Research On Separation Natural Prethrins' Six Composition And Immunological Detection Method Of Chrysanthemum Cineraraeflium

Pyrethrum is a species of perennial herbage plants (Chrysanthemum Cineraraeflium), and a sole industrial insecticidal plant all over the world. Because of excellent insecticidal characteristics of pyrethrins, including high efficiency, low toxin, broad-spectrum, low residues, and a low impact on the human being and environment, pyrethrins have been become one of the most important biologic insecticides. Now, natural pyrethrins have already been widely applied in many fields of agriculture, forest, biological pesticide, foods and so on. Therefore, it is very important to create a simple and rapid immunological method to detect pesticide residue on the spot. The immunological method has a lot of advantages of rapid,simple,sensitive and high selectivity, compared to apparatus analytical method, especially present strongpoint. Besides, the immunological method is a preferential technique to be investigated and exploited, which can be used to detect pyrethrins and pesticide residue. However, the application of it has not been reported in the world yet.The method of detecting pyrethrins would have been built. Firstly, the pyrethrins'six components whose purity were above 99% must be gotten was very difficult. Pyrethrins active ingredients contain six esters, including pyrethrinⅠ, cinerinⅠ, jasmolinⅠand pyrethrinⅡ, cinerinⅡ, jasmolinⅡ. Because of their similarity in structures, simultaneous separation of the six components is very difficult. Secondly, pyrethrins can not be induced to produce antibody, so preparation of man-made antigen must make pyrethrins'six components and carrier protein to be connected, but pyrethrins have no position to be directly connected with protein, so active functional molecule must be imported such as derived ones from pyrethrins or import ones by organic chemistry method. Thirdly, Pyrethrins'six single component man-made antibody were blended to immunise BALB/c female mice, distill the mRNA from mice'spleen and reverse transcription to cDNA, extend respectively between heavy chain(VH) gene library and light chain(VL) gene library and connect them by linker, then build single-chain variable fragment antibody library of Natural Pyrethrins. It will provide a material basis for filtrating six single clone antibodies.Main research contents and results are as follows:⑴We separated and purified the six compounds by reversed-phase HPLC from the pyrethrins oil. The purity of the six compounds was above 99%. The structure of the six compounds was confirmed by gas chromatography-mass spectrometry (GC-MS). The research makes use of optimal condition of separating and purifying the six compounds by reversed-phase HPLC from the extract of pyrethrins to succeed in getting six single component's production whose purity reach above 99%.The structure of the six compounds was confirmed by gas chromatography-mass spectrometry (GC-MS). Compared with chromatogram library may get each apex's homologous structure by analyzing these TIC chromatograms. The first component is cinerinⅡ, the second is pyrethrinⅡ, the third is jasmolinⅡ, the fourth is cinerinⅠ, the fifth is pyrethrinⅠ, the sixth is jasmolinⅠ.⑵Through optimizing the half-preparation column condition of separating and purifying, the six compounds were separated successfully from the extract of pyrethrins by reversed-phase HPLC, with their purity above 99%. The proportion of loss of six dried productions was from 2% to 30%. More than 100 mg single solid component could be obtained from 1 ml 70% pyrethrins oil, after separating and purifying by reversed-phase HPLC. This method has a good separating effect and simple operation that is going to fit to purify pyrethrins'six components in industry.⑶Preparation of man-made antigen of pyrethrins'six components are on the basis of organic chemistry principle and proper structure of pyrethrins. Because pyrethrins can not be induced to produce antibody, preparation of man-made antigen must make pyrethrins'six components and carrier protein to be connected. But pyrethrins have no position to be directly connected with protein, so we need import active functional molecule such as derived ones from pyrethrins or import ones by organic chemistry method. Importing active functional molecule is very difficult and are not applied to be reported. Making use of pyrethrins'six components(cinerin I,jasmolin I,pyrethrin I,cinerin II,jasmolin II,pyrethrin II) is elementary material, and make them react with p-hydrazinobenzoic acid on the condition of feebleness acid(pH 4.0~5.0), and heat up and circumfuse them, and will get pyrethrins'six components that consist of the active carboxyl, and connect antigen to Bovine Serum Albumin by the method of CDI in order to get six man-made antigen of pyrethrins'six components. Lastly, using ultraviolet theory can identify whether connection is success or not.⑷Construction of single-chain variable fragment antibody library of Natural pyrethrins must depend on immunological theory and the technique of polymerase chain reaction. Pyrethrins'six single component man-made antibody were blended to immunise BALB/c female mice. After one month, blood serum effect was detected by Enzyme-linked immunosorbent assay. mRNA from mice'spleen was seperated and reversely transcripted into cDNA. Heavy chain (VH) gene library and light chain (VL) gene library were extended respectively, connected by linker, thus the single-chain variable fragment antibody library of Natural Pyrethrins was build up.