Cloning,Expression And Antibody Preparation Of Goat Angiotensin Converting Enzyme 2 (ACE2)

This study aimed at cloning goat angiotensin angiotensin converting enzyme 2(ACE2)gene and construct expression vector for protein expression and antibody preparation.The research includes the following three aspects:1 Clone and Bioinformatic Analysis of Angiotensin Converting Enzyme 2(ACE2)Gene from GoatsTo determine ACE2 gene sequence,we used homogeneous clone and RT-PCR method.First,primers were designed on the basis of the mRNA sequence of ACE2 in cattle.Second,specific fragments of ACE’s coding sequence were amplified from the kidney tissues of goats.These specific fragments were then cloned into pMD19-T carrier using T-A method and transferred to DH5a competent cells.In the end,the ACE2 gene sequence was determined after the colonies of DH5α were validated.The result showed the cDNA coding sequence of ACE2 in goats covered 2,415 nucleotides and coded 804 amino acid residues.Sequence homology analysis showed that the sequence in goats shared the highest homology(99%)with that in sheep and the lowest homology(60%)with that in zebra fishes.Genetic evolution analysis showed that this gene in goats has the shortest genetic distance with such gene in sheep,but resides in a totally different branch from the ACE2 gene in zebra fishes,which accords with the general genetic evolution rules.Analysis of protein structure and physicochemical properties predicted that ACE2 protein is a stable type I transmembrane protein with its signal peptide sequences locating between laa and 18aa.In this experiment,the full length of ACE2 gene in goats was first successfully cloned,and its protein structure and physicochemical properties were predicted.The sequence has been uploaded to the GenBank.Its gene sequence number is KF921008.1.2 Protein Expression of the goat ACE2 in E.coliAccording to the sequence of goat’s ACE2 gene,the primers of ACE2 was designed.Specific bands was amplified from pMD19-ACE2 extracellular domain(19-738aa)gene through the primers.The amplified products were digested by BamH I and Hind III enzymes respectively,and connected with pET28a and pET32a vectors,then transformed into E.coli BL21(DE3).We successfully constructed prokaryotic expression vector pET28a-ACE2 and pET32a-ACE2.The expression of ACE2 protein was induced by IPTG 0.25mM at 28℃.After 16h,the protein was tested by SDS-PAGE and Western-Blot for its molecular weight and immunogenicity.The results found that the ACE2 protein was expressed in the form of inclusion body in the precipitation of the bacteria.the protein was washed,denatured,purificated and renaturated.Results showed that the protein from pET28a-ACE2 was 90kDa by SDS-PAGE,and while ACE2 protein from pET32a-ACE2 was 100 kDa.Both of them was mainly existed in the form of inclusion bodies.Western-blot analysis showed that the protein have immunogenicity.In this experiment we successfully constructed pET32a-ACE2 and pET28a-ACE2 expression vector,and proved that the expression of ACE2 extracellular region protein mainly existed in a form of inclusion body,The molecular weight of the expression protein was about 90kDa and 100kDa.3 The Preparation and Biological Identification of ACE2 Polyclonal Antibody in GoatsThe ACE2 polyclonal antibody can be obtained using the purified antigen protein expressed by the recombinant gene,pET28a-ACE2.In this study,ten rats of hygienic quality were selected.They were injected subcutaneously with the recombinant protein expressed by pET28a-ACE2 repeatedly,0.8mL/protein per rat.After 4 weeks of injection,the antibody tests all showed positive.Then the rats were killed and the ACE2 antibody was separated out from the serum of these mice.The titer of the antibody was determined by ELISA method so as to determine the optimal antigen coating concentration and the best dilution to use.The prepared antibody was tested by Western-blot and immune histochemistry method for its specificity,which also showed roughly the location of the antibody in a cell.Results:the ELISA showed that the optimal antigen coating concentration was 0.6 μg/ml.Using this concentration,the immune tests of the obtained serum all showed positive.The ELISA also showed the optimal dilution was 1:200.Western-blot analysis showed that the prepared antibody had a good specificity since a single ACE2 protein band was found.The immunohistochemical detection found ACE2 protein positive reactions in the rumen,reticulum,omasum,abomasum,duodenum,jejunum and colon of goats,which was also an indirect evidence of the specificity of the prepared antibody.In this study,through gene clone,gene expression and repeat immunization,the ACE2 polyclonal antibody was obtained with a good titer and specificity.Thus it can be used for further study.