Cloning And Expression Of Chicken Angiotensin Converting Enzyme 2(ACE2) And Preparation And Identification Of Polyclonal Antinody

Angiotensin converting enzyme 2(ACE2)acts as the renin-angiotensin system.Since it was found in 2000,the pathophysiological function of RAS,especially as the functional receptor of SARS,SARS-coV-2 and other coronaviruses invading cells,has shown great potential,but the related research on ACE2 in chicken has not been carried out.In this study,the chicken ACE2 gene was cloned,the prokaryotic and eukaryotic expression system of chicken ACE2 was established,the polyclonal antibody of ACE2 was prepared,and the active protein of ACE2 was obtained.The research includes the following three aspects:1.Cloning and bioinformatics analysis of ACE2 gene in white feather broilerWhite feathered broilers were selected as the research objects.Firstly,the expression of ACE2 gene and protein was determined by RT-PCR and Western blot.Then,the specific fragment of ACE2 gene coding region was amplified from jejunum tissue of white feathered broiler.T-A Cloned into pMD-19T vector and transferred to DH5α.The sequence was analyzed by bioinformatics.Results:1)ACE2 was found to exist in the body of white feathered broiler,and the expression of ACE2 was tissue-specific.2)The whole gene sequence of ACE2 coding region of white feathered broiler was cloned successfully,which contains 2444 nucleotides and 808 amino acid residues.The comparison of homology shows that the highest homology of the nucleotide sequence of ACE2 gene in chicken is 99%with that in Hongyuan chicken,and the lowest homology is 50%with that in human.Genetic evolution analysis showed that the ACE2 gene was the closest genetic distance between pig,and was in two different branches with cattle and sheep.3)Bioinformatics analysis showed that the ACE2 gene coding protein was an unstable and hydrophilic protein,and the protein signal peptide sequence was between 1-17aa.The protein transmembrane helix was predicted to be type I transmembrane protein.The cells were mainly distributed in cell membrane,endoplasmic reticulum and cytoplasm.There are 87 phosphorylation sites in the whole polypeptide chain,including 37 serine sites,32 threonine sites and 18 tyrosine sites.There was no O-glycosylation site,mainly N-glycosylation site.According to the analysis of 18 key amino acid residues related to the binding of SARS S protein,except the 330 and 353 amino acids of ACE2,the other 16 amino acids of ACE2 were not consistent.In this experiment,the complete gene sequence of ACE2 coding region of broiler was cloned successfully for the first time,and its protein structure and physical and chemical properties were preliminarily predicted.The obtained sequence has been uploaded to GenBank,and the GenBank accession No.is MK560199.2.Prokaryotic expression of ACE2 and preparation and identification of its polyclonal antibodyThe pMD19T-ACE2 plasmid obtained in the previous chapter was digested and recovered.The purified target fragment was linked with the prokaryotic expression vector pET32a which was digested by double enzymes.The prokaryotic expression vector pET32a-ACE2 was constructed,and the linked product was transformed into the competent cells.The monoclonal colonies were selected and sequenced after PCR and enzyme digestion.The expression of ACE2 protein was induced by IPTG and the conditions of induction were optimized.The expressed protein was purified by KCL staining,and the specificity and molecular weight of the expressed protein were identified by SDS-PAGE and Western blot.Isoelectric point was determined by isothermal focusing with tubular gel.The purified recombinant ACE2 protein expressed in pET32a-ACE2 was used as antigen to immunize rats.After 4 weeks of immunization,the antibody was determined to be positive,and then the rats were slaughtered.The ACE2 polyclonal antibody was obtained from the serum.The titer of antibody was measured by indirect ELISA,and the best concentration and dilution of antibody were determined.Western blot,immunohistochemistry and immunofluorescence were used to detect the specificity and localization of the antibody.Results:1)The prokaryotic expression vector of pET32a-ACE2 was successfully constructed.The optimal expression condition of the target protein was IPTG concentration of 1mmol/L,induction time of 10h.The expression of ACE2 protein was mainly in the form of inclusion body.2)The recombinant protein of chicken ACE2 was obtained by KCL staining and gel cutting,which was single chain protein with molecular weight of 105kDa and isoelectric point of 5.01.3)The rat-anti-chicken ACE2 polyclonal antibody was successfully prepared.By ELISA,the optimal concentration of the antigen was 1.6ug/mL,and the antibody titer was 1:400.Western blot analysis showed that the polyclonal antibody against ACE2 had good specificity,and a single band of ACE2 protein appeared.The results of immunohistochemistry showed that ACE2 protein was positive in lung,duodenum and testis,which indirectly proved the specificity of the antibody.Immunofluorescence analysis showed that ACE2 was expressed in LMH cells,and most of ACE2 was distributed on the cell membrane.Conclusion:the prokaryotic expression vector of chicken pET32a-ACE2 was successfully constructed,and the recombinant protein with good antigenicity and specificity was obtained.The chicken ACE2 polyclonal antibody was successfully obtained through cloning,expression and multiple immunizations.The antibody has good potency and specificity,and can be used for further research of ACE2 in white feather broilers and poultry.3.Establishment of eukaryotic expression system of chicken ACE2 and screening of stable cell lineThis chapter aims to construct a stable eukaryotic expression cell line and obtain the active protein with ACE2 activity.In this study,the jejunal tissue of white feathered broiler was used for the following experiments:1)the full-length sequence of chicken ACE2 was amplified by RT-PCR.After the enzyme digestion identification was correct,the ACE2 gene fragment was connected to pcDNA3.1(+)by homologous recombination.After the identification of bacterial solution PCR,enzyme digestion and sequencing,it was transfected to CHO cells by liposome method.2)The expression of ACE2 gene in CHO cells was detected by RT-PCR,Western blot and immunofluorescence.3)The enzyme activity of ACE2 active protein was determined by double antibody sandwich method.Results:1)Eukaryotic expression vector pcDNA3.1(+)-ACE2 was successfully constructed in vitro,7872bp fragment was found by single enzyme digestion,and the sequencing results were consistent with the expectation.2)The recombinant vector pcDNA3.1(+)-ACE2 was successfully transfected into CHO cells,and RT-PCR results showed that there was high mRNA expression in CHO cells;immunofluorescence results showed that pcDNA3.1(+)-ACE2 recombinant protein was evenly distributed on the cell membrane,and had high transfection efficiency;Western blot results showed that pcDNA3.1(+)-ACE2 recombinant protein was highly expressed in CHO cells,and the protein size was about 105kDa.3)Four cell lines with stable expression of ACE2 protein were successfully screened,named pcDNA3.1(+)-ACE2-A,pcDNA3.1(+)-ACE2-B,pcDNA3.1(+)-ACE2-C and pcDNA3.1(+)-ACE2-D.4)The results of double antibody sandwich method showed that the activity of ACE2 protease extracted from CHO cells was 601.77U/L,and the enzyme activity was high.ACE2 protein with biological activity was obtained,which laid a foundation for further study on the biological role of ACE2 in chicken.