Ma Xian-Liang, Liu Qin, Zhang Yi
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Key Laboratory of Parasite and Vector Biology, MOH, WHO Collaborating Centre of Malaria, Schistosomiasis and Filarasis, Shanghai 200025, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2012 Aug 30;30(4):262-7.
To clone and express full-length thioredoxin peroxidase (TPx) gene of Oncomelania hupensis and study on the peroxidase activity of the recombinant protein.
Total RNA was obtained from the cultivated O. hupensis and a cDNA sequence of the TPx gene was cloned by RT-PCR. The TPx cDNA ends were amplified by the SMARTer RACE cDNA Amplification Kit. After sequencing, blasting and matching, the full-length cDNA of the TPx gene was obtained. The TPx cDNA was ligated with the pGEM-Teasy and transformed into E. coli DH5alpha. After sequencing and blasting, the characteristics of biological information of the TPx gene was analyzed. The positive recombinants with pGEM-Teasy/TPx and expression vector pET-28a were digested by the double restriction enzymes, ligated each other, transformed into E. coli BL21 (DE3), and induced by IPTG for expression. The recombinant TPx was expressed as a histidine fusion protein and was purified with Ni chromatography and NTA cation exchange chromatography. The expressed and purified TPx was analyzed by SDS-PAGE. The different concentrations of TPx recombinant protein (10, 20, 30, 40, and 50 microg/ml) were added into hydrogen peroxide (H2O2) reduction test in vitro to calculate the clearance rate of H2O2, each concentration with parallel control of dithiothreitol sugar alcohol (DTT). In the protection test of super-coiled DNA, the TPx protein was added with a concentration of 2.5, 5.0, and 10 microg/ml, respectively, to observe the protective effect of super-coiled DNA in metal-catalyzed oxidation (MCO).
The complete cDNA encoding TPx was 992 bp. ORF was 747 bp with GenBank accession number of JN831437. The ORF encoded 249 amino acids, and the relative molecular weight (M(r)) of predicted protein was 27 000. The recombinant plasmid pET28a/TPx was built, and the soluble recombinant protein was obtained by induction and purification. The results of SDS-PAGE showed that the M(r) was 27 000. H2O2 reduction test in vitro showed that the H2O2 clearance rate of reaction system containing DTT was significantly higher than the clearance rate without DTT (P < 0.05), the difference among various concentrations was not statistically significant (P > 0.05). The protection of super-coiled DNA showed that the protective effect of 5.0 microg/ml group was better than 2.5 microg/ml group, but there was no difference between the protective effect of 5.0 microg/ml group and 10 microg/ml group.
The full-length cDNA of the TPx gene of O. hupensis is obtained, and the recombinant TPx protein shows a certain antioxidant activity.
克隆并表达日本血吸虫硫氧还蛋白过氧化物酶（TPx）全长基因，研究重组蛋白的过氧化物酶活性。
从培养的日本血吸虫中提取总RNA，采用RT-PCR克隆TPx基因的cDNA序列。利用SMARTer RACE cDNA扩增试剂盒扩增TPx cDNA末端。经测序、比对和匹配，获得TPx基因的全长cDNA。将TPx cDNA与pGEM-Teasy连接并转化至大肠杆菌DH5α。经测序和比对后，分析TPx基因的生物信息学特征。用双酶切pGEM-Teasy/TPx阳性重组体和表达载体pET-28a，相互连接后转化至大肠杆菌BL21（DE3），用IPTG诱导表达。重组TPx表达为组氨酸融合蛋白，通过镍柱层析和NTA阳离子交换层析进行纯化。用SDS-PAGE分析表达并纯化的TPx。在体外过氧化氢（H2O2）还原试验中加入不同浓度的TPx重组蛋白（10、20、30、40和50μg/ml），计算H2O2清除率，各浓度均设二硫苏糖醇糖醇（DTT）平行对照。在超螺旋DNA保护试验中，分别加入浓度为2.5、5.0和10μg/ml的TPx蛋白，观察其在金属催化氧化（MCO）中对超螺旋DNA的保护作用。
编码TPx的完整cDNA为992 bp。开放阅读框（ORF）为747 bp，GenBank登录号为JN831437。该ORF编码249个氨基酸，预测蛋白的相对分子质量（M(r)）为27 000。构建了重组质粒pET28a/TPx，经诱导和纯化获得可溶性重组蛋白。SDS-PAGE结果显示M(r)为27 000。体外H2O2还原试验表明，含DTT的反应体系H2O2清除率显著高于不含DTT的清除率（P < 0.05），各浓度间差异无统计学意义（P > 0.05）。超螺旋DNA保护试验表明，5.0μg/ml组的保护作用优于2.5μg/ml组，但5.0μg/ml组与10μg/ml组的保护作用无差异。
获得日本血吸虫TPx基因的全长cDNA，重组TPx蛋白具有一定的抗氧化活性。
