Construction Of Prokaryotic Expression Vector And Preparation Of Recombinant Proteins Of Porcine LIN28?KLF4?c-MYC And OCT4

Being firstly achieved in 2006 and having similar characteristics with embryonic stem cells?ESCs?,which can self-renew,proliferate unlimitedly and form almost any cell types,induced pluripotent stem cells?iPSCs?are of great value in regenerative medicine,conservation of domestic animal genetic resources and biological basic researches,and became one of the hottest topics in frontier fields of life science.Recently,successful generation of iPSCs from domestic animals,such as pig,broke the dilemma of long-term fail in derivation of de novo ESCs from pig and other livestock.However,iPSCs still face many problems,such as poor quality,low efficiency and uncertain safety.Use of recombinant proteins or small molecules to generate iPSCs is promising in overcoming safety hazards that might resulted from integration of exogenous genes.But pig i PSCs induced by recombinant protein have not well established yet.This study was to construct prokaryotic fusion expression vector haboring porcine pluripotent transcription factors LIN28?KLF4?c-MYC and OCT4 associated with cell penetrating peptide,and explore optimized conditions of production of pig soluble LIN28-11 R recombinant proteins.Besides,expression conditions of the other three transcription factors recombinant proteins were also investigated,in order to lay a good foundation for future generation of i PSCs using recombinant proteins.The experiments and results are as follows:1.Construction and identification of prokaryotic expression vectors of porcine genes LIN28?KLF4?c-MYC and OCT4.Firstly,using Optimum Gene software,codons of pig OCT4?NP₀01106531?,KLF4?NP₀01026952?,c-MYC?NP₀01005154?and LIN28?NP₀01116605?sequences were optimized,and in the 3' terminal the sequences of coding Linker and cell penetrating peptides 11 R were added.Then the of-interested genes were artificially synthesized and cloned into vector pET30 a.The results of enzyme restriction and sequencing showed that pET30a-NdeI-Oct4-linker-11R-XhoI-His,pET30a-Nde I-Lin28-linker-11R-XhoI-His,pET30a-Nde I-Klf4-linker-11R-XhoI-His and pET30a-NdeI-c-Myc-linker-11R-Xho I-His were successfully obtained.2.Preparation of porcine LIN28-11 R recombinant protein and the other three transcription factors.Recombinant plasmid was transformed into E.coli BL21.LIN28-11 R was induced with different temperature and time in order to explore optimal conditions to produce recombinant proteins.After purification and identification,recombinant protein ofinterest was added for in vitro culture of pig fibroblast cells in order to evaluate if the fused protein has capacity of cell membrane penetration.Moreover,the small scale production condition of the other three transcription factors was preliminarily explored.Porcine LIN28-11 R recombinant protein,with function of penetrating cell membrane,was successfully obtained.The optimal expression condition for OCT4-11 R,KLF4-11 R and c-MYC-11 R are: KLF4-11 R has a higher expression in supernatant of bacteria cracking liquid by 4 hours induction with IPTG at 37?;c-MYC-11 R expressed in the deposition and bacteria cracking liquid by 4 hours induction with IPTG at 37 ?;and OCT4-11 R has no expression in different conditions at both 30? and 37?.In all,prokaryotic fusion expression vector containing LIN28?KLF4?c-MYC and OCT4 associated with cell penetrating peptide are constructed,and LIN28-11 R with cell penetration ability is successfully achieved.Furthermore,optimal expression condition of OCT4-11R?KLF4-11 R and c-MYC-11 R are preliminarily established.The study offers a basis for subsequent preparation recombinant proteins,which somehow lays a foundation for future generation of pig iPSCs using proteins.