| The biggest problem of organ transplantation in the treatment of disease is a serious lack of donor organs.Patients with multiple organ failure died due to inability to obtain timely donor organs.Xenotransplantation has been recognized as an efficient way to solve the shortage of donor organs by the scientific community and the medical profession and the pig has been considered the most suitable non-primate xenotransplantation donor.The major technique barrier for pig human xenotransplantation is the hyperacute rejection(HAR),in which ? 1,3 galactose(?1,3Gal)is the major antigen that causes HAR.In clinical practice,rejection action may still occur as pig organs with ?1,3 Gal knocked were transplanted into baboons,indicating that there are still other heteroantigens besides? 1,3 Gal.In order to realize clinical application of organ transplant pig-people as soon as possible,the majority of medical and scientific researchers are focused on non-?1,3 Gal genes.In this experiment,the unique material BMI,known as an excellent donor for pig?human xenotransplantation,was selected as the experimental material,and 5 non-?1,3Gal genes,CMAH?OXSR1?CD80?ASGR1 and B4GALNT,related to pig?human xenotransplantation rejection reaction were selected as candidate genes.The correlation between the 5 genes and pig?human xenotransplantation rejection reaction was verified in the molecular,protein,and cellular level simultaneously.Firstly,these genes were isolated from the mRNA and the complete coding region were obtained.Secondly,the multiple immune-related tissues expression levels were checked by qPCR at the mRNA level,and the results were verified by the Western Blot at the protein level and were confirmed at the cell level employing fluorescence detection which purpose gene were fused into green fluorescent protein GFP fusion expression vectors.Thirdly,those proteins' functional biological information including basic information,feature information,structural domains,functional sites,higher-order structure was analyzed.The results showed that full-length CDS sequences of pig CMAH,OXSR1,CD80,ASGR1 and B4GALNT2 gene were 1734 bp,1590 bp,894 bp,861 bp,1509 bp,respectively.The results of qPCR analysis and Western Blot showed that the highest expression of CMAH,OXSR1,CD80,ASGR1 and B4GALNT2 was in submandibular gland tissue,testis,spleen,liver and tonsillar,respectively.The results revealed the differentially expressions of the same gene in different tissues and different genes in the same tissue.It also indicated the important genes,organizations and target tissues will be focused on further.These genes were fused to AcGFP1 and were transfected into porcine kidney epithelial PK15 cells for transient expression,respectively.The positive results were detected in PK15 cells.To evaluate the correlation between these pig genes and human immunological rejection reaction,these fused genes were transfected into human umbilical vein endothelial cells HUVEC and green fluorescence stable expression were obtained indicated these genes related to pig human xenotransplantation related immune rejection.The results helped to elucidate the molecular mechanism of these genes in pig?human xenograft rejection.To understand further the relevant function of the proteins encoded by the genes,functional bioinformatics analysis was performed including basic information such as amino acid composition,molecular formula,molecular weight,isoelectric point,extinction coefficient,half-life period,atomic composition,instability coefficient,fat index,and hydrophilic coefficient;feature information such as hydrophobicity,transmembrane structure,coiled coil,signal peptide,inherent disorder,leucine-rich nuclear export signal,and subcellular localization;structural domain and function site(phosphorylation and glycosylation);secondary structure and tertiary structure.These results provide a theoretical basis not only for further functional studies and verification but also for knockout and modification of genes involved into pig?human xenotransplantation related immune rejection.This study fills a gap in non HAR beside ? 1,3Gal.The results provide not only the basic data material for the application of BMI in pig?human xenotransplantation,but also scientific basis for the development and utilization of BMI. |
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