Preparation And Identification Of The Pleiotrophin Monoclonal Antibody

The pleiotrophin(PTN)is a kind of heparin binding growth factor of the same family as Midkine(heparin binding cell factor).PTN is also known as HB-GAM,HBNF,OSF-1 and NEGF1,etc.As the growth of the age,the expression position of PTN also change accordingly,from nerve and embryonic mesoderm in embryonic development period to the adult expression in confined areas such as the mammary gland,heart,and testicles.Domestic and foreign research shows that PTN has a variety of biological functions,such as promoting mitosis,promote tumor growth,inflammation,angiogenesis and bone development and so on.In progress in a wide variety of tumor diseases,particularly,PTN was detected in a certain degree,typically such as breast cancer,gastric cancer,liver cancer,and pancreatic cancer.This suggests that PTN can be used as a potential marker in the process of tumor detection,and at the same time it also can be used as a sign of the prognosis of patients with tumor.Therefore,the preparation of PTN antibody can be applied in the area of experimental science and clinical application,which has certain value and significance.First,a pET-28a-his-PTN plasmid is constructed in this paper,and the expression condition of PTN protein induced by IPTG in the Escherichia coli is studied.The main influencing factors include the biomass before inducing,induced oxygen flux,the temperature of induction,the concentration of inducer and the induction time.And the optimal conditions of expression is successfully obtained by use of uniform design experimentation.A large number of PTN protein with six histidine(his)tag are achieved in nickel and cobalt affinity chromatography experiment by eluting with different concentrations of imidazole buffer,and the protein freeze-dried powder is finally acquired.Second,in the immunological experiment of BALB/C mice,using the purified PTN as antigen which is emulsified by complete freund adjuvant and incomplete freund adjuvant repeatedly immunes mice.Taking the blood through the tail and using indirect ELISA method,PTN polyclonal antibody in the serum of mice is detected.By PEG 4000,the splenocytes of mice was successfully fused with NS-1 myeloma cells within qualified mice spleen which polyclonal antibody has eligible titer.The hybridoma cell lines with stable expression and high antibody expression were obtained through two screening twice in HAT and HT selective medium.And a large number of PTN monoclonal antibodies are obtained by injecting hybridoma cells to BALB/C mice enterocoelia so as to prepare ascites and collecting the supernatant of hybridoma cells.Finally,we qualify the monoclonal antibody has qualified titer and immugenicity by ELISA and Western blotting.To sum up,we investigated the optimal conditions of pleiotrophin protein expression induced by IPTG in e.coli,and purified a lot of pleiotrophin protein by using nickel and cobalt affinity column.Also we obtained PTN antibody through the mice immune experiment,hybridoma preparation experiment and ascites preparation.Eventually we identify PTN monoclonal antibody can recognize pleiotrophin protein specifically which can be primary antibody used for ELISA and Western blotting and it can provide experimental foundation for the construction of the PTN,and can be used as a basis for further research on PTN.