1.Clone, Expression And Purification Of Ricin A 2.The Research Of The Effect Of Ulinastain On Injury Of Mesentery, Liver And Kidey Of Rats With Obstructive Jaundice

Although organ transplantation has become an effective treatment to save lifes and prolong the survival time of patients, the rejection after transplantation is the main cause which affects the final outcome. In order to suppress rejection and increase the survival rate of transplantation, expensive immunosuppressants have to be used in the remainder of patients' life. However, large amount of immunosuppressants can cause not only the side-effects, such as hyperpiesia and hepatorenal toxity, but also severe infection and malignant carcinoma, especially in a long run. Nowadays the key methods to avoid or alleviate these side-effects are to induce immune tolerance or low immunoreaction state, and sustain the ability of immune response against other antigens (including anticancer, anti-infection and so on ). According to the theory of genetic engineering, wedesigned the experiment to construct ricin A chain gene vector and express and purify ricin A protein which can be used as a tomb to kill actived T cell in order to suppress rejection.Ricin A protein is toxic part of ricin which can be used a tomb of killing cells specially. In our study, we cloned, expressed and puried ricin A.In the study, firstly extract total mRNA from castor bean and reverse transcription. Design the primer with computer. Ricin A chain gene was cloned in vector pcDNA3.l by PCR and analyse the sequence. The nucleotide sequence of the ricin A cDNA was the same as the paper reported in genebank. Then it was inserted into the prokaryotic fusion expression vector pRSET A to construct recombinant expression vector pRSET A-ricinA. pRSET A-ricinA was tranformated E.coli BL21, then induced by IPTG. The SDS-PAGE showed E.coli BL-21 containing pRSET A-ricinA could express a fusion protein in which exsisted in a soluble form. The soluble protein was purified by Ni-NTA spin column. We found the ricin A could killing the cells SMMC-7721.The study results suggested that toxicity of ricin A can be used as a tomb to kill cells specially. The study was the basis of subsequent study.