Genetic Research Of Porcine Coagulation Related Factors

Pig is a kind of valuable biomedical model organism, widely used in avariety of medical researches. In recent years, pig is considered as the mostpromising candidate of xenotransplantation to resolve the great donor graftsshortage, due to its advantages in size, breeding, less risk of virus diseasetransmission, and ethical consideration. In the last ten years, the rapidprogresses in xenotransplantation research have promoted the development ofporcine genomics and proteomics.A series of immunological and physiological barriers must be overcome forthe successful clinical application of xenotransplantation. The acute phases ofxenograft rejection have been prevented or at least attenuated by a variety ofinterventions including treatment of the recipient and genetic modification ofthe donor. The production of alpha1,3-galactosyltransferase gene-knockout(GT-KO) pigs and human complement regulation protein transgenic pigs hasovercome the barrier of preformed anti-Galalpha1,3Gal (Gal) antibodies.Survival of pig organs in nonhuman primates is currently limited by thedevelopment of a thrombotic microangiopathy that results in increasing ischemic injury of the transplanted organ over weeks or months. Potentialcausative factors include vascular endothelial activation from preformedanti-nonGal antibodies or cells of the innate immune system. But it may alsobe facilitated by the coagulation dysregulation associated with molecularincompatibilities between pig and primate, which means porcine coagulationregulators couldn't efficiently regulate the primate coagulation cascade. Inrespect of liver xenotransplantation, if pig derived coagulant, anticoagulantand fibrinolytic proteins could take place of human proteins efficiently andhave normal interactions with coagulation regulators on the surface ofendothelia cells and in peripheral blood are critical questions presented beforexenotransplantation researchers.Banna minipig inbred line (BMI), developed in Yunan AgricultureUniversity of China by consanguineous mating for more than twenty years, isthe unique actual highly inbred minipig of the world, and is considered as theideal candidate for pig-to-human xenotransplantation. Therefore, investigatingthe genetic background of BMI would shed light on xenotransplantationresearch.In this research, we successfully construct a cDNA expression library fromBMI liver tissue. The library contains 1.05×10~6 indepentant clones.The titersof the primary and amplified libraries are 0.7×10~6 pfu/ml and 4.87×10~9 pfu/mlrespectively. The percentages of recombinants are 96.6% in primary libraryand 97.5.0% in amplified library. The lengths of most inserts are between 500bp and 2.0 kb.To gain an insight into the expression profile of BMI liver tissue, werandomly single-pass sequence 200 clones of the cDNA library. The results ofsequence alignments show that 89 clones encode previously known porcine genes, including some high abundance genes such as albumin, cytochromeP450, and apolipoprotein. 70 clones match no porcine genes, while have highsequence similarities with known genes of other species, and most of themshare high similarities with human. The other 33 clones showing nosimilarities with genes in GenBank would be the new ESTs specific to BMIliver. Through protein alignments, 22 of the 192 clones were determined withfull length encoding regions, 38 with complete 5'terminent sequences, and 140have complete 3'terminent sequences. These new discovered expressedsequences would be important resource for research on physiological characterand medical usage of inbred pig and contribute to porcine genomic sequencingproject.Coagulation factor(CF)Ⅱ,Ⅶ,Ⅹ, antithrombinⅢ(AT-Ⅲ) andplasminogen (PLG) have played pivotal roles in coagulant, anticoagulant andfibrinolytic systems. However, there are few report about their nucleotidesequences and physiological, functions in pig. To gain better understanding oftheir roles in xenotransplantation, we cloned the full length eDNA of thesegenes through library screening, RACE technique and electronic cloning. Inresult, the full length eDNA of BMI CFⅡ, CFⅦ, CFⅩ, AT-Ⅲand PLG are2027 bp, 1416 bp,1856 bp, 1498 bp, and 2768 bp, respectively. All of thesesequences were submitted to GenBank, and the accession numbers areDQ530370 to DQ5303704.In the forth part of this dissertation, we analyzed the nucleotide and proteinsequences of cloned BMI CFⅡ, CFⅦ, CFⅩ, AT-Ⅲand PLG bybioinformatics tools. Comparisons of the primary sequences andthree-dimentional protein structures between BMI and human genes give ussome significant suggestions about the function similarities and potentialmolecular incompatibilities. The results show: the nucleotide similaritiesshared by BMI and human CFⅡ, CFⅦ, CFX, AT-Ⅲand PLG are 83.84%,78.34%, 79.81%, 87.67% and 80.21%, respectively; the amino acid similarities of them are 83%, 74.08%, 73.10%, 89.06% and 80.67%,respectively. Another important discovery through sequence assignments isthat each Cys of these proteins is rigidly conserved among different species,and the catalytic triad His-Asp-Ser is also conserved in all of the serineproteinases. Homology modeling of the three-dimentional structure of BMICFⅡ, CFⅦ, CFⅩ, AT-Ⅲand PLG by Swiss-Model program provide thepossibility to investigate the protein function and molecular incompatibilitiesin more natural forms. From comparisons between the modeled 3-D structureof BMI proteins and crystal structures of human proteins, we could see theconsiderably resemble conformations of their main atom backbones, and manyimportant function and activity related binding sites, which suggests that thesecoagulation factors may share similar physiological function and catalyticactivity across species. However, the sequence differences of some specialsites between BMI and human might contribute to different conformationchanges of some key atoms and construct different hydrogen bonds ormolecular surface to hinder the binding of special xeno-ligands, and result inimpaired affinity of these pig derived coagulation factors with the humanderived substrates and regulator molecules.In the last part, we did some work to study the in vitro expression system ofBMI CFⅡ. We successfully constructed the recombinant prokaryotic E. coliexpression vector PET22b-cfⅡ, Pichia pastoris expression vector pPIC-9k-cfⅡand eukaryotic expression vector pcDNA3.1~+-cfⅡ. When prokaryoticexpression in E. coli, cfⅡis expressed as inclusion bodies. In Pichia pastorisexpression system, through SDS-PAGE and Western-Blot with anti-His tagantibody, target protein is detected in the plasma of His~+ and Geneticin~+recombinants. But the result suggests the expression product is at very lowconcentration and has no detectable biological activity. Finally, CHOmammalian cell expression system is introduced. After screening thetranfected cells, we generate the stable integrant cell line with anti-G418 property. Unfortunately, no target protein is detected in culture media byWestern Blot. The causative interpret for the failure is that the large moleculeweight, complex structure of CFⅡand indispensable post-translationalmodification for its biological activity increase the difficulty of in vitroexpression. More work should been done to optimize the expression system tofind a better way for coagulation factors expression with good quantity andgood quality.In summary, our research successfully constructs a cDNA library of BMIliver tissue, primarily studies the character of gene expression profile of BMIliver, clones full length cDNA of important coagulation related factor CFⅡ,CFⅦ, CFⅩ, AT-Ⅲand PLG; analyzes the difference between BMIcoagulation factors and human homologues, and gets some experiences in invitro expression of BMI CFⅡ. These work will benefit further study of proteinfunction of porcine coagulation factors, and shed light to investigation ofmolecular mechanism of coagulation disorder, function compatibility andgenetic modification in xenotransplantation.