Expression Of Peiotrophin (PTN) Protein And Preparation Of Its Polyclonal Antibody

Peiotrophin has the functions of promoting angiogenesis and mitogen activity,and is involved in various biological processes.Because it is expressed in many kinds of tumors,the mechanism of action is still not clear,which has become a research hotspot in recent years.Natural heavy chain antibodies are found in camels and are also called nanobodies.Nano-antibodies have attracted attention due to their small molecular weight,high tolerance,and wide range of antigen-binding activities,as well as their good role in monitoring and treatment of zoonosis..In this experiment,the His6-SUMO-PTN recombinant plasmid was constructed by artificially synthesizing PTN gene,and it was induced to express correctly in T7 Shuffle E.coli.Purified PTN fragment protein was used as the complete antigen to immunize multiple spots near the lymph nodes of alpaca.The basic immunization was the emulsification of the same amount of antigen with Freund's complete adjuvant,one week of booster immunization,and the booster immunization with equal amounts of the antigen and the same amount of Freund's incomplete adjuvant emulsified for a total of six times.15 ml of blood was taken from the jugular vein before each immunization,and 15 ml of each blood was collected at 6 h,12 h,and 24 h after the second immunization.The serum that had separated out was collected after standing for 24 h at4°C.The experimental results showed that:(1)The secretory expression system of recombinant PTN was successfully established by fusing His6-SUMO-labeled PTN with pelB signal peptide;(2)After collecting bacterial cells,Ni-NTA was used to purify and recover recombinant PTN protein;(3))Serum IgG titer was detected by indirect ELISA.The detection was 1:100.(4)Observe the changes of various cytokines in the alpaca at different times after injection.The results show that after immunization of the alpaca with the PTN antigen,the changes of the cytokines in the alpaca serum are as follows: TNF-?,IL-2,IL-6.The levels of IL-5 and IL-10 increased at first and then decreased,but the changes of IFN-? and IL-4 were not obvious.There was a significant positive correlation between IL-4 and IL-6,IL-5 and TNF-?.The correlation between other cytokines was not obvious.