| Malaria is a severe and harmful parasitic disease which broadly spreads in tropical and sub-tropical regions of the world 0 The statistical results by WHO indicate that about 300-500 million people suffer from malaria each year among which 3 million patients die of this disease, Malaria also occurs in most parts of China, of which the incidence of this disease in Southern China such as Yunnan, Guangxi and Hainan has been increasing0 Hence, Diagnosis and treatment of malaria has become one of the most critical roles in parasitic disease researcho The current diagnostic technique of malaria is proceeding to the aspect which is simple, rapid, sensitive specific and easy to be determined The Dipstick immunoaffinity chromatography, a technique which is based on the monoclonal antibody, possesses the advantages above and is a promising and effective way for the diagnosis of malaria. The rationale of this study is to utilize the plasmodium LDH( pLDH) as the targeting antigen to prepare the monoclonal antibodies, and to develop a diagnosis kit, which recognizes both falciparum malaria and tertian malaria using the exclusive method. The method is fast, affordable and effective, which meets the requirement for the malaria diagnosis that the tertian malaria is the dominant type in China .In our present study, we clone LDHpf cDNA into PET-23a(+) expression vector, obtaining PET23-LDH recombination construct which is highly expressed in BL21 bacteria. The molecular weight of this protein is about 33kDa, which is consistent with the reported 31-36kDa of this protein in plasmodium . The expression product is dissolved in the supernatant after-4-the bacteria is lysed by ultrasonic, and is 41.2 percent of the total bacteria proteins o Upon the purification of the combination protein by Q-Sepharose High Performance negative ion-exchange chromatography, the purity is about 89.1 percent measured by SDS-PAGE and gel image analysis system ?And the concentration is 400ng/ml0 We further immunize mouse using the recombination protein to prepare the antiserumo ELASA result shows a very strong reaction between the antiserum and the pET23-LHD expression product hi contrast, there is no reaction between the antiserum and the expression product from the pET-23 empty vector control o Western blot analysis indicates that there exists a 33kDa specific band in pET23-LDH containing bacteria lysis, however, there is no positive band in pET-23a vector containing bacteria 0 Polyclonal antibodies thus prepared could specifically recognize a 33kDa protein from P.falciparum and P.vivax as revealed by Western blot, without reacting to normol hunman red blood cell, which suggests that the pET-LDH recombination protein possesses a high antigenicityolimmunize mouse using the recombination protein , We adopt its spleen cell mouse marrow SP2/0, Amalgamation cell use HAT culture medium carry through cultive The identification results for Ig type and sub-type indicate that 2BIK 3C9 3DIO 6D3 7D2 1E7 6G7C6 and 2D7 are IgGl for heavy chain and K for light chain; 2F12 is IgG2 for heavy chain and K for light chain; and 2C6 is IgGl for heavy chain and A for light chain o The ELASA titers of the culture cell supernatant from all the 11 clones range from 1:100 to 1:400 and those of ascites are from 1:6400 to 1:51200.GICA method which recognizes both falciparum malaria and tertian malaria is then established using the monoclonal antibodies purified by saturated ammonium sulfateo The results from two rounds of screen show an optimal reaction pattern, in which the 3C9 represents the coating antibody while the 1E7 is the marker antibody ?This reaction pattern is applied to detect different concentration of recombination antigen: Ing/mK 5ng/mK-5-l0ng/mK l00ng/ml and 500ng/ml, in which the lowest concentration that could be detected is 5ng/ml, and the results show a fine gradient which suggests a direct proportional relationship between the reaction color and the antigen amount 0 Thirty cases of falciparum...
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