Preparation Of Recombinant Leptin And Leptin Receptor Protein Of Tibet Mininpig

BackgroundWith the improvement of people's living standard and changes in dietary structure, obesity is becoming prevalent around the world. Not only an independent disease, obesity is also a predisposing factor of diabetes, fatty liver, hypertension and other diseases. Studying the causes of obesity and developing methods for control and treatment of obesity has become very urgent. An ideal animal model of obesity is the foundation of research in obesity mechanisms and development of obesity drugs. Rodents have been the most used animals in modeling obesity syndrome and the general methods include high-fat and sugar diet, electrolysis, sodium glutamate, and Kim S glucose method. Additionally, genetic obese rats and diabetes animal models have also been developed. A group of U.S. scientists found that leptin and its receptor (OBR) dysfunction and leptin resistance is one of the main causes of obesity. This suggests studying leptin and its receptor function may provide a new way in establishing animal model of obesity. Minipigs, having many similarities with human in metabolism, organ morphology and physiological functions, and with advantages like small in size and convenient for operation, are ideal animals in preparing metabolic disease models. Tibet minipig, a new kind of laboratory animals, is one of the smallest and lightest pigs. The clarity in genetic background,100% gene purity, lean-meat type and easiness for testing and observation after modeling, make Tibet minipig an ideal animal for establishing obesity syndrome model.ObjectiveThe purpose of this work is the cloning of leptin and its receptor genes, and prokaryotic expression and preparation of fusion protein of the mature peptide of leptin and leptin receptor, so as to collect fundamental materials and set up ground for further study in establishment of obesity Tibet minipig model. The traditional methods can not simulate the pathophysiology of human obesity and are limited to rodents or extreme conditions. Few reports have been found on establishment of obesity model, control and treatment of obesity by immunization methods using leptin and leptin receptor gene engineering vaccine. Therefore, create a new animal model of human obesity based on Tibet minipig, using leptin and its receptor gene engineering vaccine, is of great importance.Methods and ResultsThis paper includes mainly three parts: (1) Cloning of leptin and leptin receptor from Tibet minipigThe primers were designed according to the leptin and leptin receptor gene sequences founded in Genbank (Accession number:GQ240885.1 and AF092422.1). The amplified leptin cDNA was obtained by RT-PCR using the total RNA purified from Tibet minipig's fat tissues. The amplified leptin receptor cDNA fragments of OBR intracellular and extracellular domains were obtained by RT-PCR using the total RNA purified from Tibet minipig's liver tissues. The full cDNA sequence of OBR gene was then obtained by linking the leptin receptor cDNA fragments together by overlapping PCR. The leptin and leptin receptor cDNA were ligated into pMD18-T vector after purification and then the recombined vectors were transformed into E.coli.DH5αfor PCR identification and sequencing. Those positive bacteria were conserved after sequencing. Homology and phylogenetic analysis of evolutionary trees were performed against other swine breeds or species reported in GenBank.The result indicated that the cloned leptin and leptin receptor cDNA have a sequence length of 504bp and 3498bp, respectively. The leptin cDNA sequencing result was compared against the Tibet minipig leptin gene sequences reported in GenBank (accession number:GQ240885.1) using the software DNA Star. A silent mutation was found to occur in the 246 (Tâ†'C) site. The homology and phylogenetic analysis of evolutionary trees between the Tibet minipig OBR gene and those of other swine breeds or species reported in GenBank showed the Tibet minipig has a highest affinity to wild hogs and the Guangxi Bama mimi-pig (99.6%), the affinity to cows, dogs and human are 91.1%,90.0% and 88.3%, respectively.(2) Constructing recombinant prokaryotic expression vectors of leptin mature peptide and leptin receptor extracellular domain of Tibet minipigLeptin mature peptide (64-504bp), signal peptide extracellular domain of OBR genes (322-969bp), LBD gene area of OBR extracellular domain (1072-1641bp) and the corresponding cDNA fragments of the OBR gene near the extracellular transmembrane domain (1705-2364bp) were amplified respectively by nested PCR, using Leptin and the OBR transformed positive bacteria as templates and designing four pairs of specific primers (named OBm, OBRp, OBRm and OBRa) with BamHâ… and Hindâ…¢restriction sites. After purification, the amplified fragments were cloned to the pMD18-T vector and transformed into E.coli.DH5αcompetent cells. After overnight culture, the recombinant plasmids were extracted and identified by PCR and BamHâ… and Hindâ…¢restriction enzyme digestion. The digested BamHâ… and Hindâ…¢restriction enzyme has the restriction fragment sizes of 450bp,660bp, 580bp,670bp, which are consistent with the theoretical sizes. The four goals sequences were cloned to the pre-digested (BamHâ… , and Hindâ…¢) pRSETA plasmid directly and constructing fusion expression vectors, named as pR-OBm, pR-OBR,pR-OBRm and pR-OBRa respectively. The four constructed expression vectors were transformed into E.coli BL21 (DE3) competent cells. Then the recombinant plasmids of pR-OBm, pR-OBRp, pR-OBRm and pR-OBRa were extracted for identification using PCR, restriction enzyme (BamHâ… , Hindâ…¢),and sequencing analysis. The positive recombinant plasmids were sent to Shanghai Invitrogen Biotechnology Company for sequencing to make sure the target genes are correctly inserted into recombinant plasmids and the reading frames and bases have not been changed.The results showed that the Leptin mature peptide, signal peptide extracellular domain of OBR genes, LBD gene area of OBR extracellular domain and the corresponding cDNA fragments of the OBR gene near the extracellular transmembrane were successfully cloned. The recombinant prokaryotic expression vectors of pR-OBm, pR-OBRp, pR-OBRm, pR-OBRa were identified by PCR and BamHâ… ,Hindâ…¢restriction enzyme digestion. The identification results demonstrated that the sizes of amplified fragments and restriction fragments are matched with cDNA of OBm, OBRp, OBRm, OBRa coding sequences, and the length of the vector pRSETA also matches. The positive recombinant plasmids (pR-OBm, pR-OBRp, pR-OBRm and pR-OBRa) were sent to Shanghai Invitrogen Biotech Company for sequencing. The analytic results by the software DNA Star demonstrated that the reading frame and bases had not been changed after recombination, suggesting the sequences of OBm, OBRp, OBRm and OBRa cDNA have been successfully inserted into the pRSETA vector, and constructed corresponding expression vectors.(3) Expressing and purifying fusion protein leptin mature peptide and leptin receptor extracellular domain of Tibet minipigThe recombined plasmid pR-OBm, pR-OBRp, pR-OBRm and pR-OBRa were transformed into E.coli BL21(DE3). The transformed bacteria pR-OBm, pR-OBRp, pR-OBRm and pR-OBRa were induced with 1mol/L IPTG and produced recombinant proteins with relative molecular mass of 17.5 kDa,27.6kDa,23.5 kDa and 27.6kDa, respectively. The pRSETA empty cell has no expression of the corresponding protein. These showed that pR-OBm,pR-OBRp,pR-OBRm and pR-OBRa can express recombinant Tibet minipig leptin mature peptide and partial OBR ECD of Tibet minipig in E.coli. strain BL21(DE3). Tibet minipig leptin mature peptide protein was also confirmed by western blotting. This work provided the basis for further research on pig leptin and OBR.This work also explored optimal expression conditions of the recombinant plasmid pR-OBm, pR-OBRa induced by IPTG (including cultural time and concentration). The optimal time of the recombinant plasmid pR-OBm, pR-OBRa induced by IPTG:E.coli BL21 (DE3) transformed with recombinant plasmid pR-OBm, pR-OBRa was shaking-cultured in LB liquid medium at 37℃, when the D600nm was above 0.6,0.5mmol/L IPTG was added into the cultural medium. The induction time was Oh, 1h,2h,3h,4h,5h,6h, and 7h respectively. The optimal IPTG concentration:E.coli BL21 (DE3) transformed with recombinant plasmids pR-OBm, pR-OBRa was shaking-cultured in LB liquid medium (containing Amp100μg/mL) at 37℃, when the D600nm was above 0.6, IPTG with concentrations of 0,0.05,0.1,0.3, 0.5,0.8,1 and 1.5 mmol/L respectively was added into the cultural medium and induced for 4 hours. The results showed, with increase of the culture time, the expression levels of fusion protein improved, especially when the induction time was over 3 hours, the fusion protein came to achieve the highest expression level, containing 40.8%,16.6% of total cell protein respectively, but IPTG concentration had no affect to the expression of fusion protein.Leptin mature peptide and the extracellular domain of OBR gene near the transmembrane domain of recombinant fusion protein express with higher level, therefore they were selected for large scale preparation and purification. A sufficient amount of recombine leptin mature peptide and partial OBR ECD which near transmembrane domain fusion protein expressed in inclusion body form were denatured in guanidine hydrochloride solution and purified by 50% Ni-NTA chromatography. The purified recombined fusion proteins were refolded by dialysis.ConclusionIn this study, we have successfully cloned leptin, leptin mature peptide (64-504bp), OBR, near signal peptide extracellular domain of OBR genes (322-969bp), LBD of OBR extracellular domain (1072-1641bp) and the corresponding cDNA fragments of the OBR gene near the extracellular transmembrane domain (1705-2364bp) cDNA, and pR-OBm, pR-OBRp, pR-OBRm and pR-OBRa expressed recombinant Tibet minipig leptin mature peptide and partial OBR ECD of Tibet minipig in E.coli. strain BL21(DE3). Leptin mature peptide and the extracellular domain of OBR gene near the transmembrane domain of recombinant fusion protein had higher expression level and were selected for large scale preparation and purification. This work has prepared fundamental materials and ground for further studies in animal immunization, biological function of leptin and OBR, establishment of Tibet minipig animal models of human diseases such as human adiposis, diabetes and diseases of cardiovascular system by active immunity method, obesity mechanism study and development of methods for control and treatment of obesity.