Polyclonal Antibodies Preparation And Expression Analyses Of GlnRS And ArgRS

Aminoacyl-tRNA synthetase(AARS), one of the key enzyme in protein synthesis, can attach special amino acid substrate to its cognate tRNA. In addition to classical catalytic function, it also possesses noncanonical functions of example transcription regulation, cell metastasis, protection against DNA damage and angiogenesis suppression, antiapoptotic effect. Glutamine-tRNA synthase(GlnRS) and arginyl-tRNA synthetase(ArgRS) have many noncanonical functions which are related with specific diseases. The study of structures and functions of GlnRS and ArgRS can provide theoretical basis for generating mechanisms of related diseases. Objective:The purpose of this experiment is to prepare polyclonal antibody against GlnRS and antibody against ArgRS and then use the preparation polyclonal antibodies to detect GlnRS and ArgRS expression in different cells to lay foundations for the study of functions of GlnRS and ArgRS. Methods:1. Preparation and detection of polyclonal antibody against GlnRS(ArgRS)Bioinformatics approach was applied to determine the antigenic sites in GlnRS(ArgRS). The target gene was amplified by PCR. The amplified product was inserted into PET28 a between EcoRI and XhoI to construct expression vectors. Protein expression was induced by IPTG in BL21. The expressed protein was purified by NI-NTA affinity chromatography. The mice were injected subcutaneously with purified proteins to obtain polyclonal antibody. The specificity of the antibody was assayed by western blot.2. Expression analysis of GlnRS(ArgRS) in different cellsSelect cells in logarithmic growth phase and use prepared polyclonal antibody as primary antibody to detect the expression of GlnRS(ArgRS) in different cells by western blot. Results:1. Preparation and detection of polyclonal antibody against GlnRS(ArgRS)By bioinformatic analysis, we selected the GlnRS N-terminal 236 amino acids(N(1-236)-GlnRS) as an antigen for preparation antibody of GlnRS and ArgRS N-terminal 72 amino acids(N(1-72)-ArgRS) for preparation antibody of ArgRS. The target gene encoding N(1-236)-GlnRS(N(1-72)-ArgRS) was obtained by PCR and the expression vector PET28a-N-Gln RS(PET28a-N-ArgRS) was successfully constructed. The recombinant protein N-GlnRS(N-ArgRS) expressed efficiently and was purified by NI-NTA affinity chromatography. The polyclonal antibody against GlnRS(ArgRS) was prepared successfully with high specificity.2. Expression analysis of GlnRS(ArgRS) in different cellsIn expression analysis experiment, GlnRS expression is upregulated in He La, not in HepG2 and LO2. In lymphoma of Eμ-Myc high expression, the level of GlnRS expression is high than that in normal cells, the higher degree of malignancy, the higher level of GlnRS expression. ArgRS expression is upregulated in HepG 2, not in HeLa and LO2. Conclusion:1. The polyclonal antibody agaist GlnRS and polyclonal antibody agaist ArgRS were prepared successfully and show high specificity. They lay foundations for the study of functions of GlnRS and ArgRS.2. The expression of Gln RS(ArgRS) is different in cancer cells of different kinds or different generating mechanisms. It is not all right that the level of GlnRS(ArgRS) expression in cancer cells is higher than that in normal cells.