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Cloning and Expression of the Porcine TrxR2 and Selenoprotein H Genes and the Selenoprotein Antibody Preparation

Author: WuZuoHong
Tutor: XiaXinJie；WuDe
School: Sichuan Agricultural University
Course: Comparison of animal and human nutrition
Keywords: TrxR2 SelH Clone Gene Expression Antibody
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 21
Quote: 0
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Abstract


Selenium is an essential trace element in life activities, plays a key role in a variety of physiological functions in organisms. Selenium mainly in the form of selenoproteins play a physiological function. So far, the study found that the pig selenoprotein least 25 species, but which most of the selenoprotein physiological function has not been determined. The the study TrxR2 and SelH pigs not yet clear gene sequences reported in this study by means of bioinformatics cloned pig the body TrxR2 and the SelH gene sequences, and rabbit anti-polyclonal antibody prepared on this basis, help to study the selenoprotein the physiological function and mechanism. This experiment related genes in the NCBI GenBank EST sequences, primers were designed from fresh lung tissue by RT-PCR method clone of selenium the the protein TrxR2 and SelH gene cDNA sequence (GenBank: GU181287/GenBank,,: HM018602) including complete signal peptide sequence contains selenocysteine ??the SECIS stem loop structure. By sequence alignment analysis the pig TrxR2 genes colleagues, cattle, dogs, monkeys and chimpanzees, etc. TrxR2 gene homology of more than 85%, the colleagues of pig SelH gene, horse, monkey, dog and gorilla, etc. SelH gene homologous has reached 93% to 95%. Selenoprotein genes connected to the prokaryotic expression vector pET-30a () to obtain the recombinant plasmid pET-30a ()-TrxR2 pET-30a ()-T-SelH, the the CaCl2 method into expression strain BL21 (DE3) pLysS IPTG was added to induce three hours, successfully prokaryotic expression of selenoproteins nickel affinity chromatography purified recombinant protein by SDS-PAGE gel electrophoresis to determine the a purified protein size of approximately 67 KD and 23 KD consistent with the intended purpose of the protein product of the size. And use as immunogen successfully prepared rabbit anti-pig polyclonal antibody titer by ELISA analysis and Western blotting analysis to prove that the antibodies are positive, good specificity. Detected by ELISA using antibody serum pig body the various organizations protein extract, concluded: pigs TrxR2 hypothalamus highest concentrations, followed by content in the lung, the remaining tissue content was not significant; the pig body SelH protein in second highest content in skeletal muscle and lung content of the thyroid. In this study, by the antibody preparation, distribution, and simplified ELISA titer analysis and Western blotting analysis method laid the foundation for further study of the function of the selenoprotein, provided technical support for the purification of natural pig of selenoproteins TrxR2 and pig SelH.

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