Construction Of A CDNA Library From Liver Tissue Of Rhesus Macaque And Genetic Research Of Rhesus Macaque Coagulation Related Factors

Rhesus monkeys (Macaca mulatta) are human's closest evolutionary relatives next to Chimpanzees, which makes them widely used in biomedical researches. As the great development of xenotransplantation, pig to Rhesus monkey transplantation has become the most important animal model to investigate the various problems in this field. However, there are still some intrinsic evolutionary differences in monkey and human, so it is a serious issue to pay attention to, that if the experimental results from pig to monkey transplant could veritably reflect the conditions after porcine tissues or organs were transplanted into human body. Experiments in animal models have suggested that coagulation disorder is one of the most important hindrances of xenotransplantation, and the immunologic injure by hyper acute rejection and molecular incompatibility of coagulation related factors may be the main contributors. Nevertheless, the mechanisms have not been well understood. Therefore, it is of great importance to deeply investigate the genetic backgrounds and specialties of Rhesus monkey coagulation related factors, and compare its coagulation system with that of pig and human. It will benefit to construct animal model more effectively and discover the potential mechanisms and treatments for coagulation disorder in xenotransplantation. Genetic study of Rhesus monkey could also contribute to correctly understand and apply the experimental results of Rhesus monkey models to guide research or therapy in human, and promote the safe and effective xenotransplantation to be used in clinic practice.cDNA library construction is an important technique in molecular biology. Screening and cloning new genes from cDNA library is an effective and time-saving strategy of gene cloning. However, it do need some technological bases for quickly and effectively constructing a cDNA library with high quality and getting useful sequence information of target genes. In our study, we firstly constructed a peripheral lymphocyte cDNA library of Banna Minipig Inbred Line (BMI) withλZAP Express vector andλTripIEx2 vector, then developed a convenient, quick and high-quality cDNA library construction method with some improvements of the basic protocol, and successfully constructed a liver cDNA library of BMI. Secondly, we cloned porcine coagulation factorⅦ(FVⅡ) from BMI liver cDNA library by PCR and phage hybridization, and successfully developed a cDNA library screening strategy.Most of the coagulation factors are synthesized by liver. To investigate the Rhesus monkey coagulation related factors, we successfully constructed a liver cDNA express library of Rhesus monkey withλZAP vector. The capacity of primary library is 1.2×10~6 clone/ml, and the capacity of amplified library is 7.7×10~9 clone/ml with 99% recombination rate. The average length of inserts is 2.5 kb, which is proper for further study.Coagulation factor (CF)Ⅰ,Ⅶ,Ⅹ, and anti-thrombinⅢare important in coagulation, anticoagulation and fibrolysis systems, we cloned the full-length cDNA of Rhesus monkey AT-Ⅲ, CFⅡ, CFⅦ, and CFⅩgenes by combined methods of screening the Rhesus monkey liver cDNA library with PCR and 5' RACE reaction. The cDNA of these four genes are 1545 bp, 2029 bp, 2424 bp and 1683 bp, respectively, which have been submitted to GenBank and the Accession Number are EF057492, EF057490, EF061407 and F057491, respectively.We preliminarily analyzed the nucleotide and deduced amino acid sequences of Rhesus monkey AT-Ⅲ, CFⅡ, CFⅦ, and CFⅩgenes. Through comparing the sequences and modeled protein structure with corresponding genes of pig and human, we got some information of the similarities and possible differences in the three species. The results showed that the nucleotide identities of AT-Ⅲ, CFⅡ, CFⅦ, and CFⅩbetween Rhesus monkey and human were 95.22%, 95.72%, 91.65% and 94.68%, respectively, while the amino acids identities were 96.01%, 91.11%, 89.83% and 93.65%, respectively. Sequence comparisons between these genes of pig and Rhesus monkey showed that the nucleotide identities were 89.33%, 86.14%, 76.71% and 76.63%, respectively, while the amino acids identities were 89.33%, 89.03%, 70.61% and 72.02%, respectively. The results also suggested that these coagulation factors of the three species shared the same domain pattern and cys sites. The main catalytic sites were highly conserved in sequences and positions, while some macromolecular recognition sites and some functional sites were different in the three species, such as glycosylation sites, phosphorylation sites and enzyme digestion sites. When three dimensional protein structures of coagulation related factors of Rhesus monkey, pig and human were concerned, the backbones and conformation of these proteins were considerably resemble, which suggested they might share the similar physiological functions and catalytic activities. Otherwise, some sequence differences at special sites might result in some significant changes in conformation and protein energy, and led to different binding capacity to the substrates or other interacted macromolecules.To verify the cloned coagulation factors of Rhesus monkey, and benefit the further production of massive monkey protein for in vitro and in vivo studies, we constructed the recombined prokaryotic express vector pET-22b/mAT-Ⅲand pET-DsbA/mAT-Ⅲ, recombined eukaryotic express vector peDNA3.1(+)/mAT-Ⅲand recombined Pichia pastoris express vector pPIC9K/mAT-Ⅲto express Rhesus monkey AT-Ⅲin vitro by the three kinds expression systems. We successfully got the target protein with enzyme activity from Pichia pastoris express system, which would provided some useful experience to further preperation of recombinated protein of other coagulation factors.In summary, our study has constructed proper strategies for cDNA library construction and gene screening; successfully constructed Rhesus monkey liver cDNA express library; cloned the full-length cDNA of important coagulation related factors AT-Ⅲ, CFⅡ, CFⅦ, and CFⅩof Rhesus monkey; preliminarily compared the sequences and protein structures of Rhesus monkey coagulation factors with that of pig and human by bioinformatics tools; investigated the in vitro expression of Rhesus monkey AT-Ⅲ, and acquired the functional production that demonstrated the sequence correctness. The achievements of this study would provide valuable genetic information to develop and utilize the effective animal model of primates in xenotransplantation, and impulse the protection, exploitation and application of the precious resources of Rhesus monkey in our country.