| Primary carcinoma of the liver, with high death rate, whose mobility is inferior to gastriccancer and esophagus cancer, has ranked the third among the wide-spread tumors. A study ofM.D. An-derson Research Institution of Cancer recently demonstrates that41%patients ofprimary carcinoma of the liver are related to drinking massive alcohol,24%come fromHepatitis C,10%are induced by Hepatitis B and the remaining25%have no clear causes.Although the morbidity of liver cancer in European countries and USA is comparably low andHepatitis B has become one of the greatest factors which induce liver cancer, the mobilitythere will has a sharp increase in these years.Compared with western countries, liver cancer in China has been the first in the world.And the patients who died from this illness accounts for nearly half of the whole world. Andits mobility is as ten times as it in Euro-countries. Several factors contribute to the great livercancer incidence in China:（1）. Hepatitis B and Hepatitis C are wide-spread in China and arethe great incentive to liver cancer.（2）. The long tradition of drinking stirs the cirrhosis whichis also an important factor to liver cancer.（3） Bad sanitary condition plays an important roletoo, because mildewed food contains a powerful incentive to liver cancer, aflatoxin.The best treatment of liver cancer is the surgical excision which is hard to executebecause of the easy recurrence. Around60%-70%patients will undergo metastasis orrecurrence in5years. The most available medicines for this illness are fluorouracil and Adriiamycin but with serious negative effects like vomiting, alimentary reflections and so on. Andthese medicines have no clear impact and a very low efficiency on lengthening the patients’lives who have undergone the surgical excision. So it will be of great significance in workingat the medicines and therapies for the clinical research in China.Viscum album is a general name of hemiparasites relying on deciduous species. There isa long history for Chinese to apply it to curing verole, rheumatism and hypertension, etc. And in Europe, Viscum album Agglutinin（VAA） has been used as a supplementary medicine fortumors. And there are3subdivisions of it, i.d. I、II、II. VAA-I is the most active and has beenreported for many times. VAA-I is constructed by subunit A, which recognizes the specificantibodies of some tumors and subunit B, which goes into the cells of tumor to excert itsanti-tumor function.VAA-I is obtained from viscum album traditionally, but quite confined because of itscomplicated extracted method. Somebody uses colicin to express. But colicin itself hasseveral demerits, such as the difficulty of purifying,large quantity of endotoxinexsitingbetween the memberane of colicin cells and so on. Microscale endotoxin will result in pyogenaction. While Pichia is a kind of lower eucaryons which is good for expressing andmoderating the space fold. Meanwhile, yeast cells can be easily obtained, manipulated andcultivated, so it has drawn more attention and applied to the production of protein reunion. Sofar, Pichia expression has been successfully applied to more than200proteins, and amongthem, the reunion of human insulinum,human serum protein and hepatitis B vaccine havebeen industrialized marketed. In order to lay a relatively safe and efficient foundation for thedevelopment of VAA-I,3aspects are being studied.1. The establishement of VAA reunion and Pichia secretion expressing systemThis section begins from studying the protein oder of VAA-I, optimizing the amino code,to synthesize the proper gene order which is suitable for Pichia expression. KpnI,EcoRI,SDS-PAGE and Western Blot are used to analyze the cloned VAA-I, the output in thetest tube can be up to39mg·L-1. KTA purifier is used, the fineness is nearly97%and theformula weight correspond to the expectation.2. Fermentation and purified method in VAA reunionPH, carbinol induction time, fermentation temperature, accelerated speed of carbinol,DO,medium component and defoamer are all further investigated to observe the impact.FM21is selected as the culture medium, the most suitable pH is5.5, the most propertemperature is28-30℃, revolution,650r·min-1，DO,25%，accelerated speed of carbinol is9.3mL·h-1·L-1, the induction time is96h, and finally the fineness can be up to92%and theextracting precentage is around33%. 3. Mechanism study of VAA in anticancer of liverIt is studied that rVAA-I can suppress the tumor cells according to the doses. IC50in24hcan be16ng/mL. rVAA-I can induce SMMC7721to destitute. And it is verified that Caspaseis closely related to the perish of cells. MAPK message passage way plays a vital role inkilling SMMC7721and it is verified by PCR.So it is concluded that this research establishes the rVAA-I expression of Pichia andoptimizes the fermentation skill. And meanwhile it is verified that rVAA-I is related to thedestruction of SMMC7721, and MAPK is involved in the process that rVAA-I killsSMMC7721cells. |
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