Genetic Research Of Macaca Mulatta Coagulation Factors Of Ⅶ, Ⅸ And Ⅺ

Coagulation disorder is a critical matter in xenotransplantation, especially inliver xenotransplantation. Macaca mulatta is a kind of valuable biomedicalmodel organism, widely used in xenotransplantation researches. If the datafrom the pre-clinical study of pig-to-rhesus liver xenotransplantation could beas a suggestion applied in pig-to-human, is not known yet. To resolve thisproblem, it is important to give a research on coagulants of rhesus, and thedifference of coagulants between rhesus and human. So here we show theanalysis of EST from cDNA library of rhesus liver tissue, two coagulantcDNA sequences cloned from the cDNA library, and in vitro expressionsystem of rhesus Fâ…¦.To gain an insight into the expression profile of rhesus liver tissue, werandomly single-pass sequenced 401 clones of the cDNA library. Thesequences were treated by Cross_match and Phrap, and finally produced 221unigenes. The results of protein alignments showed that 16 unigenes encodepreviously known rhesus proteins. 171 unigenes matched no rhesus proteins,while had high sequence similarities with known proteins of human. Theresults of sequence alignments showed that, 26 of the other 33 unigenesshowing no similarities with proteins in Swiss_prot, encoded previouslyknown rhesus genes. 3 unigenes (Contig28,Singlet165,243) would be the new ESTs specific to rhesus liver by dbEST matching.Coagulation factors of Fâ…¦, Fâ…¨, and Fâ…ªhave played pivotal roles incoagulant system of rhesus. To gain clear understanding of their roles in bloodclotting, we cloned the full length cDNA of these genes through libraryscreening, PCR technique. In result, the full length cDNA of rhesus Fâ…¨, was2668 bp, and partial length cDNA of rhesus Fâ…ªwas 1646 bp. The sequence ofrhesus Fâ…¨was submitted to GenBank, and the accession number isEU128170.In following part of this dissertation, we analyzed the nucleotide and proteinsequences of cloned rhesus Fâ…¨by bioinformatics tools. Comparisons of theprimary sequences and three-dimentional protein structures between rhesusand human genes gave us some significant suggestions about the functionsimilarities and potential molecular compatibilities. The results showed: thenucleotide similarities shared by rhesus and human Fâ…¨is 96.21%, and theamino acid similarities of them was 97.18%. A detail analysis showed theamino acid similarities shared by rhesus and human Fâ…¨domains of Gla,EGF1, EGF2 and Trypsin were 96.92%, 100%, 92.31% and 97.38%,respectively. Another important discovery through sequence assignments wasthat each Cys, glycosylated and carboxylated site of the two proteins was rigidlyconserved in human and rhesus, and the catalytic triad His-Asp-Ser was alsoconserved in them. From comparisons between the modeled 3-D structure ofrhesus protein and crystal structure of human protein, we could see theconsiderably resemble conformations of their main atom backbones, and manyimportant function and activity related binding sites, such as autolysis loop,the 99-1oop, and Ca²⁺ binding region, which suggested that the two proteinsmay share similar physiological function and catalytic activity across species.In the last part, we did some work to study the in vitro expression system ofrhesus Fâ…¦. We successfully constructed the recombinant prokaryotic E. coliexpression vector pET-DsbA-MCFâ…¦and Pichia pastoris expression vector pPIC-9K-MCFâ…¦. When prokaryotic expression in E. coli, was mainlyexpressed as inclusion bodies, and little as soluble, through SDS-PAGE andWestern-Blot with anti-His tag antibody. Unfortunately, no target protein wasdetected in Pichia pastoris expression. More work should been done tooptimize the expression system to find a better way for rhesus Fâ…¦expressionwith good quantity and good quality.In summary, our research successfully constructed a cDNA library ofMacaca mulatta liver tissue, primarily studied the character of gene expressionprofile of rhesus liver, cloned full length cDNA of important coagulation Fâ…¨,and partial length cDNA of Fâ…ª, analyzed the difference between rhesus Fâ…¨and human homologues, and got some experiences in vitro expression ofrhesus Fâ…¦. These works will benefit further study of protein function ofrhesus coagulation factors, and shed light to investigation of molecularmechanism of coagulation disorder and function Compatibility inxenotransplantation.