Generation,Application Of BAF-L Polyclonal Antibody And The Role Of BAF-L In Pancreas And Testicular Development Of Mice

Barrier to autointegration factor(BAF)is a small,ubiquitous and highly conserved protein,which is mainly located in the nucleus.It is reported that it mainly plays a role in chromatin structure,separation and nuclear assembly after nuclear division.BAF-L is a new homologous gene of BAF,and its protein sequence has 53% similarity with human BAF.BAF-L is a nuclear protein,but unlike BAF,BAF-L does not bind DNA.In addition,the expression patterns of BAF-L and BAF are also different.It has been reported that BAF-L m RNA is highly expressed in pancreas and testis,but there is a lack of research on the function of BAF-L protein in pancreas and testis.Therefore,based on the preparation of high titer and specific BAF-L polyclonal antibody,this study first clarified the temporal and spatial expression characteristics and cell localization of BAF-L,then studied the effect of BAF-L on the development of mouse pancreas and testis,and then verified its interaction with important nuclear proteins,so as to lay a foundation for exploring the function of BAF-L in mouse pancreas and testis.The specific research results are as follows:1.High titer and specific BAF-L polyclonal antibody was successfully preparedBy amplifying the full-length sequence of BAF-L CDs region in mouse testis,the recombinant plasmid was constructed and named pgex-4t-1-BAF-L.the prokaryotic expression of the recombinant protein was induced under appropriate conditions,and the obtained recombinant protein was purified.The emulsified recombinant protein was immunized subcutaneously on the back of New Zealand white rabbits for 4 times,and finally the polyclonal antibody of BAF-L was obtained by bloodletting.The agar diffusion titer of BAF-L polyclonal antibody was 1:8;Western blots results showed that the antibody could specifically recognize the overexpression of BAF-L protein in 293 T cells.2.Clarify the expression characteristics of BAF-L in mouse pancreas and testisBased on the purchased BAF-L knockout mice,the deletion of BAF-L protein was verified by western blots,which further clarified the specificity of BAF-L polyclonal antibody;Using the prepared antibody,it was found by western blots that BAF-L protein was mainly expressed in testis,pancreas,spleen and other tissues,among which the expression of BAF-L protein was the highest in pancreas and testis.In terms of development time,the expression of BAF-L in pancreas did not show obvious age-dependent from birth to adulthood;The expression of BAF-L in testis increased continuously from two weeks after birth and remained at a high level after adulthood;In terms of cell localization,using the prepared antibody for immunohistochemical staining,it was found that BAF-L was mainly located in the nucleus in testicular tissue.3.The absence of BAF-L has no obvious abnormal effect on the pancreas.In testicular tissue,BAF-L regulates the exchange process of histone and protamine by interacting with histone and protamineThrough he and immunohistochemical staining of pancreatic tissue of mice at different ages,it was found that the lack of BAF-L did not affect the development of pancreas and insulin secretion,indicating that BAF-L was not involved in the development process and functional regulation of pancreas;In testicular tissue,due to the characteristics of nuclear localization of BAF-L,in order to verify whether BAF-L interacts with histones,the interaction between BAF-L and histones and protamine was verified by immunoprecipitation experiment.Immunofluorescence staining found that there were still a large number of histones in the abnormal sperm cells of knockout mice.Combined with q RT-PCR,it was found that the expression levels of protamine and transition protein in knockout mice decreased significantly.Aniline blue staining analysis found that the sperm of knockout mice contained a large number of histones.The above results showed that BAF-L regulated its exchange through the interaction with histones and protamine,thus playing a key role in spermatogenesis.Western blots results showed that the acetylation modification levels of histone H3 and H4 decreased significantly in knockout mice,indicating that the lack of BAF-L caused the defect of histone acetylation,which led to the abnormal development of sperm cells.In conclusion,the specific BAF-L polyclonal antibody was successfully obtained in this study,and the application of this antibody showed that the expression of BAF-L protein was the highest in pancreas and testis,but it was not involved in the development process and function maintenance of mouse pancreas after birth.In mouse testis,BAF-L regulates the exchange of histone and protamine by interacting with histone and protamine,thus playing an important role in spermatogenesis.