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China.E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2017 Mar 20;37(3):312-316. doi: 10.3969/j.issn.1673-4254.2017.03.06.
To express and purify polyphosphate kinase (PPK) from Proteus mirabilis and prepare the polyclonal antibody against PPK.
The antigenicity and hydrophobicity of PPK were analyzed using software. The N-terminal conservative sequence containing 309 amino acids was selected as the target peptide, and its corresponding gene sequence with modification based on prokaryotic cells-preferred codon was synthesized and inserted into plasmid pET28b(+). The constructed recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and induced with IPTG. The expressed fusion protein was purified using Ni-affinity chromatography. The purified protein was injected along with adjuvant in rabbits to prepare the polyclonal antibodies against PPK.
PPK fusion protein expressed by E. coli was purified successfully using Ni-affinity chromatography. ELISA result demonstrated that the harvested rabbit anti-sera against PPK had a high titer of 1:512 000, and Western blotting showed a good specificity of the antibody, which can be used further study of the role of PPK in the pathogenesis of Proteus mirabilis infection.
表达并纯化奇异变形杆菌的多聚磷酸激酶（PPK），并制备抗PPK的多克隆抗体。
利用软件分析PPK的抗原性和疏水性。选择包含309个氨基酸的N端保守序列作为靶肽，合成其基于原核细胞偏好密码子修饰的相应基因序列，并插入质粒pET28b(+)。将构建的重组质粒转化至大肠杆菌BL21(DE3)，并用异丙基-β-D-硫代半乳糖苷（IPTG）诱导。利用镍亲和层析纯化表达的融合蛋白。将纯化的蛋白与佐剂一起注射到兔子体内，制备抗PPK的多克隆抗体。
利用镍亲和层析成功纯化了大肠杆菌表达的PPK融合蛋白。酶联免疫吸附测定（ELISA）结果表明，收获的抗PPK兔抗血清效价高达1:512 000，蛋白质免疫印迹法（Western blotting）显示该抗体具有良好的特异性，可用于进一步研究PPK在奇异变形杆菌感染发病机制中的作用。
