Heat Shock Gene Expression And Regulation

Heat shock protein ( HSP ) plays an important role in stress protection, development and differentiation. Because heat shock gene ( hsp ) encoding HSP are expressed in response to a wide range of physiological or chemically induced stresses including some clinical diseases, it has dural importance in theory study and clinical practice for us to research hsp expression levels in different conditions. Heat shock factors ( HSF ) are major trans-factors for hsp transcriptional regulation. The study of its regulation mechanism will lead to a better understanding of their biological functions.In this study we estabolished a quantitative RT-PCR system successfully which can be used for detecting expression levels of three major human hsp ( hsp70, hsp90α, hsp90β). The changes of hsp expression in five human cells were analysed with this system. In order to study the function of HSF, two human HSF prokaryotic expression plasmids ( pET-11/HSF1 and pET-11B/HSF2 ) were constructed and expressed efficiently in E.coli BL21(DE3)polysS. Expressed products were researched for th binding ability with HSE, the cis-element of HSF action.1. It has been difficult to quantitate the amount of specific mRNA without an proper internal standard. In our study, a complex internal control cDNA ( Ic-DNA ) was synthesized through a complicated process including thirteen transition clone. This synthetic gene with 1644 bp consists of six DNA fragments from hsp70, hsp90α, hsp90β cDNA connected in the sequence 70₁ - β₁ - α₁ - 70₂ - β₂ -α₂ . Direction of all DNA fragments is in 5' -3'. the DNA sequences for transcription and reverse transcription are located in the two flanks of Ic-DNA separately.