Singh Vandana, Kaushal Deep C, Rathaur Sushma, Kumar Niraj, Kaushal Nuzhat A
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226001, India.
Protein Expr Purif. 2012 Aug;84(2):195-203. doi: 10.1016/j.pep.2012.05.008. Epub 2012 Jun 7.
Plasmodial lactate dehydrogenase, key enzyme of anaerobic glycolysis, has been shown to be a potential immunodiagnostic marker as well as a novel target for chemotherapy. We have cloned, overexpressed and immunochemically characterized the recombinant lactate dehydrogenase of Plasmodium knowlesi, the fifth human malaria parasite. The P. knowlesi lactate dehydrogenase (PkLDH) gene was PCR amplified and 0.9 kb PCR product was cloned into pGEM-T Easy vector. Sequencing and BLAST analysis revealed open reading frame of 316 amino acids of PkLDH showing 96.8% homology with Plasmodium vivax LDH and around 90% with Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale LDHs. The PkLDH gene was subcloned into pGEX-6P1 expression vector and the SDS-PAGE analysis revealed that about 70% of fusion protein was present in the soluble fraction. The fusion protein was cleaved with PreScission protease and recombinant PkLDH (34 kDa) was affinity purified to homogeneity. The purified PkLDH exhibited high reactivity with polyclonal and monoclonal antibodies against plasmodial LDH. The polyclonal antibody produced against purified recombinant PkLDH in rabbits showed high ELISA reactivity with both native and recombinant PkLDH and could detect parasite LDH in malaria infected blood samples by sandwich ELISA. The purified recombinant PkLDH can be used to produce P. knowlesi specific monoclonal antibodies for specific diagnosis of P. knowlesi infection in humans.
疟原虫乳酸脱氢酶是无氧糖酵解的关键酶，已被证明是一种潜在的免疫诊断标志物以及化疗的新靶点。我们已经克隆、过表达并对诺氏疟原虫（第五种人类疟原虫）的重组乳酸脱氢酶进行了免疫化学特性分析。通过PCR扩增诺氏疟原虫乳酸脱氢酶（PkLDH）基因，并将0.9 kb的PCR产物克隆到pGEM-T Easy载体中。测序和BLAST分析显示，PkLDH的开放阅读框为316个氨基酸，与间日疟原虫乳酸脱氢酶的同源性为96.8%，与恶性疟原虫、三日疟原虫和卵形疟原虫乳酸脱氢酶的同源性约为90%。将PkLDH基因亚克隆到pGEX-6P1表达载体中，SDS-PAGE分析表明约70%的融合蛋白存在于可溶部分。用PreScission蛋白酶切割融合蛋白，亲和纯化重组PkLDH（34 kDa）至均一性。纯化的PkLDH与抗疟原虫乳酸脱氢酶的多克隆抗体和单克隆抗体表现出高反应性。在兔体内针对纯化的重组PkLDH产生的多克隆抗体与天然和重组PkLDH均表现出高ELISA反应性，并且可以通过夹心ELISA检测疟疾感染血样中的寄生虫乳酸脱氢酶。纯化的重组PkLDH可用于生产诺氏疟原虫特异性单克隆抗体，用于人类诺氏疟原虫感染的特异性诊断。
