Expression Of RTNSALP-FcD10 In CHO-K1 Cells

Background: Hypophospatasia(HPP)is a rare autosomal genetic disease,which is caused by a mutation in the liver/bone/kidney alkaline phosphatase gene,which leads to a reduced activity of the tissue nonspecific alkaline phosphatase(TNSALP)encoded by the gene.TNSALP plays a key role in normal bone calcification,lack of TNSALP will cause the body of calcium and phosphorus metabolism,thus show the hard tissue dysplasia of bone and teeth,and serum alkaline phosphatase,alkaline phosphatase,ALP)activity is low,prone to fracture,muscle atrophy,bone and joint pain and other symptoms,severe effects on the quality of life.In 2015,FDA approved the recombinant alkaline phosphatase antibody drug(rTNSALP-FcD10)for the treatment of HPP,but the rtnsalp-fcd10 antibody drug has not been marketed in China.rTNSALP-FcD10 has the characteristics of prolonging the half-life and promoting the targeting of osteoblasts,but its low yield and high cost impose a great burden on the needs of HPP patients and their families.Objectives: This project aimed at above problems,according to a report in the literature,plans to build human recombinant of TNSALP gene eukaryotic expression vector,trying to extract more efficient recombinant expression plasmid,can get stable rTNSALP-FcD10 protein expression and the expression of relatively high amount of Chinese hamster ovary cells CHO-K1 cell lines,for future large-scale production in China rTNSALP-FcD10 protein to build foundation.Methods:(1)Remove the signal peptide sequence at the N-terminal of TNSALP molecule and the GPI anchor signal at the C-terminal,recombine the Fc fragment of human antibody and 10 aspartic acids into the fusion protein rTNSALP-FcD10,and clone the rTNSALP-FcD10 gene into the eukaryotic expression vector pcDNA3.1 and pEF1/Myc-his B to construct the recombinant expression vector.CHO-K1 cells were transfected with Lipofectamine 2000,and G418 was used for pressure screening.CHO-K1 cell lines with stable expression of rTNSALP-FcD10 protein and higher expression were obtained.(2)rTNSALP-FcD10 contains both IgG antibody affinity fragment,which is a glycosylated protein,was purified by affinity chromatography,and alkaline phosphatase activity of the purified protein was detected and analyzed.Results:(1)The double enzyme digestion identification results of recombinant expression plasmids pcDNA3.1-rTNSALP-FcD10 and pEF1-rTNSALP-FcD10 correctly showed the carrier bands and the target bands,respectively.The sequencing results showed that the two recombinant expression plasmids were successfully constructed.(2)Real-time fluorescence quantitative PCR detection results showed that the transcription level of rTMSALP-FcD10 gene was significantly increased in CHO-K1 cells transfected with recombinant expression plasmid.Western blot results showed that rTNSALP-FcD10 protein was successfully expressed in CHO-K1 cells,and the protein expression in the transfected pEF1-rTNSALP-FcD10 plasmid group was higher than that in the transfected pcDNA3.1-rTNSALP-FcD10 plasmid group.(3)rTNSALP-FcD10 protein was purified by affinity chromatography with a concentration of 4.2 mg/L.(4)The results of alkaline phosphatase activity test showed that the ALP activity of the proteins transfected with pcDNA3.1-rTNSALP-FcD10-fcd10 and pEF1-rTNSALP-FcD10 recombinant expression plasmid group was significantly higher than that of the blank control group.Conclusions:(1)The recombinant expression plasmid pcDNA3.1-rTNSALP-FcD10 and pEF1-rTNSALP-FcD10 were successfully constructed,and a stable CHO-K1 cell line was established for the successful expression of the target protein.(2)Purification of rTNSALP-FcD10 protein.(3)The successful construction of rTNSALP-FcD10 gene eukaryotic expression vector and the establishment of stable transfected CHO cell line laid a good experimental foundation for further protein purification.