Studies On Expression Of Human Tissue Factor Pathway Inhibitor-2 In Pichia Pastoris

Tissue factor pathway inhibitor-2 (TFPI-2) is one of the serine protease inhibitors recently discovered , and is substance the same as the placental protein-5 (PP5) and the matrix-associated serine protease inhibitor (MSPI). Test in vitro has found that TFPI-2 has broad inhibitory spectra that inhibits plasmin, trypsin matrix, matrix metalloproteinase-1 (MMP-1), MMP-3 and FactorⅦa - tissue factor (FⅦa -TF) and so on. It has been found TFPI-2 plays an important role in in keeping cell matrix remodeling and angiogenesis, regulating invasion and metastasis of tumour cells and adjusting blood coagulation and apoptosis.The relation between TFPI-2 and invasion and metastasis of tumour has been frequently researched recent years. Serine protease such as fibrinolysin, trypsin,collagenase and cathepsin which is generated and secreted by some cancer cells can degradate the major of stroma ground substance, destroy the structure of extracellular matrix (ECM) and profit invasion and metastasis of tumour cells. Through studying the cells of chorionic carcinoma, melanotic carcinoma, prostatic carcinoma, pulmonary carcinoma and neurospongioma, we found that TFPI-2 can restrain these tumor cells to invade. The research on the relation between TFPI-2 and the tumor cell's soakage and transfer would supply a new policy for oncotherapy in future. Some TFPI-2 are necessary in research the relation of both even more if TFPI-2 could treat cancer.It is a research directon worth to be attended that we search the means to prepare TFPI-2 of high dose. It is reported that TFPI-2 protein is expressed through reform by Bacillus coli, but this system has some demerits: 1) it can't modify and elaborate protein post-translation of eucaryon, as shorn, glycosylation, disulfide bond form; 2)the expressed proteins which most form insolubile cytorrhyctes demand complicated renaturation to restore constellation and activity; 3)the background protein are difficult to purify;4)the expression outcome is not very large. And it is also reported that one of Kunitz of TFPI-2 is expressed by Pichia pastoris.On this research, we select pichia pastoris eukaryon expression system which has good operation of prokaryotic system and post-processing of eucaryon system to express reform human TFPI-2 protein having bioactivity.1 Construction of tfpi-2 Cloning VectorOn the basis of tfpi-2 cDNA sequence published in genebank, we designed primers, and extrcted total RNA from human fresh liver tissues, and obtained tfpi-2 cDNA by RT-PCR, then cloned tfpi-2 cDNA into pMD18-T vector, and established recombinant cloning vector pMD18-T-tfpi-2 with signal peptide sequence and total encoding sequence of tfpi-2. The designed sequence identified with published sequence by enzyme digesting and DNA sequencing, structuring a base for establishing tfpi-2 expression vector later.2 construction of tfpi-2 expression vectorAccording to the principle of primer design, we used cloning vector with correct sequence as a template to design expression primers: inserting enzyme cutting sites, XhoI and XbaI at the end of 5'terminal of forward prime and backward prime. We amplified encoding sequence of tfpi-2 fusion gene by PCR, and cloned it into pPICZαexpression vector. DNA sequencing through Sanger dideoxy chain termination method verified that we obtained correct pPICZα- tfpi-2 recombination expression vector.3 Expression and Identification of TFPI-2 in Pichia pastorisWe electrotransformated linearrizated plasmid pPICZα-tfpi-2 into competent cell, Pichia pastoris X-33, then smear the cells on YPD agar plate containing Zeocin. After cultured them 48h-72h at 28℃,We choosed 9 clones with Zeocin-resistance to carry on verification. In the process genome DNA was extracted used as the template , in addition to expression primers to test.We amplified positive 9 converters verifying by PCR to shake cultivation at 28℃and induced them to express protein with 0.5% methyl alcohol. Then took supernatant after fermentation, and screen strain having trypsin inhibition activity through remain enzyme-substrate coloration reaction basing the inhibition of TFPI-2 to trypsin. Through identifying supernatant protein with SDS-PAGE analysis, we found a chromosome strap of 32kDa molecular weight of strain by activity screening.Trypsin activity can be tested absorbance at 410nm of nitroaniline producing by hydrolysis substrate DL-BAPNA. In our study, we use residual trypsin activity after TFPI-2 protein react trypsin to detect the activity of TFPI-2. Taking supernatant of 1~7 days produced by the highest activity strain, then take 100μL to test TFPI-2 activity after diluting 5 fold. Through observing the relation between the expression of recombination TFPI-2 and induction time, we found that TFPI-2 had a crest value 41750 TIU/L at 5th days. To sum up: in the present experiment we successfully constructed TFPI-2 eukaryotic expression vector pPICZα-tfpi-2 by the technology of genetic recombination, and successfully obtained expression product recombination TFPI-2 having biologic activity in eukaryotic expression system Pichia pastoris. This is a creative research as no related report in domestic and abroad. Our study settle the base to future researching the relation of TFPI-2 and cancer.