Efficiently Expression Of Antioxidant Peptide VECYGPNRPQF In Pichia Pastoris And Its Functional Identification In Vitro And In Vivo

In the present study,P.pastoris KM71H FZM_{2014 AOP} was constructed to efficiently secrete AOP which contains antioxidant peptide VECYGPNRPQF?VEC?,and the bioactivity of the produced AOP was determined in vitro.D-galactose induced oxidative stress rats was used to study the effects on growth performance,redox status,intestinal health,and AAs transportation and metabolism from AOP were evaluated.1)Construction of eukaryotic expression strain(P.pastoris KM71H FZM_{2014 AOP})of antioxidant peptide VEC and antioxidant activity assay of AOP secreted from P. pastoris KM71H FZM_{2014 AOP} in vitro.The corresponding polynucleotide sequence of VEC was designed to construct the expression cassettes P_AOX1-?Factor-VEC-Zeocin^r based on the codon-usage bias of P.pastoris.The linearized pPICZA?-VEC-Zeocin^r plasmid was transferred into P.pastoris KM71H by electroporation.The colonies proved to be positive by zeocin screening and Tricine SDS-PAGE.PCR was used for further fermentation.The purified sample has the same molecular weight as the natural polypeptide VEC.The P.pastoris KM71H FZM_{2014 AOP}014 AOP fermentation supernatant?AOP?showed high antioxidant activity in vitro via the TEAC?Trolox Equivalent Antioxidant Capacity?assay.2)AOP secreted from P.pastoris KM71H FZM_{2014 AOP} alleviates oxidative stress in rats.Fifty male SD?Sprague-Dawley?rats with an average body weight of 246.73±1.48 g were used in the experiment.Oxidative stress was induced by intraperitoneal injection of D-galactose?300mg/kg BW?dissolved in 1ml normal saline at 4:00 pm every day while the control group was intraperitoneal injected with 1ml normal saline.The normal control?CON?group and oxidative stress?OS?group were given an intragastric administration of 2 ml normal saline at 9:00 am every day.The test groups?EQ group,KFS group,and AOP group?were given an intragastric administration of 2 ml solution with ethoxyquine?1mM?,P.pastoris KM71H fermentation supernatant?FS?and P.pastoris KM71H FZM_{2014 AOP}?Constructed in our lab?FS,respectively.The experiment lasted for 10 weeks.The weight gain and feed intake were recorded every three days.At the end of experiment,all rats were executed after overnight fasting.10ml of blood were firstly collected into heparin-coated tubes,centrifuged,and then stored at-80°C for further analysis.The samples of jejunum,ileum,liver,kidney and muscle were collected,immediately snap-frozen in liquid nitrogen and stored at-80°C until analysis.The results indicate that:?1?dietary intragastric administration with AOP increased the average daily gain?ADG?and average daily food intake?ADFI?,while EQ and KM71H had no obvious effect on growth performance of rats.?2?Compared with the OS group,AOP significantly decreased the serous concentration of MDA and LPS,EQ and KM71H significantly decreased the serous concentration of LPS?P<0.05?;the levels of CuZnSOD,MnSOD,GPX2,and CAT in liver were not significantly changed among all treatment groups except KM71H group?P>0.05?.?3?EQ had no clear influence on proinflammatory cytokines in intestine;KM71H promoted the expression of IL-2 and IL-6 in ileum;AOP obviously decreased the expression of IL-1?,Claudin-3 and Occludin in jejunum while increased the expression of Claudin-3 and Occludin in ileum?P<0.05?.?4?Compared with the OS group,EQ showed an increase tendency in the concentration of serous AAs while there was no significance;KM71H reduced the serous concentration of Ile;however,the concentration of serous AAs in AOP group showed a significant reduction?P<0.05?.EQ clearly up-regulated the expression of y⁺LAT1,xCT,LAT1,and EAAT2 in kidney and the expression of CAT-1,xCT,and LAT2 in intestine.EQ showed a clear increase in the expression of LAT1 in muscle while decreased the expression of EIF4G2 in liver?P<0.05?.KM71H mainly reduced the expression of AA transporters in intestine while enhanced the expression of partial AA transporters in kidney and muscle.Supplementation of AOP down-regulated the expression of partial AA transporters in kidney and jejunum and obviously up-regulated the expression of TAT1,LAT4,ASCT2,IMNO,rBAT,EAAT3,xCT,and y⁺LAT1 in ileum?P<0.05?.In liver,AOP improved the expression of ASCT2 while reduced the expression of EIF4E,4E-BP1,EIF4G2,and mTOR.In muscle,both the expression of AA transporters?ASCT2,PAT1,SNAT2,CAT-1,and LAT1?and protein synthesis and degradation related genes?4E-BP1,EIF4G2,DDO,and LOX?were dramatically enhanced by AOP?P<0.05?.These results show that both antioxidant peptide AOP and synthetic antioxidant EQ alleviated the oxidative stress on rats,and AOP is a better antioxidant compared with EQ.In conclusion,P.pastoris KM71H FZM_{2014 AOP} was successfully constructed to effectively secrete AOP in the present study.The bioactivity of the produced AOP was determined in vitro and in vivo.The results show that AOP secreted from P.pastoris KM71H FZM_{2014 AOP} enhanced the growth performance,redox status,intestinal health,and AAs transportation and metabolism,which is expected to be a natural and safe alternative antioxidant in livestock industry.