Studies On Cloning, Expression, Purification And Activity Of Curcin And Chitinase Of Jatropha Curcas

Jatropha curcas L, belonging to Euphorbiaceae and thriving in many areas of the tropics and sub-tropics around the world, is a multipurpose plant with many attributes and considerable potentials. For the first time, Stripe (1976) extracted the toxin protein from the seeds of Jatropha curcas which was proofed to be curcin, a single strain RIPs. RIPs, exist in many higher plants, is a kind of toxin protein which can act on rRNA and inhibit the function of ribosome. RIPs can inhibit the growth of tumor cells and HIV, and it can be made into immunotoxin which will be the development direction of the anticancer medicament. Much work had been done on curcin and it was found that curcin could inhibit the growth of some tumor cells. However, the quantity of Jatropha curcas is limited and it will spend a long time to grow up. In order to utilize it conveniently, much research would be bone on it. Under the foundation of former study, we cloned the gene encode the mature protein of curcin and express it by Escheria coli. The gained recombinant protein was purified by Ni-NTA affinity chromatography and refolded by dialyse. The in vitro activity of the recombinant protein was tested by cell free translation system and some tumor cells. It was shown that the gene encoding the mature protein of curcin could be amplified by PCR from the total RNA of the seeds of Jatropha curcas, and it was also shown from the results of bacteria PCR and substrate of double enzyme that the gene encoding the mature protein could be express in pQE-30 and pET-32. But the further research of SDS-PAGE, clone Western and Western-blot showed that the recombinant protein of curcin could be successfully expressed in the vector of pQE-30. It meant that the vector of pET-32 was not suitable for the express of curcin. It was found in the study that the recombinant protein being expressed by the form of inclusion body.Different revulsants, different temperature, different concentration of Amp and different adding time of revulsant were designed to study the expression condition of the recombinant strain in the experiment. It was demonstrated in the study that the recombinant strain could not be induced to express the recombinant protein by xylose and galactose, but by IPTG and it was also shown in the study that the recombinant strain could produce the recombinant protein at a very high production at the concentration of 0.5mM of IPTG. The production of the recombinant protein changed with the temperature, the higher the temperature, the higher the production. The recombinant strain could produce the most protein at 28℃and at the concentration of 200mg/L of Amp.The recombinant protein was expressed in the form of inclusion bodies and purified by Ni-NTA affinity chromatography. The purified protein was refolded by dialyse and dried by low temperature. The in vitro activity of the refolded protein was tested by the cell free translation system and such tumor cells as NCL-H446, S180, SGC7901 and VSV. It was shown in the test that the refolded protein showed intense inhibitory activity in the cell-free translation system at very low concentration when the concentration was increased to some degree the inhibitory effect became higher. And it was shown that the refolded protein could inhibit the growth of several tumor cell lines, such as cellular pulmonary cancer NCL-H446, gastric cancer SGC-7901 and S180. But it had no effect on WISH cells or the vesicular stomatitis viruses. It was also shown that, at the concentration of 5μg/ml, the protein could inhibit the growth of tumor cells and when the concentration was increased to some degree, the inhibitory effect became higher. Under the fluorescence microscope, we could find some of the tumor cells (S180) killed by the recombinant protein compared with the control and stained by acridine orange. All the results have encouraged us to continue studying the recombinant protein of curcin in the fields if immunotoxin and anti-tumor medicine.Chitinase (EC 3.2.1.14) catalyze the hydrolysis of chitin, a linear homopolymer ofβ-1, 4-1inked N-acetylglucosamine (GlcNAC) residues. Chitinase can be found in plants, animals and microorganisms and in which the plant chitinases have been studied by many researchers because of its special role in pathogene defense. It was found that plant chitinase play an important role not only in plant defense but also in plant growth. By land been particularly studied, some of the proteins are of antitumor activity and somer, many proteins have been extracted from the seeds of Jatropha curcas and some are esterase, fatty acid enzyme and et al. But no research had been done on the chitinase of it. In order to probe into the foundation of the pathogen defense, we studied the basic characteristic of the chitinase of Jatropha curcas. Buffers of different pH were used to extract the chitinase of the seeds of Jatropha curcas, and it was found that the chitinase would reach the highest activity at pH 7.0. The gained chitinase were tested at different temperature for different time and it was shown that the optimum temperature of chitinase was 55℃and the optimum time of it was 60min. Further study showed that the special activity of chitinase of different parts of Jatropha curcas were different. It showed that the special activity of leaf is the highest and that of root is the lowest.