Preparation Of Amphi-ERK5,Amphi-p38Antibodies And Preliminary Study On The Expression Of The Two Proteins In Amphioxus

ERK5, p38are important members of MAPKs, they both play an important role in biological activities. MAPK family is a highly conserved family of serine/threonine protein kinases in evolutionarily, and it can be activated by several extracellular signals. MAPK family plays an important role in embryonic development, cellular differentiation, proliferation and apoptosis. In addition, MAPKs have been shown to be extremely relevant in tumor and inflammation and MAPKs are important parts of innate immune system.Our lab has acquired ERK5gene of amphioxus (Amphi-ERK5), p38gene of amphioxus (Amphi-p38) before. In this paper, some fragments that had better antigenicity were analyzed by bioinformatics methods and selectively cloned, inserted into vectors to get protein expression vectors and the peptide fragments expressed were used to prepare polyclonal antibodies. Expression of Amphi-ERK5> Amphi-p38proteins in adult amphioxus were detected by immunohistochemistry. At the same time, expression of Amphi-ERK5、Amphi-p38proteins in adult amphioxus were detected by immunohistochemistry using the ERK5, p38heterologous antibodies bought, to validate the credibility of the immunohistochemistry results obtained by using the Amphi-ERK-5Amphi-p38antibodies prepared by our own lab. This research will be the foundation of the further study of the functions of Amphi-ERK5、Amphi-p38.In this study, Amphi-ERK5、Amphi-p38protein expression vectors were constructed by using pet-29b(+) as protein expression vector, BL21(DE3) as expression strain and the prokaryotic expression method was used to get the target proteins. Two methods were used to prepare the mouse anti-amphioxus polyclonal antibodies:1) To prepare the Amphi-ERK5antibody, some mice were immuned with amphioxus ERK5protein fragments purified by Gel Slices. Western blotting was carried out using Amphi-ERK5protein fragments to test if the antibody was prepared successfully and the antiserum was then obtained.2) To prepare the Amphi-p38antibody, some mice were immuned with amphioxus p38protein fragments that got through Ni-sepharose purification and dialysis renaturation. Western blotting was carried out using Amphi-p38protein fragments to test if the antibody was prepared successfully and the antiserum was obtained.The results which used homemade Amphi-ERK5antibody to carry the immunohistochemistry out showed that Amphi-ERK5expression was a large number of columns in the expression of skin, gill filaments, hepatic caeca, intestine and gonad, little in muscle and notochord. When Polyclonal Goat Anti-human p-ERK5antibody was used to repeat the above experiments, the consistent results were obtained. The results which used homemade Amphi-p38antibody to carry the immunohistochem-istry out showed that Amphi-p38expression was a large number of columns in the expression of skin, gill filaments, hepatic caeca, intestine and gonad, a small amount of expression in muscle and endostyle, little in notochord. When Polyclonal Mouse Anti-human p38antibody was used to repeat the above experiments, the consistent results were obtained.