Cloning And Expression Of Angiotensin-converting Enzyme 2(ACE2) In Pigs And Its Fuction In LPS-induced Intestinal Inflammation

The research object of angiotensin converting enzyme 2(ACE2)covers human beings and also animals.But so far,the research on ACE2 is mainly focused on rodents and humans.Other animals,such as cattle,sheep and pigs,have little or no information.Many studies have shown that ACE2 plays important anti-inflammatory and anti-injury roles in different tissues and organs.However,its role in intestine has not been fully clarified.The aim of this study was to establish the ACE2 prokaryotic and eukaryotic expression system of pig,prepare ACE2 polyclonal antibody,obtain ACE2 active protein,and to discuss the basics ACE2's anti-inflammation effect.The study includes the following three aspects:1.Prokaryotic Expression,Polyclonal Antibody Preparation and the Expression of ACE2 gene in pigsWe used the sujiang pig as the experiment animal.The pig-specific recombinant proteins were obtained through prokaryotic expression,and temperature for induction and concentration of IPTG were optimized in order to improve the expression level of target protein.The inclusion bodies of recombinant pET-32a-ACE2 fusion protein were obtained and purified with KCl gel slices and Ni2+-NTA chromatography,respectively,in order to be used as the polyclonal antibody of ACE2.Then ACE2 was prepared using immunizing wastar rats with the purified proteins,and antibody titer was detected using ELISA.The specificity of the antibody was monitored using immunofluorescence and Western blot.The expression and distribution of ACE2 protein in Pig tissues were detected using Western-Blot and Immunohistochemistry.The results were list as follows:1)The sequence of ACE2 of the pigs was obtained by RT PCR and it covered 2418 nucleotides,coded 805 amino acid(aa)residues.Sequence homology analysis showed that the homology of ACE2 sequence in pigs is highly conservative.Genetic evolution analysis showed that this gene in pigs has the shortest genetic distance with goat.Analysis of protein structure showed that ACE2 protein was a trans-membrane secreted protein with high hydrophilicity and a signal peptide sequence locating between 1aa and 17aa;2)Two prokaryotic expression vectors for pET-28a-ACE2 and pET-32a-ACE2 were constructed.The SDS-PAGE result showed that the ACE2 fusion protein was mainly expressed in inclusion body under the induction with 1.0 mmol·L-1 IPTG for 10 h.The product molecular weight was approximately 100 kDa,as predicted.3)While both KCl gel slices and Ni2+-NTA chromatography can purify recombinant pET-32a-ACE2 fusion to obtain soluble recombinant,the KCl gel slices produced better purity and concentration.Western Blot confirmed that soluble recombinant pET-32a-ACE2 fusion proteins had satisfactory antigenicity;4)The mouse anti-porcine ACE2 polyclonal antibody was prepared successfully by immunizing wastar rats with the purified fusion protein.The result of ELISA showed that the titer of the rats' anti-ACE2 antiserum was 1:3200.Western blot analysis showed that the antibody could specifically bind both the recombined expression protein and the endogenous ACE2 from the pigs;5)Immunohistochemical results showed that ACE2 protein was expressed in every kind of tissues of pigs,especially notable at the bottom of the stomach,at the brush edge parts of intestinal epithelial cells and in glands of the large intestine.2.Construction of Eukaryotic Expression Plasmid pcDNA3.1(+)-ACE2 and Determination of its Enzyme.ActivityThis part aims at constructing a stable eukaryotic expression cell line and obtaining active proteins with ACE2 enzyme activity,which is a foundation for further research.The duodenum of pig was used for the following experiments:1)ACE2 gene was amplified by reverse transcription polymerase chain reaction(RT-PCR),and the product was cloned into pcDNA3.1(+)vector after it was identified by PCR and double restrictive edonuclease digestion and sequence analysis.Then the recombinant expression plasmid pcDNA3.1(+)-ACE2 was transfected into CHO cells.The transfected cells were screened first with G418 and then single clone so that the CHO cell line could consistently express gene by monoclonal screening;2)The ACE2 protein expression was identified using Western-blot and Immunofluorescence;3)The activity of ACE2 was determined using colorimetric assay.Results were listed as follows:1)The pcDNA3.1(+)-ACE2 eukaryotic expression plasmid was successfully established;2)7 CHO cell lines that can overexpress ACE2 protein were successfully selected by Western-blot,and were comfirmed by Immunofluorescence;3)The results of colorimetry showed that ACE2 protein extracted from CHO cells had enzyme activity at 7.42 nmol FAP/min.Conclusion:The pcDNA3.1-ACE2 eukaryotic expression plasmid was constructed successfully,and 7 strains of pcDNA3.1(+)-ACE2 stable cell strains were selected.The results showed that ACE2 expressed in CHO cells had good enzyme activity,which lays the foundation for further study of the immunological functions of ACE2 protein.3.Effect of ACE2 against LPS-Induced Inflammatory injury in IPEC-J2 cellsThis study used LPS to establish an inflammatory injury in IPEC-J2 cells and discussed the anti-inflammatory effect of ACE2.IPEC-J2 cells were used for the following experiments:1)Immunofluorescence was performed to determine ACE2's location in IPEC-J2 cells;2)LPS of different concentrations were applied to induce inflammatory injury in the IPEC-J2 cells.The optimal induction time was established using cell viability,the release of inflammatory cytokines and changes in cell mitochondrial membrane potential as indicators;3)After LPS treatment,expression levels of ACE2,MasR,ACE and ATIR in cells were measured for different LPS concentrations respectively(1ng/mL?10 ng/mL?100ng/mL?1000ng/mL?10000ng/mL),using Western Blot.The content of Ang?and Angl-7 in cell supernatant were determined using RIA and ELISA.Results:1)The immunofluorescence result showed ACE2 protein intensively located on the cell membrane;2)The inflammatory injury was successfully established in IPEC-J2 cells with the induction time being 4 h according to the changes in cells relative viability,inflammatory factors and mitochondrial membrane potential;3)After cells were exposed to LPS for 4 h,with the rise of the LPS concentration,the levels of ACE2,MasR and the content of Angl-7 showed an decreasing trend,while the levels of ACE,AT1R and the content of Ang?showed an increasing trend.It is to be noticed that the ratio of ACE/ACE2 first decreased and then increased.These results suggest that a valid inflammatory injury was induced after the cells were treated by different concentration of LPS for 4 h.Meanwhile,the ACE/Ang?/AT1R and ACE2/Ang 1-7/MasR axis were both activated.The ACE2/Angl-7/MasR axis plays the major role under low concentration LPS stimulation,where ACE2 gives anti-inflammation effect through degrading Ang? and mediating Angl-7/MasR,while under high concentration LPS stimulation,the ACE/An?/AT1R axis and its pro-inflammation effect predominates.