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Research on Two FKBP12 Genes from Silkworm, Bombyx Mori

Author: FeiJingJing
Tutor: ZhangYaoZhou；JinYongFeng
School: Zhejiang University of Technology
Course: Biochemistry and Molecular Biology
Keywords: Silkworm FKBP12 Expression Analysis Gene structure Promoter analysis
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 45
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Abstract

FKBPs immunosuppressant FK506 intracellular receptor proteins, these proteins are involved in a variety of physiological effects, including protein folding, inhibition of T cell activation regulation, as well as cell cycle inhibition. The 12 kD FKBPs protein is the representative member of the widely expressed in the cells of each species and highly conserved PPIase activity. Just multiple FKBP12 subtypes in vertebrates, and our through NCBI silkworm gene pool to find two complete coding silkworm FKBP12 protein the nRNA sequence, named for the the Bm-FKBP12A and the Bm-FKBP12B GenBank number DQ443197 and DQ443423. Multi-species sequence found FKBP12 homologous genes and genes homologous heterosexual having a high degree of similarity at the amino acid level the the Bm-FKBP12A protein with human FKBP12 protein homology for the 77% Bm-FKBP12B protein with human FKBP12 protein 73%, the Bm-FKBP12A and Bm-FKBP12B similarity between the 83% homology. The speculated BM-FKBP12A protein molecular weight, isoelectric point were 11.82kD and 7.86 the Bm-FKBP12B protein of molecular weight, isoelectric point, were 11.63kD 8.79, these characteristics similar to known protein FKBP12. The the two FKBP12 gene cDNA sequence of silkworm genome sequence to compare two gene consists of three exons and two introns, but the Bm-FKBP12A gene intron sequences than Bm-FKBP12B long. Through the online site of the the Bm-FKBP12A and Bm-FKBP12B promoter sequences of two genes were chromosomal localization prediction and found that two genes are located on chromosome 20, the distance difference 1500kbp. Of Bm-FKBP12A in a promoter sequence 5 'end of the upstream (-361) - bp region (-311), Bm-FKBP12B at the 5' end A Raw (-161) - (-121) bp region promoter sequence. Tissue-specific cis-acting element is not found in the genes of the Bm-FKBP12A and Bm-FKBP12B, Bombyx mori FKBP12 protein is widely expressed in various tissues. Bm-FKBP12B ORF gene was cloned into the vector pGEX-4T-3 fusion protein was expressed in E. coli BL21 GST-Bm-FKBP12B by GST affinity chromatography, to give relatively pure fusion protein GST-Bm-FKBP12B , and then through a molecular sieve SephadexG-thrombin fusion protein GST ADX 75 further purification. After purified BM-FKBP12B as antigen to immunize New Zealand White Rabbit polyclonal antibody ELISA detection of the antiserum titer of 1:12800. Pair our purified proteins the BM-FKBP12B of the PPIase activity was measured, the results show higher activity. By the similar EST search, real-time fluorescence relative quantitative PCR and Western blotting analysis of the distribution of the two types of FKBP12 protein expression of the various periods of the silkworm and organizations. Found Bm-FKBP12 exist in various organizations and various periods of the silkworm, BM-FKBP12A transcription of the organizations than the Bm-FKBP12B of transcription of the of the and Bm-FKBP12B the highest in the moth and the embryonic period. Western blotting shows the highest expression of FKBP12 in the silk gland and intestines, hinted at silking pre-FKBP12 protein may be an important role in the regulation of gene expression of the silk gland and silk protein formed. GST-Pull down silkworm FKBP12 Hmo1 of protein interaction can not occur in vitro. These results lay the foundation for further study of silkworm FKBP12 gene.

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