Wang Lu-Lu, Lu Shi-Ying, Hu Pan, Fu Bao-Quan, Li Yan-Song, Zhai Fei-Fei, Ju Dan-Di, Zhang Shi-Jun, Su Bing, Zhou Yu, Liu Zeng-Shan, Ren Hong-Lin
Key Laboratory of Zoonosis Research, Institute of Zoonosis/College of Veterinary Medicine, Jilin University, Xi An Da Lu 5333, Changchun 130062, China.
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Public Health of the Ministry of Agriculture, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
J Vet Res. 2019 Mar 22;63(1):99-105. doi: 10.2478/jvetres-2019-0004. eCollection 2019 Mar.
Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase activity and phospholipase A2 activity. Previous studies have shown a significant positive correlation between the intracellular survival ability of and Prdx6. Here, the Prdx6 enzyme with a single activity was constructed to facilitate study of the relationship between the single function of Prdx6 and infection.
The target open reading frame (ORF) DNAs of Prdx6 with a single active centre were prepared using gene splicing by overlap extension PCR (SOE-PCR), and the recombinant eukaryotic expression plasmids inserted by Prdx6 with the single activity centre were constructed and transfected into murine Raw264.7 macrophages. The glutathione peroxidase activity and phospholipase A2 activity of the constructed Prdx6 were examined.
The core centres (Ser and Cys) of Prdx6 were successfully mutated by changing the 94 nucleotide from T to G and the 140 nucleotide from G to C in the two enzyme activity cores, respectively. The constructed recombinant plasmids of Prdx6 with the single active centre were transfected into murine macrophages showing the expected single functional enzyme activity, which MJ33 or mercaptosuccinate inhibitors were able to inhibit.
The constructed mutants of Prdx6 with the single activity cores will be a benefit to further study of the biological function of Prdx6 with different enzyme activity.
过氧化物酶6（Prdx6）是一种具有谷胱甘肽过氧化物酶活性和磷脂酶A2活性的双功能蛋白。先前的研究表明，[某种物质]的细胞内存活能力与Prdx6之间存在显著的正相关。在此，构建了具有单一活性的Prdx6酶，以促进对Prdx6单一功能与[某种物质]感染之间关系的研究。
使用重叠延伸PCR（SOE-PCR）基因剪接制备具有单一活性中心的Prdx6的目标开放阅读框（ORF）DNA，并构建具有单一活性中心的Prdx6插入的重组真核表达质粒，然后将其转染到小鼠Raw264.7巨噬细胞中。检测构建的Prdx6的谷胱甘肽过氧化物酶活性和磷脂酶A2活性。
通过分别将两个酶活性核心中的第94个核苷酸从T改变为G以及第140个核苷酸从G改变为C，成功地对Prdx6的核心中心（丝氨酸和半胱氨酸）进行了突变。将构建的具有单一活性中心的Prdx6重组质粒转染到小鼠巨噬细胞中，显示出预期的单一功能酶活性，而MJ33或巯基琥珀酸抑制剂能够抑制该活性。
构建的具有单一活性核心的Prdx6突变体将有助于进一步研究具有不同酶活性的Prdx6的生物学功能。
