| Iron is closely linked with human nutrition and health. Iron deficiency would result in many diseases, such as anaemia, runtishness, immunomechanism damage, nerve malpractice. Plant is one of main sources of human nutrition. However, iron content in plant food is very low and its efficiency of assimilation is not as high as that of animal food. Therefore, iron nutrition of human is mainly depended on the latter. And there is a serious problem of iron deficiency in which plant food is the main food resource, especially in developing country or area. Ferritin, widely distributing in plants, animals and microorganisms, is a key iron storage, protein constructed from 24 subunits. The researches acquired suggest that manipulating ferritin expression and other soluble components of seed iron in soybeans and possibly other seeds, using Mendelian genetic technique and biotechnological approaches, could contribute to a sustainable solution to the global problem of iron deficiency.Utilizing the technique of immunoassay to detect the expression of transgenic plant is simple, sensitive, fast and credible. However, it is necessary to acquire the antibody or the antiserum, which could specially react with the expression protein of die objective gene transferred into the transgenic plant According to the characteristics of high homology and immune cross-reaction among plant ferritin, using the special immune serum of pea ferritin, the content of plant ferritin could be detected for studing the ferritin expression of transgenic plant by the technique of immunoassay such as immunoprecipitation, ELJSA and Western blotting. In this paper, the pea ferritin was purified from pea seed, and its antiserum was raised from rabbits for investigating the expression of transgenic plant. The main experimental results were summarized as following:1. Establishment of ferritin detection system.During the course of purification, the method of orthophenanthroline staining is used to detect the ferritin. 30|il of the fractions eluted from the AcA^gel column were pulled into the cells of the minim incubation plate. Then SOpil 20% dithionite and 40|il 0.1% orthophenanthroline solution were pulled into respectively. According to the colour change, the content of ferritin is detected. In addition, the quantitative analysis of ferritin is measured based on the absorbance of Fe2*/orthophenanthroline at 510 nm.2. Purification of pea seed ferritin.The crude extraction of dry pea seed, which was obtained through marination, homogenization, filtration, centrifugation and other methods, was precipitated by 50 mmol/L (final concentration) MgQ2. The pellet was chromatographed on AcA^ gel filtration and DEAE-cellulose 52 (DE 52) anion-exchanger. Single eluting peak containing ferritin was obtained finally. After manipulated as above, the productive rate of purified pea ferritin is 20-30mg/kg.3. Identification of purified pea ferritin.Subjected to PAGE under non-denaturating conditions, the purified pea ferritin migrated was single band, which indicates that the purified ferritin of pea has attained electrophoresising purity. Under the conditions of SDS-PAGE, it depolymerized into only one subunit of 28 kD, which reveal that the subunit of purified ferritin of pea is undivided4. Preparation of antiserum of pea ferritinThe purified pea ferritin was mixed with Freund's complete adjuvant or Freund's incomplete adjuvant, and then administered through palmula, back, abdominal cavity, vena auricularis and other routes for rabbits. Seven weeks later, the litre of antiserum reached 1 I 32. The antibody was then purified by fractionation with (NH^SC^ and its litre still keep at 1 '. 16. These results suggested that high litre antiserum had been acquired.5. Immunoassay of plant ferritin.Using agarose gel double diffusion method, the antiserum of pea ferritin can cross-react with the crude extraction of soybean seed Ferritin. The result indicated that high specific antiserum had been acquired and could be used~in the detection...
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