Cloning and Expression Pattern of Ribosomal Protein S18 Gene in Musca domestica

doi:10.13560/j.cnki.biotech.bull.1985.2016.06.019

Abstract

Abstract: This research is to confirm the expression stability of ribosomal protein S18（RPS18）gene in Musca domestica. The complete coding sequence（Registration number in NCBI：KT006855）of RPS18 gene was obtained via PCR amplification using the cDNA of RPS18 gene from the cDNA library of M. domestica larva as a template，further the gene and its encoding protein were predicted and analyzed by bioinformatics method. The constructed recombinant plasmid of pET28a/RPS18 was transferred into Escherichia coli Transetta（DE3）for the induced expression and protein purification，and the polyclonal antibody was prepared as well as the specificity of antiserum was analyzed. The transcription and translation expressions of RPS18 gene at different growth stages of the M. domestica larva and in the different tissues of 3-year larva were analyzed by RT-PCR，qPCR and Western blot. The results showed that the RPS18 ORF was 459 bp in length encoding 152 amino acids with a predicted molecular mass of 17 590.5 Da and pI of 10.48. The recombinant plasmid was transferred into E. coli Transetta（DE3），and the purified protein RPS18 was acquired. The purified protein was immunized to rabbits，then the polyclonal antibody was harvested，and the single band was visualized by both His single resistance identification and antiserum specificity analysis. The analysis by RT-PCR，qPCR and Western blot indicated that RPS18 genes expressed stably in different growth stages and different tissues of the larva. In conclusion，comprehensive analysis suggests that the gene may maintain solid stability in different growth stages of M. domestica and different tissues of larva.

Key words: ribosomal protein S18, Musca domestica, reference genes, prokaryotic expression, expression pattern

HU Ya, LU Cheng, WEI Chuan-chuan, XIU Jiang-fan, WU Jian-wei. Cloning and Expression Pattern of Ribosomal Protein S18 Gene in Musca domestica[J]. Biotechnology Bulletin, 2016, 32(6): 135-142.

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