Fusion protein mixture for inducing human pluripotent stem cell and preparation method there of

1. A mixture comprising fusion proteins,

wherein each of the fusion proteins comprises the structure: PTD-NES-SUMO-Specific Protein, in that specific order;

wherein the PTD is a Protein Transduction Domain, and wherein the PTD is the TAT PTD region of HIV TAT;

wherein the NES is a Nuclear Export Sequence, and wherein the NES is human IkBa Nuclear Export Sequence;

wherein the SUMO is small ubiquitin-like modifier, wherein the SUMO is yeast SMT3p;

wherein the Specific Protein is any one of the proteins selected from the group consisting of C-myc, SOX2, KLF4 or OCT-4;

wherein the PTD-NES-SUMO moiety of the fusion protein is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; and

wherein an applied concentration of each of the fusion proteins in the mixture is 1 ng/ml-1 mg/ml.

2. The mixture of claim 1 , wherein the SUMO is a cleavage site and enables the PTD to be cleaved from the fusion protein.

3. The mixture of claim 1 , wherein the Specific Protein is SOX2, and the fusion protein is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3.

4. The mixture of claim 1 , wherein the PTD-NES-SUMO moiety of the fusion protein comprises the amino acid sequence of SEQ ID NO: 2.

5. The mixture of claim 1 , wherein the Specific Protein is OCT4 and the fusion protein is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4.

6. The mixture of claim 1 , wherein the Specific Protein is KLF4 and the fusion protein is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5.

7. The mixture of claim 1 , wherein the Specific Protein is C-myc and the fusion protein is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6.

8. A method for preparing a mixture of fusion proteins, wherein the method comprises the following steps:

(1) constructing an expression plasmid encoding a fusion protein comprising the structure PTD-NES-SUMO-Specific Protein, wherein:

PTD is a protein transduction domain and is the TAT PTD region of HIV-TAT,

NES is a Nuclear Export Sequence and is human IkBa Nuclear Export Sequence,

SUMO is small ubiquitin-like modifier and is yeast SMT3p, and

Specific Protein is selected from the group consisting of OCT-4, SOX2, KLF4, and C-myc,

wherein constructing the expression plasmid comprises:

synthesizing a polynucleotide encoding the PTD-NES-SUMO moiety of the fusion protein by synthesizing 8 oligo primers which are 75 base pairs in length and mutually overlap in 20 base pairs and performing overlapping PCR three times;

synthesizing a polynucleotide encoding the fusion protein by amplifying OCT-4, SOX2, KLF4, or C-myc cDNA from total RNA of human embryonic stem cells, whose 3′-end has an Xhol site and assembling the polynucleotide encoding the PTD-NES-SUMO-Specific Protein by PCR with the synthesized PTD-NES-SUMO encoding sequence, wherein said PTD-NES-SUMO encoding sequence comprises the nucleotide sequence of SEQ ID NO: 1; and

cloning the polynucleotide encoding the fusion protein into the Ndel/Xhol site of pET-24a(+) vector;

(2) screening an expression strain by:

transforming host strain BL21 with the expression plasmid from step (1);

and, screening for a clone that expresses the fusion protein;

(3) performing large-scale expression of the fusion protein by:

inoculating a culture medium with the clone that expresses the fusion protein and culturing until OD 0.6 followed by addition of IPTG and induction for 3 hours; and

(4) separating and purifying the fusion protein by hydrophobic chromatography and ion exchange chromatography.