Sequence Analysis,Polyclonal Antibody Preparation And Construction Of Recombinant Lentivirus Vector Of Porcine Aminopeptidase N Gene From Local Pig Breeds In Three Local Pig Breeds In Guangxi

Viral diarrhea is the main cause of early death in piglets,among which Porcine transmissible gastroenteritis virus(TGEV),Porcine epidemic diarrhea virus(PEDV)and Porcine deltacoronavirus(PDCo V)are the most serious.Luchuan pigs,Bama miniature pigs and Longlin black pigs are famous local pig breeds in Guangxi province,but the sustainable development of these breeds is affected by viral diarrhea.Virus-receptor interaction is the first step of virus infecting host cells,related studies have confirmed that Porcine aminopeptidase N(pAPN)is related to the invasion of coronaviruses such as TGEV,PEDV and PDCo V,and plays an important role in virus infection.At present,there are few reports on pAPN related to local pig breeds in Guangxi.Therefore,this study focuses on three local pig pAPN in Guangxi.1.Cloning,sequencing and bioinformatics analysis of pAPN gene of local pig breeds in Guangxi.In this study,the pAPN genes of Luchuan pig,Bama miniature pig,Longlin pig and Duroc × Landrace × Yorkshire(DLY)pigs were obtained by RT-PCR amplification.The open reading frame are all 2892 bp,encoding 963 amino acids,and the homology was 99.2%～100%.Based on bioinformatics analysis,it was found that the obtained pAPN was located at positions between 12 to 34 amino acid.There was no O-glycosylation sites,and there are 7 high specific Nglycosylation sites.The secondary structure was dominated by α-helix and the tertiary structure was homodimer.Compared with NM-214277,the mutations of F82 N,L107F and L108 I were found in the pAPN genes of Luchuan pig,Bama miniature pig,Longlin pig and DLY pig,in which the mutation of F82 N were added with a highly specific N-glycolylation site.2.The establishment of real-time fluorescence quantitative PCR method for pAPN genes and the study of tissue expression.The Real-time fluorescence quantitative PCR(RT-q PCR)technology of pAPN was established in this study,the correlation coefficient of the standard curve was 0.9917,which showed a good linear relationship.RT-q PCR was used to quantify pAPN genes in the heart,liver,spleen,lung,kidney,duodenum,jejunum,ileum,colon and caecum of DLY pigs,and the results showed that pAPN was detected in all tissues and had the highest content in jejunum.3.pAPN protein expression and preparation of polyclonal antibody(Pc Ab).In this study,the pAPN gene of Longlin black pig was cloned into the prokaryotic expression vector of p ET-32a(+),to construct recombinant expression plasmid p ET-32a-pAPN,after it has been verified by sequencing,and then was transformed into E.coli Rosetta(DE3)cells.The recombinant pAPN expressed by IPTG induction,and was identified by Western blot.After soluble analysis of the recombinant protein,the expressed fusion protein was purified by denaturation,Ni-column affinity purification and renaturation.The polyclonal antibody was prepared by the immunization to the rabbit,and the good reactivity of antibody was identified by Western blot,which lays a foundation for further research and development of reagents and antiviral drugs.4.Construction,packaging and identification of pAPN recombinant lentivirus vectors.In this study,the pAPN gene of Longlin black pig was used to replace the GFP label on the plenti-CMV-GFP-hypro vector,and the recombinant lentivirus plasmid Plenti-CMV-pAPN-hypro was constructed.The recombinant virus was obtained by co-transfection into 293 T cells with the recombinant lentivirus plasmid,ps PAX2 and VSVG,which could infect LLC-PK1 cells,laying the foundation for the construction of stable expression cell lines.The pAPN gene was amplified from local pigs in Guangxi and the mutation sites were screened out,providing a theoretical basis for the subsequent screening of effective genetic markers of Co V for disease-resistant toxic diarrhea in pigs in Guangxi.The pAPN fluorescence quantitative PCR method was established in this study provides effective support for subsequent detection experiments.pAPN polyclonal antibodies and recombinant lentivirus were prepared in this study,laying the foundation for further biological function research and the pathogenic mechanism research of related viral diarrhea CoV.