LZ-8 Protein Activity Detection And Its Ligand Peptide Screened By Phage Display Technology

Ganoderma lucidum has been a precious medicinal material in traditional Chinese medicine since ancient times.Some small molecular substances extracted from Ganoderma lucidum,such as protein and polysaccharide,also have the functions of immune regulation and anti-tumor.Ganoderma lucidum protein-8（LZ-8）is a fungal immunoregulatory protein extracted from Ganoderma lucidum mycelium.Its structure and function are similar to immunoglobulin,and it has a wide range of immunoregulatory activities and antitumor activities.Therefore,LZ-8 can be used as an anti-tumor drug and has great potential in research and development.Due to the low extraction amount of wild Ganoderma lucidum,at present,the preparation of LZ-8 mostly adopts the method of genetic engineering,that is,recombinant Ganoderma lucidum-8（rlz-8）.rlz-8 as a genetic engineering drug,its preparation has become increasingly mature,but so far there is no set of standard,rapid detection method.Therefore,this paper adopts phage display technology to screen rlz-8ligand short peptide,and establishes a method to detect rlz-8 protein,instead of monoclonal antibody,to realize rapid,convenient and real-time detection of rlz-8,which provides a new product and biological detection method for the detection of anti-tumor drugs.In this study,we first used phage display technology,after four rounds by screening,we got the recombinant phage which specifically combined with rlz-8.Randomly selected clones were identified by ELISA,and their ssDNA was extracted,sequenced and sequenced.A peptide L-T-P-H-K-H-H-K-H-L-H-A with strong specific binding with rlz-8 was obtained.The peptide was synthesized and biotinized and KLH protein modified.The binding ability of the polypeptide and rlz-8 was verified by biomembrane interference.The KD value was9.5×10^-7M.The methodology for the detection of rlz-8 was established by coupling KLH peptides.The stability,repeatability and recovery of rlz-8 were tested to evaluate the feasibility of the methodology.The results showed that the standard curve of the peptide and rlz-8 protein was y=0.412x+0.1361.The recovery rate is between 95%and 110%,repeatability:CV%of high and low value samples is 3.53%and 6.12%respectively,stability:CV%of high and low value samples is 1.30%and 2.10%respectively,all within the acceptable range.The method was applied to the monitoring of rlz-8 fermentation broth,and the results were consistent with those of electrophoresis.It provides a new detection method for protein drug development.