Expression, Purification Of Plasmodium Vivax Lactate Dehydrogenase(LDH) And Preparation Of Its Monoclonal Antibodies

In China, Malaria, especially Plasmodium vivax, is the most epidemic and harmful during the parasitic disease. It was estimated that malaria killed 2.7 million people worldwide annually. In recent years, the report shows that in statutory reporting of infectious diseases, the incidence of malaria will always be in the ninth, the mortality rate ranks between twelfth and fourteenth; In all of the natural infectious and arbovirus diseases, the incidence of malaria is 28.25% to 32.62%, always living in the second, only after epidemic hemorrhagic fever. One reason that morbidity and mortality of malaria is difficult to control is short of fast, easy and accurate methods for the diagnosis and treatment in epidemic areas. Diagnosis of malaria using microscopy examination of stained thick and thin smears remains the gold standard and also a most cost-effective method.However, not only it is labor-intensive, requiring a well-functioning, high-quality microscope, but also the interpretation of data demands considerable expertise, particularly at low levels of parasitaemia. Although two new recently developed diagnosis methods, i.e. the QBC and Kawamoto test, are shown to be rapid and accurate, they do need highly trained specialists to operate. Moreover, these two methods also require specialized centrifugation and viewing instruments, and electricity supply, thus making their uses in the field rather difficult. Though PCR-based tests are extremely sensitive, they are limited by the needs for considerable expertise, specialized equipment, and by the high cost of reagents. These limitations, therefore, have prevented the widespread applications of PCR-based methods in the field.The new-generation antigen-capture tests, including ParaSight-F (Becton Dickinson, Cockeysoville, Maryland, USA), ICT Malaria P.f and ICT Malaria P.f/P.v (ICT Diagnostics, Sydney, Australia) and OptiMAL (Flow Inc, Portland Oreg), are capable of detecting fewer parasites and of producing a result more rapidly. They are commercially available as kits, which include all the necessary reagents, and do not require extensive training or equipment to perform or to interpret the results. Moreover, these kits are reported to perform well in the field. Unfortunately, these kits are still too expensive for widespread applications in the developing countries. Patients who suffer from malaria normally live in remote areas and lack the necessary resources to go through the current diagnosis procedure. Hence, it is very obvious that a cheaper, but accurate and rapid diagnosis kit is urgently required.Previous studies have shown that Plasmodium lactate dehydrogenase (LDHp) is significantly different from the host LDH in biochemical, immunological and enzymatic properties. One of the biochemical characteristics that distinguishes LDHp from human LDH is the ability of LDHp to rapidly utilize 3-acetylpyridine NAD(APAD) as a coenzyme in the reaction leading to the formation of pyruvate from lactate. Human erythrocyte LDH could not make use of APAD. This unique feature makes it feasible to differentiate these two types of LDH either by spectrophotmetry or by electrophoresis. In addition, it has been suggested that Plasmodium LDH possess species- and genus-specific antigens, which are considered to be promising candidates for the diagnosis purpose. As LDHp is only produced by live parasites, and a correlation between the level of parasitemia and the activity of parasitic LDH was demonstrated in previous studies, consequently, LDHp activity assay could be developed to specifically detect a particular plasmodium clone, and to monitor the effectiveness of drug therapies.Compared to Histidine-Rich Protein-2 (HRP-2), which is the first application in malaria rapid diagnosis, the specificity of LDH is 100% in the diagnosis. Because pLDH can clear from the blood quickly after treatment. It is to avoid drug abuse for the long-term result of circulating antigen caused by the presence of false positive .Therefore more responsive to the needs of a large-scale epidemic region.In this study, the ultimate goal is to establish immune chromatography assay based LDH as diagnostic antigen, which is able to carry out specific diagnosis of Plasmodium vivax (P.v), at the same time the differential diagnosis of Plasmodium falciparum (P.f). Our laboratory has been successfully prepared P.f monoclonal antibodies and established colloidal Gold-immunochromatography assay (GICA). But at that time, because the parasite didn't live in vitro and P.v gene didn't access yet, it can not be overcome between the Plasmodium vivax antigen problems. In 2004 Brown WM Salvador I first reported the LDH of the partial gene sequences. In 2005 Balik D Turgut reported Belem strain of LDH gene sequence, which has never been reported in our country .Therefore, access to the monoclonal antibody against P.vivax specifically is the key to the establishment of the technology. On the basis of the parasite LDH Salvador I of the partial gene sequence, we successfully cloned the PvLDH gene., we made them expressed in E.coil. By renaturation and chromatography, the fusion protein was purified. Monoclonal antibody was prepared against goal protein by hybridoma technology and identified.We successfully amplified the gene encoding lactate dehydrogenase from P. vivax strain by PCR. The PCR products were purified and cloned into the pGEX-4T-l expression vector. Recombinant plasmids pGEX-LDH were screened and identified by PCR and restriction analysis. The recombinant plasmid pGEX-LDH was induced to express a 56KDa fusion protein in the form of inclusion bodies in E.coli. Compared to dilution refolding and dialysis refolding following dissolve, we got the better refolding method of 20 mmol/L Tris-HCl,1 mmol/L EDTA,1 mmol/L GSSG,0.1 mmol/L GSH,pH8.0. Upon the purification of the combination protein by Glutathione Sepharose 4B affinity chromatograph, the concentration of the purified LDH is 0.5mg/ml. The expressed protein induced the specific humoral responses in mice and the titer of the specific antibody was 1∶51200 by ELISA. Western-blot analysis indicates that there is a strong reaction between the antiserum and the blood of patient with P. vivax; however, there is no reaction between the antiserum and the normal human blood, which suggests that the LDH/GST recombination protein possesses a high antigenicity.BALB/c mice were immunized with purified recombinant LDHpv and McAbs against LDHpv were prepared according to the protocol of hybridoma technique. The McAbs were characterized by ELISA and Western-blot analyses. Five McAbs against LDHpv antigen were obtained, 6D1, 6D2, 5A10, 5C7, 4F8 respectively. The titers of five McAbs were between 1:256 and 1:512 in supernatant.These results suggest that, we first prepared monoclonal antibodies which is against LDH of P. vivax in our country. This lays foundation for developing more superior, practical diagnostic kits of malaria.