The Expression,Purification And Application Of IHF-? Protein For Enhancing The Efficiency Of PCR

Polymerase Chain Reaction or PCR for short is a biological technique that can rapidly amplificate specific DNA fragments in vitro,is one of the most basic techniques of molecular biology and is widely used in the fields of biology research,clinical diagnosis etc.PCR is simple,fast and has strong specificity thus has been widely used.However,it also has some deficiencies that need to be improved.For example,when the template concentration in the PCR reaction system is too low or the template has impurity proteins,the PCR results may show false-negative,when the primers of the PCR are designed unreasonably,show false-positive results.The main reason for these false-negative or false-positive result is the limited sensitivity or specificity of the current PCR technique.In order to better use the PCR technique,researchers continuly optimize and improve it.Usually,PCR optimization methods include optimization of the reaction conditions and change the strategies of the reaction.Beside,adding an additive to the PCR system is also a method to improve the efficiency or specificity of PCR amplification.PCR additive is a material added to PCR reaction system additionally to increase the efficiency of PCR amplification or prevent invalid amplification.So far,researchers have found a variety of PCR additives such as some polymers,small molecules,nanomaterials and proteins,which impact the PCR system in different aspects by their respective properties.Among these,proteins are noticed and developed for their participating in important events in vivo such as DNA replication,repair,recombination,etc.However,there are many similar proteins in vivo,as long as they have heat resistance and other characteristics,they also can be considered to research and develop.This paper choices one of such proteins called integration host factor(IHF)as the research object.Integration host factor is a heat resistance DNA-binding protein that participates in the process of DNA replication,transcription,integration in prokaryotic.IHF consists of two heterologous subunits that have 30% homology.It has been reported that the IHF-? subunit is capable of making stable complexes with DNA in vitro.Therefore,based on these features,we predict that IHF-? subunit can influence the PCR system,which imitates the replication of DNA in vitro,in some respects.In this paper,the IHF-? is recombinated,expressed and purified and then applied to the PCR system to analyze its impact on the effeciency of the PCR amplification and the PCR-based gene synthesis.The main results of this research are as follows:1.The obtaining of the IHF-? geneHas designed 12 primers which are needed to synthesize the IHF-? gene based on the PCR based gene synthesis and assembled the IHF-? gene by one-step PCR method.Has confirmed the synthesized IHF-? gene sequence 100% homology with the target gene sequence by sequencing and Blast aligning.2.The recombination of the IHF-? gene to the expression vector,the expression of IHF-? protein and its optimizingHas constructed the prokaryotic expression vector pET-22b-IHF-?,and transformed into E.coli expression host strain BL21,then induced by IPTG.The induction condition has been optimized to overnight induction under 18? so the expression can get relatively large amounts of soluble IHF-? protein.3.The purification the IHF-? proteinThis study has used three different purification methods to purify the soluble and inclusion body IHF-? protein respectively.The results show that the method combining the PEI precipitation with ammonium sulfate precipitation to purify the soluble IHF-? protein is simple,fast,but poor reproducibility;while the method of combining denaturating and dissolving IHF-? inclusion bodies by urea with refolding the IHF-? protein by dialysing can get plenty of purified active IHF-? protein but is more complicated and takes longer time;but the purify of the soluble IHF-? protein with QIAGEN's Ni-NTA kit has failed to get active proteins.4.The preliminary research on the application of IHF-? proteinPreliminary results of this study have showed that the IHF-? protein can effectively improve the efficiency of PCR amplification and the efficiency of PCR-based gene synthesis in a certain concentration range.