Fundamental Research Of Molecular Breeding Based On Sheep Functional Gene GCSF Regulation By Codon Extension Technique

The purpose of this paper is to study the molecular genetic breeding method of sheep functional genes regulated by gene codon expansion.Through the expression,bioinformation and functional research of granulocyte colony-stimulating factor(GCSF),which is an immune related protein,and the functional analysis of its mutant,which was expressed by N?-(tert-Butoxycarbonyl)-L-lysine's participated orthogonal system in sheep primary cells,the feasibility of molecular genetic breeding method using gene codon expansion technology to control sheep functional genes was confirmed.The thesis consists of three parts as follow:1 Characterization of isoforms of the ovine granulocyte colony stimulating factorThe granulocyte colony-stimulating factor regulates the maturation,proliferation,and differentiation of precursor cells of neutrophilic granulocytes,and has been widely studied in several species.To investigate the function of variants of sheep GCSF(sGCSF),this study compared difference in their mRNA expression levels.The results showed that both the activity and mRNA expression level of GCSFv2 were higher than those of GCSFv1.Their sequences were aligned,which showed that they had the highest homology with bovine GCSF.Then,predicted ovine GCSF isoforms and their constant C-terminals(named as GCSFwt)were cloned and expressed,which were stably expressed in mammalian cells.After purification,proliferation and differentiation stimulation functions of all GCSF were different both in vitro and in vivo,and the GCSFwt was the best.These results indicated that all GCSF variants owned the ability to stimulate both the proliferation and differentiation of progenitor cells and to activate the maturation of neutrophils,and could be used for research of efficacious non-antibiotic protein drugs.Furthermore,GCSF can be used as candidate target of genetic breeding to specifically improve sheep immunity.2 Construction of orthogonal expression system with participation of BocKThe orthogonal expression system with participation of BocK has been reported in a variety of prokaryotic and eukaryotic cells,but it was not reported in sheep primary cells.In order to verify the usful of the constructed system,GFPwt/GFP151TAG was as report gene in the expression system of unnatural amino acids incorporation,to confirm the reliability of the system with BocK participating in protein expression.In the aim of increase the expression efficiency of protein synthesis with BocK,the constructed plasmids were optimized.ExpiCHO-S cells,HEK293F cells,sheep granulosa cells and sheep embryonic fibroblasts were used in transformation.The results showed that BocK could be used in all these cells with constructed plasmids transformation to participate in GFP151 TAG-BocK expression.In this study,we successfully constructed an orthogonal expression system that can use BocK in protein expression,and proved the feasibility of the application of the orthogonal expression system in sheep granulosa cells and sheep embryonic fibroblasts,which would be the foundation for the study of unnatural amino acids participation in the functional protein expression and the corresponding expression regulation in sheep.3 The study of BocK participate functional protein orthogonal expression in sheep primary cellsIn order to study the feasibility of the orthogonal expression system of BocK participating in the functional protein expression of GCSF3TAG-BocK in sheep cells,three protein expression plasmids,pRTL1-GCSF3TAG,pLou-PylRSwt-8xRU-GCSF3TAG-BocK,and pLou-PylRSwt-8xRU-GCSFwt were constructed,to detecte the expression of mutant proteins with unnatural amino acid,BocK.The expressed and purified GCSF3TAG-BocK protein was bioassay by M-NSF60 cells proliferation.Then GCSF3TAG-BocK protein was injected into the mice for in vivo test,including metabolism and kinetics test of stimulate the production of granulocytes and their progenitors.The results showed that the same as the GCSFwt,GCSF3TAG-BocK protein concentration reached the maximum in 8 h and then rapidly metabolized to the low level,and it could be detected that protein stimulates bodies to produce a large number of granulocytes and their progenitors at 24 h.The purified GCSFwt/GCSF3TAG-BocK proteins were added in granulosa cell and sheep embryonic fibroblast culture medium in experiment groups with non-added as control.Alarmarblue was used to measure cell proliferation,and flow cytometry was used to detect cell cycles and apoptosis.RT-PCR was used to detecte expression level of GCSF after transformation in granulosa cells,and alarmarblue was used to measure GCSFwt/GCSF3TAG-BocK bioactivity of secretery expressed protein in supernatant.The result showed that granulosa cell activity was raise with GCSFwt/GCSF3TAG-BocK concentration from 0.06 to 600 ng/ml at 24 h and 48 h.Cell cycles were significantly different between experiment and control groups at 24 h.The apoptosis ratio of experiment group was significantly reduced(P<0.05)at 48 h.The expression level of GCSF after transformation plasmids with GCSF gene in granulosa cells raised more than 200 000 folds at 72 h.The bio-activity of GCSF could be detected with very significant difference at supernatant of granulosa cells,which were transferred with GCSFwt or GCSF3TAG with BocK adding in culture medium.The result showed that fibroblast cell activity was non-significantly affected with GCSFwt/GCSF3TAG-BocK concentration from 0.03 to 300 ng/ml.Cell cycles were significantly different between experiment and control groups.The apoptosis ratio of GCSFwt group was very significantly reduced(P<0.01)at 24 h and 72 h and non-significantly difference at 48 h.The apoptosis ratio of GCSF3TAG-BocK group was very significantly reduced(P<0.01)at 24 h,48 h and 72 h.The expression level of GCSF after transformation with GCSF gene in fibroblast cells raised more than 50 000 folds at 72 h.The bio-activity of GCSF could be detected with supernatant of transformation fibroblast cells,which were transferred with GCSFwt or GCSF3TAG with BocK adding in culture medium.In conclusion,GCSF could enhance cell proliferation,inhibit apoptosis,and regulate cell cycles on in vitro cultured sheep granulosa cells.Both GCSFwt and GCSF3TAG-BocK could be expressed in sheep granulosa cells with bio-activity.GCSF could regulate sheep fibroblast cells cell cycles and inhibit apoptosis.GCSF could be expressed in sheep fibroblast cells with bio-activity.At the molecular and cellular level,BocK participates in the expression of function protein,GCSF3TAG-BocK,which can be used in molecular genetics and breeding.