Identification of protein that interacts with adhesion protein of Mycoplasma genitalium from T7-phage display cDNA library / 中国免疫学杂志

Objective:To screen and identify the protein that interacts with the adhesion protein of Mycoplasma genitalium (MgPa)from T7-phage display cDNA library of human uroepithelial cells(SV-HUC-1).Methods:Recombinant adhesion protein of My-coplasma genitalium(rMgPa)was used as target molecule to biopan the T7 phage display cDNA library of SV-HUC-1 cell,the selected positive clones were analysed using DNA sequencing and BLAST analysis and identified by means of indirect ELISA,Dot immunoblot and Far-western blot.Results:After four rounds of biopanning,positive phages were obviously enriched.According to the results of DNA sequencing and BLAST analysis,the selected randomly 32 positive clones included 7 kinds different sequences,of which the number of RPL35 repeats was the most.The results of indirect ELISA,Dot immunoblot and Far-western blot showed that 7 representative phages could bind specifically with rMgPa.Conclusion:60S ribosomal protein L35(RPL35) may be the interacting protein of MgPa,which lays the experimental foundation for understanding the function of MgPa and the pathogenesis of Mycoplasma genitalium.

A phage display combined with DNA affinity magnetic system can be applied to a screening of DNA binding proteins, such as transcription factors / Un fago pantalla combinado con un sistema de afinidad magnético al ADN se puede aplicar al exámen de las proteínas de unión al ADN, tales como los factores de transcripción

Here we introduce a new approach for the screening of DNA binding proteins, using a phage library based on a phage display technique. In principal, a complementary DNA (cDNA) library based on the recombinant bacteriophage T7 expressing target proteins on its capsid (phage display) is constructed. These phage particles are hybridized with a biotinylated target DNA fragment which is immobilized on the surface of streptavidin paramagnetic particle (SA-PMP). The phage particles are released from the target DNA fragment by a nuclease treatment and the recovered phages are used to the next round of hybridization. These processes are repeated three times to amplify the target phages in the population. This simple method is faster, and more systemic than other current methods (e.g. yeast one hybrid system). As a proof of this principle, we tried to isolate transcription factors which specifically bind to the promoter region of the Arabidopsis thaliana AtGST11 gene. Two obtained candidates, RING zinc finger protein and AtHB6, showed DNA binding activity to the AtGST11 promoter region. We could validate that our new application of phage display is a superior method for isolation of DNA binding proteins with a broad range of potential applications.

Screening of proteins interacting with hepatitis C virus core protein from T7-phage display library / 第三军医大学学报

Objective To screen and identify proteins that interact with the hepatitis C virus core protein by means of T7-phage display system. Methods The hepatitis C virus core protein was expressed by prokaryotic expression and used as selected molecule to biopan the T7 human liver cDNA library. The selected positive clones were identified by DNA sequence and analyzed with BLAST program in GenBank. Results After BLAST in all positive clones, one protein--Smad interacting protein 1 (SIP1) was found to interact with the hepatitis C virus core protein. Conclusion T7-phage display system is a convenient, rapid and effective method for screening interacting proteins.

Construction of a T7 phage display cDNA library from lung of Microtus fortis / 中国血吸虫病防治杂志

Objective To construct a T7 phage display cDNA library from the lung of Microtus fortis for further screening the schistosomiasis-resistence-related genes of Microtus fortis. Methods mRNA was isolated from total RNA extracted from the lungs of Microtus fortis by TRIzol reagent, and was used to synthesize double strain cDNA by the reverse transcription. Then the double strain cDNA was given with EcoRⅠ and Hind Ⅲ adhering ends by ligation with the directional EcoRⅠ/Hind Ⅲ linkers and digestion with EcoRⅠ and Hind Ⅲ. The double strain cDNA fragments longer than 300 bp in length were fractionated by the Mini Column, and ligated into the T7 Select 10-3b vector with EcoRⅠ and Hind Ⅲ adhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library. Results The library constructed here contained 1.5?106 clones and the titer of the amplied library was 1.1?1012 pfu/ml. The PCR identification results of 100 clones picked at random showed that 91% clones were recombinant and 90% of recombinant clones contained cDNA fragments longer than 300 bp in length. Conclusion A T7 phage display cDNA library from the lung of Microtus fortis is successfully constructed.

Screening of proteins interacting with the Hepatitis B Virus PreS1 protein from T7-Phage display library / 重庆医科大学学报

Objective:To screen and identify proteins that interact with the hepatitis B virus PreS1 protein by means of T7-phage display system. Method:Hepatitis B virus PreS1 protein was expressed by prokaryotic expression and used as selected molecule to biopan the T7 select human liver cDNA library,the selected positive clones were identified by DNA sequence and analyzed with BLAST program in GenBank. Result:After BLAST in all positive clones,one protein:Cytochrome c Oxidase Subunit I(COX1)was found to interact with the hepatitis B virus PreS1 protein. Conclusion:T7-phage display system is a convenient,rapid and effective method for screening interacting proteins. This results will provide important evidences for studying the pathogenesis and mechanism of HBV.