| The high metastasis ability of tumor cell relates to it's high adhesion ability. To block tumor cell's adhesion may be the best way to prevent tumor metastasis and recrudescence. Based on the others work, we designed the anti-adhesion peptide p3 (DLYYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMK), which had three repeat sequences of β1(DLYYLMDLSYSMK). And we tried to express the β3 using E.coli. expression system. Farther, we studied some of the anti metastasis ability of this expressed β3, such as the ability of anti tumor cell adhesion to ECM, and the. inhibitory of tumor cell mobility and invasion, and the influence to the quantity and activity of tumor cell's MMPs. So that we can find and manufacture a new anti-metastasis drug.At first we synthesized the DNA sequence of β3 according to the favorite cordon of E.coli. Then we used 6 different kinds of vectors to express β3 synchronously so that we could screen out the high-effect expression vector. Our result showed in different vector, things were different. The expressed product of pGEX4T-1, GST-β3, existed as inclusion body. It's quantum can be 40% of E.coli total protein. Taking advantage of the vector coding GST protein, GST-P3 could be separated and purified by Glutathione Sepharose 4B Bead. And we do get the protein. Using pMBP-P, pTrc-CKS and pET-DsbA, we didn't get satisfying results. Using pET22b(+), p3 could be expressed. The expressing protein is 5.5% of E.coli. total proteins. But this expressing product contained the secretion signal sequence of E.coli., pel B.We get expressing product of β3 using pET-His. The product also is inclusionbody. It is 4% of total E.coli. proteins and 10% of E.coli total infusibility proteins.On the condition of denaturalization, His-β3 can be separated and purified by metalchelated agarose 6B FF. So we could get 20 mg His-β3 peptide in 1 liter culturemedium. And it's purity is 92.2%. The sequence of expressed β3 isMGSSHHHHHHSSGLVPRGSDLYYLMDLSYSMKGGDLYYLMDLSYSMKGGD LYYLMDLSYSMKAS) . It's molecular weight is about 5.5 KD.Using FN as the ECM, we studied the influence of peptides on the adhesion of tumor cells to FN. We found the expressed 3 3, the synthetic 3 3, the synthetic 3 2, the synthetic 3 1 and GRGDS could all inhibit the adhesion of tumor cells to FN, showing dose-effect relationship. The inhibition effect of expressed 3 3 on the adhesion of HCCLM6 cells to FN was higher than which of synthetic 3 1 and synthetic 3 2, and similar as which of synthetic 3 3. And, at the low dose level (10, 20nmol/L) , the inhibition effect of expressed 3 3 was as the same as the GRGDS, but at the high dose level (50, lOOumol/L) , the effect of expressed 3 3 was higher than which of GRGDS. On the different co-incubation time, the expressed 3 3, synthetic 3 1 and GRGDS all specifically repressed the adhesion of tumor cells to FN, showing time-effect relationship. The repression effect was expressed 3 3 higher than GRGDS than 3 1. And the effect of HCCLM6 cells was stronger than which of SMMC-7721 cells.Using Transwell Boyden and matrigel, we studied the tumor cell's mobility. We found after using 100 u mol/L expressed 3 3, the moving ability and invasion ability of SMMC-7721 tumor cells and HCCLM6 tumor cells were both inhibited obviously. The inhibitory rate of mobility were 32.7% and 40.7%, of invasion were 35.2% and 51.3%.To study the influence of expressed 3 3 on the MMPs of tumor cells, we tested the quantity of MMP-1, MMP-2, MMP-9 of the medium of tumor cells by ELISA. ? We found the expressed 3 3 repressed the MMP-9 of SMMC-7721 tumor cells and HCCLM6 tumor cells, but had little influence on the quantity of MMP-2. In addition, we tested the quantity and activity of MMP-2 and MMP-9 by galatinzymography. We found the expressed 3 3 could reduce the active type of MMP-2 and MMP-9 o These results proved the expressed 3 3 inhibited the invasion ability of tumor cells through reducing the MMP-9 protein level and declining the active type of MMP-2 and MMP-9. In addition, we also found the expressed 3 3 could reduce the quantity of MMP-1 in HCCLM6 tumor cells. The role of this phenomenon was not clear.We expressed 03 using pET-His system effectively. It's very conveniently to separate and purify the product. So, this system is suit for expressing 03. The expressed 3 3 could change tumor cells invasion ability through inhibiting the adhesion ability of tumor cells to ECM, decreasing the mobility of tumor cells, and...
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