preparation of LHRH-mediated TAT protein core peptide PTD and research on its membrane transfer

MA Hongyuan;YUE Yuhuan;LI Zehong;ZHANG Guoli;TIAN Yuan;WU Guangmou;LIU Yuling;YU Xiaoying;YANG Wenqi;CHEN Yunyu;College of Life Sciences, Jilin Agricultural University;Institute of Military Veterinary, Institute of Military Medical Sciences, Academy of Military Sciences;Center for Drug Screening and Evaluation, College of Pharmacy, Wannan Medical College;

LIU Qiong%LIU Yongshi%SHANG Xiaomin%LI Guiling%JIANG Yanlong%WANG Chunfeng%College of Food Engineering,Jilin Engineering Normal University%College of Animal Science and Technology, Jilin Agricultural University/Jilin Provincial Engineering Research Center of Animal Probiotics/Key Laboratory of Animal Production and Product Quality Safety of Ministry of Education

core peptide;;luteinizing hormone releasing hormone;;target protein;;transduction efficiency;;green fluorescence;;transmembrane

In order to study the transmembrane action of the LTHH-mediated core peptide PTD mediated by luteinizing hormone releasing hormone(LHRH), determination of the transduction efficiency of the protein of interest,the plasmid pET28 a-PTD-GFP was used as a template to design a PCR primer containing the LHRH gene sequence, which was double digested by PCR and cloned. In the prokaryotic expression vector pET28 a, the recombinant expression plasmid pET28 a-LHRH-PTD-GFP was constructed and induced in E. coli BL21(DE3) competent cells. After ultrasonication, the precipitate was precipitated by ammonium sulfate gradient and the supernatant was passed through the hydrophobic layer. The column and ion chromatography were used for purification. The purified target protein was added to HeLa cells cultured in vitro. Transduction was observed and the effect of protein concentration on transduction efficiency was analyzed. The results showed that the target gene LHRH-PTD-GFP was amplified by PCR and the recombinant plasmid pET28 a-LHRH-PTD-GFP was successfully constructed. The target protein was successfully induced in E.coli BL21(DE3) competent cells by phenyl-HP chromatography. The target protein was successfully purified by column and Q anion exchange chromatography column. Under the fluorescence microscope, the green fluorescence was observed in HeLa cells. The transduction efficiency obtained by software was positively correlated with the target protein concentration. The regression coefficient was 0.096 6. R~2 is 0.937 8. It indicats that the LHRH-mediated PTD protein has a good transmembrane property, and the transduction efficiency is dependent on the target protein concentration. The transduction efficiency increases with the concentration of the target protein in a certain concentration range.