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Purification And Characterization Of High Molecular Weight Cpis From Silver Carp Egg And Its Preliminary Effect On Ishikawa Cell

Posted on:2015-05-02Degree:MasterType:Thesis
Country:ChinaCandidate:L LiuFull Text:PDF
GTID:2271330482476109Subject:Agricultural Products Processing and Storage
Abstract/Summary:PDF Full Text Request
In this study, two high molecular weight CPIs which were named as gLMW CPI-a-3-17 and gHMW CPI-b-2-14 were purified from silver carp egg by a series of chromatography. In addition, their molecular weight and purity were characterized by gelatin-SDS-reverse, TSK gel G2000 SWXL and Jupiter 300 C4 RP-HPLC. Then these two high molecular weight CPIs were characterized further by MALDI-TOF-TOF tandem mass spectrometry peptide fingerprinting. Moreover, their biochemical properties were characterized, including thermal stability, pH stability, Ki and inhibitive type. At last, the preliminary effects of silver carp egg gHMW CPI-b-2-14 on Ishikawa cell was got by determining cell proliferation, migration, adhesion, colony formation rate, cell morphology and VEGF.Purification and characterization of high molecular weight CPIs from silver carg, egg:The crude extract of CPIs were further purified by chromatography after homogenization, acid treatment and concentrated by ultrafiltration (30KDa). During this period, its inhibitive activity was detected by fluorescence spectrophotometer using Z-Phe-Arg-MCA as substrate. The peak II with high inhibitive activity from Sr Sepharose Fast Flow was collected and applied on Blue Sepharose 6 Fast Flow, then collected peak 3 and applied on Octyl-Sepharose 4 Fast Flow hydrophobic chromatography, afterwards, three active fractions (a, b and c) were obtained. The fraction a and fraction b with high specific activity were studied later in this paper.Fraction a-3 was collected from fraction a by ConA Sepharose 4B, and then applied on TSK gel G2000 SWXL. It was found that fraction a-3 had only one protein peak at 17.331min, named gLMW CPI-a-3-17. It also had one peak at 14.321min by C4 RP-HPLC, indicating that CPIs were purified effectively and the purification factor was 848.76 times, the yield was 2.13%. We named it gLMW CPI-a-3-17, and its molecular weight was 44KDa identified by SDS-PAGE and TSK gel G2000 SWXL,Fraction b-1 and b-2 were collected from fraction b by ConA Sepharose 4B, and then applied on TSK gel G2000 SWXL.It was found that fraction b-1 and b-2 had one protein peak at 13.727min and 14.421min, which were named ungHMW CPI-b-1-14 and gHMW CPI-b-2-14, respectively. The purity of gHMW CPI-b-2-14 had one peak at 14.544min by C4 RP-HPLC, and its purification factor was 468.91 times, the yield was 2.07%. The molecular weight identified by SDS-PAGE was 125KDa. MALDI-TOF-TOF tandem mass spectrometry peptide fingerprinting showed that ungHMW CPI-b-1-14 matched with an unidentified protein of Medaka(Oryzias latipes). However, gHMW CPI-b-2-14 didn’t have a matching record.It was found that gLMW CPI-a-3-17 and gHMW CPI-b-2-14 all had a wide range of thermal stability and pH stability, remaining more than 90% and 80% residuary activities at 80 ℃, respectively, and exhibited the most stable property when pH were 7-9 and 8.0, respectively. They were able to inhibit the activities of silver carp cathepsin B, cathepsin L and papain, but had no effect on trypsin and chymotrypsin. Moreover, the inhibitor constant (Ki) of gLMW CPI-a-3-17 and gHMW CPI-b-2-14 were 0.0292nM and 0.0116nM respectively when tested with the Dixon-plots method, implying that both of them are competitive inhibitors against papain.The effect on Ishikawa cell of gHMW CPI-b-2-14:The preliminary research on Ishikawa cell showed that gHMW CPI-b-2-14 could effectively inhibit the proliferation of Ishikawa cell in a dose and time-dependent, that is, with gHMW CPI-b-2-14 dose increased and incubation time prolonged, the inhibition effect was more obvious. One week later, high-dose group (5-40μg/mL) was lethal to monoclonal Ishikawa cells, while the colony formation and theamount of Ishikawa cells decreased with the low-dose group (0.5-2.5μg/mL) increased, compared with the control group, the difference between each low-dose group was significantly (p<0.01). The scratch experimental results showed that the migration of Ishikawa cells also appeared dose-and time-dependent with gHMW CPI-b-2-14. After 72h, the scratch width of control group was healing, however, the width treated with high-dose group was non-significant (p>0.05) compared with that at 0 h. With the increasing of gHMW CPI-b-2-14 doses, the inhibitory effect on adhesion ability of Ishikawa cells was more significant. The results of Hoechest 33342 staining results indicated that staining intensity of Ishikawa cell nuclear was significantly enhanced after treatment by high-dose group (5-40μg/mL), showing a bright blue, followed with nuclear condensation and nuclear fragmentation, while the low-dose group (0.5-2.5μg/mL) was not significant (p>0.05) compared with control group. ELISA method showed that gHMW CPI-b-2-14 could effectively inhibit the secretion of VEGF-A and reached the highest peak at 48h. Moreover, when the dose of gHMW CPI-b-2-14 was 1.5μg/mL, the effect of inhibition on secretion of VEGF-A was the most remarkable, and was significant when compared with control group (p<0.01).
Keywords/Search Tags:Silver carp (Hypophthalmichthys molitrix) egg, High molecular weight CPIs, Purification and Characterization, Ishikawa cell, Effect
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