Design, Cloning and Sequencing of cDNA Mutant of Human Protein C

Objective To construct human protein C cDNA mutant by point mutation and directly express the human protein C with biological activity in mammal cells. Methods Design primers according to the cDNA sequence of human protein C, introduce point mutation and extract the heavy and light chains of human protein C from the total RNA of human fetal liver by RT-PCR and recombinant PCR respectively. Link the 2 chains by recombinant PCR and clone into pGEM-T vector.The recombinant plasmid was digested with restrict -tion endonuclease, identified by PCR and sequenced. Results A cDNA fragment with a length of 1374 bp was amplified by RT-PCR and recombinant PCR, and a recombinant plasmid pGEM-T/mbPC for cloning human protein C cDNA mutant was successfully constructed. The result of sequencing proved that the active peptide of human protein C was substituted with the introduced, nucleotide sequence encoding 8 amino acids. Conclusion A clone of human protein C cDNA mutant was obtained by point mutation. It laid a foundation of further study on expression and activity of human protein C cDNA mutant.