Antibody Preparation Of Recombinant Protein Of T Cell CD3 And CD4 In Bactrian Camel

Bactrian camel is a unique animal in the northwest of China.It lives in the desert area with bad natural environment all the year round.It has been shown that Bactrian camel has a unique mucosal immune system,which plays an important role in the process of resisting the invasion of external pathogens.Among them,T cells are a kind of important effector cells involved in the protective response,but the research on T cells of Bactrian camel has not been reported.Therefore,bioinformatics analysis,prokaryotic expression of recombinant protein and preparation of polyclonal antibody were carried out for two important molecules CD3 and CD4 on T cell surface of Bactrian camel.In order to obtain the recombinant protein and polyclonal antibody of CD3? and CD4,the nucleic acid sequence and amino acid sequence of CD3? and CD4 of Bactrian camel were searched in GenBank.The transmembrane region,signal peptide,antigen index and hydrophilicity of CD3? and CD4 were predicted by bioinformatics analysis software.Then,some amino acid sequences outside the membrane of CD4 and CD3? were selected and removed The gene corresponding to the amino acid sequence was cloned into pET-28a(+)expression vector,transferred into BL21(DE3)E.coli,and then induced the expression of the recombinant protein.SDS-PAGE was used to identify the recombinant protein.The two recombinant proteins were purified by Ni column affinity chromatography and immunized to healthy male rabbits to prepare polyclonal antibodies.After four immunizations,blood samples were collected from the heart and serum was extracted.The titer of CD3? and CD4 polyclonal antibodies was detected by ELISA.The results showed that:(1)The prokaryotic expression vector containing the target gene was constructed successfully,and the recombinant proteins of CD3? and CD4 were obtained,both of which were expressed in the form of inclusion bodies.(2)The results of ELISA showed that the effective dilutions of anti-CD3? and anti-CD4 reached 1:32000 and 1:16000.(3)WB results showed that the recombinant proteins of CD3? and CD4 could specifically bind to their antibody serum.In conclusion,the recombinant proteins of CD3? and CD4 molecules of Bactrian camel can be expressed successfully in prokaryotic expression system;CD3? and CD4 have the ability to stimulate the immune system to produce antibodies in rabbits,indicating good immunogenicity;Through the prokaryotic expression of CD3? and CD4 molecules on the surface of T cells of Bactrian camel and the preparation of polyclonal antibody,we can provide reference for the study of differentiation,proliferation,function development,signal transduction of T cells of Bactrian camel and the further development of specific antibody for the treatment of diseases related to immune system of Bactrian camel.