Construction Of The Estrogen Receptor Mediated Gene Expression Regulation System

The Nucleic Receptor Superfamily has always been occupying a dominantposition in the regulation of gene expression in vivo, involved in almost all thefunction regulation of the human tissues and organs. It also played an important rolein the development as well as the occurrence of diseases such as tumor, immuneresponse, inflammation, diabetes, Alzheimer’s disease and cardiovascular disease.The Estrogen Receptor (ER), one of the nuclear receptor superfamily, is atypical ligand-dependent transcriptional factor: Combined with estrogen diffusesedinto the nucleus, the ER occured isomerization to form dimmers, then thedimerization complex was high affinity to estrogen response element ERE to triggerthe mechanism of gene regulation with the response by regulating the transcriptionof the downstream gene. It would provide a biological detection system for detectingestrogen analogues, if the estrogen response element ERE was assembled into thepromoter region of the reporter gene to construct reporter vector, which was thenco-integrated with estrogen receptor expression vector to the microorganisms. Inaddition, it would also provide a new model and method for exploiting exogenoussmall molecule compounds to directionally regulate gene expression.Pichia pastoris is commonly used eukaryotic expression system, withoutnatural plasmid in vivo, it is therefore necessary to achieve exogenous geneexpression in Pichia pastoris by integrating the exogenous gene expression boxtogether with the host chromosome. In this way, both to avoid the loss of the plasmid,while ensuring that the expression of exogenous gene was regulated by promoter andregulatory elements vector carrying its own. Based on this, in this paper, we carriedout the subject to construct the ER-mediated gene expression regulation system inPichia pastoris based on estrogen as an inducer.Firstly total RNA extracted from the human breast cancer MCF-7cells was toobtain hER cDNA by RT-PCR method. The ER genome was divided into threesections, and then the overlapping PCR method was adopt to get full-length sequences of ER gene by connecting the three sections of the PCR product. Gelelectrophoresis experiments suggested that the size of hER obtained by overlappingPCR was correct, sequence determination further proved it to be entirely correct.Subsequently, hER gene was inserted into the common carrier pPIC9of thePichia pastoris to construct hER expression vector. The ERE and reporter geneEGFP were cloned into pPIC9K to build reporter plasmid. Sequencing indicated thattwo plasmids were constructed successfully.Finally, the two plasmids were transformed into Pichia pastoris GS115hoststrain to verify the effectiveness of the regulatory system. After re-integration ofyeast genes, methanol and17β-estradiol were used respectively to induce the geneexpression. The results indicated that the reporter gene did not expressed withoutinducer， the fluorescence intensity was1.48light intensity/pixel; while the17β-estradiol concentration was10-7mol, the expression amount of fluorescencegradually increased during the24h, up to206.92light intensity/pixels.