Screening And Validation Of Pbx4 Interacting Proteins

This paper is divided into two parts: the first part is about the screening and validation of PBX4 interacting proteins;In the second part,the telomerase activity was detected by multiple quantitative PCR(Taqman probe).Describe the two parts separately.Part AThrough further family analysis,whole genome sequencing and cytological experiments,Pbx4 was identified as the functional gene that affects the development of hind limbs in mice.In mammals,Pbx genes include Pbx1,Pbx2,Pbx3 and Pbx4.PBXs is expressed during the development of mouse embryos.As a central signal mediated by integration,PBXs participates in a variety of developmental signaling pathways and affects the process of embryo development.The function of Pbx4 in vertebrates is mediated,in part,by the interaction of Pbx proteins with members of the Hox and Meis/Prep families.It is a cofactor of Hox protein and forms a transcriptional regulatory complex with Hox to regulate the expression of downstream target genes,thereby affecting the expression of developmental signals.Based on the above research background,we constructed p LL3.7-PBX4-EGFP vector and 15 p LL3.7-HOXS-HA-EGFP vectors,and co-transfected HEK293 T cells with 70% to 80%overgrowth.Pbx4 protein and 15 candidate Hoxs proteins with HA label were fused to express Pbx4 protein.Immunoprecipitation and Western Blot were used to verify the positive and negative Hox protein binding with Pbx4 protein,and finally,Hoxc12 interacted with Pbx4.Among them,three genes--Hoxa13,Hoxd11 and Hoxd10 failed to synthesize genes due to the high GC content of CDS sequences,and the interaction with Pbx4 protein could not be verified.In order to further study the gene expression profile of Hoxc12,the hind limbs of E10.5,E11.5,E12.5,E13.5 and E14.5 mouse embryos were taken for spatio-temporal expression,and it was found that the highest expression level of Hoxc12 gene was found in E13.5 mouse embryos.Finally,RNA was extracted from different tissues and organs of E13.5 mice,and then the relative expression of Hoxc12 gene was detected by SYBR Green real-time fluorescence quantitative analysis.The results showed that Hoxc12 was expressed in the hind limbs of mice with the highest expression level,and the heart was slightly expressed,but almost no expression level in other tissues and organs.Part BIn recent years,with the rapid development of cell therapy,in order to better standardize the control of cell quality,telomerase activity detection can be used as a standard for the risk assessment of tumor-forming cells,so as to improve cell biological safety.Most of the current telomerase activity detection methods are cumbersome.Some direct detection methods can reflect telomerase activity by extracting cell proteins and polyacrylamide gel electrophoresis,but they are rarely used due to certain radioactive pollution and high cost.Some indirect detection methods have been applied in the market.SYBR Green method is used to measure the expression level of telomerase catalyzed subunit TERT to reflect telomerase activity indirectly.However,due to the low expression level of TERT gene,Ct value fluctuation cannot be accurately quantified,resulting in certain pore difference and non-specific amplification,resulting in false positive interference.Therefore,how to improve the specificity and sensitivity of the response,as well as the accurate quantitative detection of cell genes is a great challenge.In view of the above problems,this study established a stable and sensitive method to detect the expression level of telomerase catalytic subunit TERT,thus reflecting telomerase activity.Specific reverse transcription primers,two pairs of quantitative primers and one fluorescent labled probes were designed for the CDS sequence of TERT.The reaction system was optimized and multiple reactions were performed with the reference GAPDH gene in a single tube using fluorescence quantitative PCR(Taqman probe method).In addition,HEK293 T positive cells from tumor-derived human cells with telomerase activity and MRC-5 negative cells from normal human differentiated cells without telomerase activity were selected,and telomerase activity was determined by TERT amplification curve and ?Ct stability between GAPDH gene and tumor cells.And the specificity,stability and reliability of the method were verified.Results: Using c DNA obtained under the action of specific reverse transcription primers as template,the two pairs of primer probes of TERT gene were mixed with GAPDH for multiple quantification,which significantly improved the sensitivity and TERT fluorescence signal quantity of the whole system by 3 times.Secondly,in a single tube,TERT gene expression can be detected efficiently and the?Ct between GAPDH and TERT remains stable after gradient dilution of positive cells c DNA.Ct value of TERT gene was not detected after c DNA gradient dilution of negative cells.The intragroup and inter-group coefficient of variation are less than 1%,which proved that the method is stable.Sixteen samples are tested by this method,and the postive rate is 44%,which met the expected requirements.The results show that the relative expression level of TERT gene is significantly different between cancer cells and normal cells(p <0.001),which proved that the system is stable and reliable.This novel telomerase activity detection protocol plays an important role in tumor prediction and clinical diagnosis.