Bir1p's Homologization-Survivin's Influence Of Growing And Molecular Rearrangement On P.pastoris

As one of the eukaryotic expression system, P.pastoris have many advantages which other expression systems have not, for example:compared to E.coli expression system, it can regulate the expression of foreign protein in high performance and strictly; modify the expression protein when it was translated;compared to mammalian cell expression system,it can ferment cultivation in high density, and it need only little nutrition, grow faster, cheap medium,be better for industrialization; expression in and out of cell conveniently; genetic manipulation is so easy, and so on. So, recently, the P.pastoris expression system's application is so widely in the filed of medical and pharmaceutical industry.However, this expression system has many disadvantages in area of application:one of them is the long time in the process of expressing the foreign protein by using the P.pastoris. In this process, many factors such as toxicity of methanol, the condition of growing, and enviorment can lead to the decrease of activity of growing, and accompany with the phenomenon of apoptosis. All of them are the disadvantages for expressing the foreign protein. To solve this problem,we consider the apoptosis mechanism and analyze the apoptosis gene to decrease the death rate for raising the growing rate and density of cell.In order to develop the P.pastoris of high activity, short grow cycle and anti-apoptosis. We choose Birlp's congener-survivin to reform the by molecular recombination for providing a basement of P.pastoris's industrial producing the foreign protein.We analysis and differentiate the yeast apoptosis gene Birlp and huamn apoptosis gene survivin are homologization analogues by bioinformatics. We found that Birlp have 2870 bases, Survivin have only 429 bases,but on the level of functional,They are homologization analogue. They all can form a structure called zinc finger which have some relationship with apoptosis. However, because the Birlp have no idea with its mechanism paths and part of its mechanism, and it have a long fragment which have difficult gene operation. So, this paper make the gene survivin integrate into the genome of P.pastoris by the inducible expression and the constitutive expression. To proof the gene survivin can raise or degrade the P.pastoris'apoptosis or death rate, providing the theory basement for the development of yeast's industry.This paper makes the gene survivin and survivin (T34A) into two expression plasmids, pGAPZA and pPICZA, and integrate into the genome for researching. To analyze the growth tendency by depicting the growth curve, we found that the grow rate and OD of X33 which been integrated the survivin is higher than the other, and the control is on the level of average. We analyze the apoptosis rate in the special time by MTT method, we found that the apoptosis rate have the tendency of going up, and the inducible form is higher than the constitutive form. By nucleic acid electrophoresis, we found that electrophoresis spectra show a typical ladder. By the flow cytometry, we proved that the two form have visible disparation.The conclusion is survivin can encourage the growth of yeast cell, and the mutant of survivin can inhabit the growth of yeast cell, and the inducible expression have better effective than the constitutive expression. Then, making the gene survivin (T34A) integrate into the yeast is useful for yeast industry development.