Human PCSK9 Expression In Eukaryotic Cells And Its Antibody Preparation

Proprotein convertase subtilisin/kexin type 9(PCSK9) is a secreted serine protease that consists of 692 amino acids. PCSK9 is synthesized in liver cells and the mature PCSK9 is able to combine with members of low density lipoprotein receptor family on the cell membrance and accelerate LDLR degradation. Thus the liver ability of plasma low-density lipoprotein cholesterol(LDL-C) metabolism is downregulated. PCSK9 inhibition can be used for hypercholesterolemia therapy and fully human monoclonal antibodies and humanized monoclonal antibody have entered Phase III clinical trial stage, but there isn’t related nanobody, which is obviously more stable, transport more easily and cost less than conventional antibody.Here, we prepared the pure human PCSK9, and obtained PCSK9 specific heavy chain antibody, which laid the foundation for PCSK9 nanobody library construction and high specificity nanobody screening. We obtained PCSK9 polyclonal antibody by mice immunization, which laid the foundation for PCSK9 monoclonal antibody preparation and producing high specific clinical testing kits. The main contents are as follows:(1) To prepare human PCSK9 in GS115 yeast cells and Chinese hamster ovary(CHO)-s cells,we constructed the plasmids named PCSK9-p PICZαA and PCSK9-pc DNA4.0 with cordon optimized, synthesized human PCSK9 genes. His-tagged PCSK9 recombinant protein was successfully expressed in both GS115 and CHO cells. With the Western Blotting method, it is easy to find that the size of the recombinant protein expressed in GS115 is 100 KDa, larger than native protein(60 KDa) and the protein is also found degraded, so it is hard to quantify and apply to the immunization. While the protein expressed in CHO cells is nearly the same the size as human naive PCSK9 protein and can be purified easily with a purity up to 90% and a yield of 5.6 mg/L.(2) The polyclonal anti-PCSK9 serum was successfully prepared by camel immunization with pure PCSK9. The serum obtained from camel after the third time’s immunization was added to the protein G column followed by the protein A column to get heavy chain antibody. Then PCSK9 specific heavy chain antibody was obtained by adding the heavy chain antibody to magnetic beads coupling PCSK9.(3) The titer of the serum obtained from mice after three times’ immunization with pure PCSK9 is 1:80000 by the enzyme-linked immuno sorbent assay.A rapid method of preparing human PCSK9 recombinant protein has been developed.Meanwhile the two PCSK9 protein expression systems were compared. PCSK9 specific heavy chain antibody and mouse anti-PCSK9 antibody were obtained, which laid the foundation for PCSK9 nanobody library construction with proteomics, high-throughput sequencing and phage display technology and eventually preparation PCSK9 specific nanobody and clinical testing kits.