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Construction of an Expression Vector and Screening a Stable Transfection Cell Line for HSF4b Protein

Author: LiXueLi
Tutor: ZhangJun；MaYuanFang
School: Henan University
Course: Immunology
Keywords: Heat shock transcription factor 4 Lentiviral vectors Cell lines
CLC: R776.1
Type: Master's thesis
Year: 2010
Downloads: 23
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Abstract

Background congenital cataract is the main reason for cause of childhood blindness, about 0.3 to 1.5/1000 children with congenital cataracts, its main pathological changes of lens growth retardation, non-transparent lens scar tissue formation and chromosomal abnormalities mainly caused disease causes. It has been found that the the various mutant gene with clinical and animal experimental congenital cataract related to the occurrence. Coded lens cell structure protein gene mutation (MIPs, CP49 and alpha-, beta-and gamma-crystallins) and regulation of lens development associated transcription factor gene mutation (Pax6, and FoxE3, Six3, Prox1, Sox2 / 3, Maf , Pitx3, AP-2a and Hsf4). 2002, BU people a congenital cataract pedigree linkage analysis, the disease gene is located on chromosome 16, identified Hsf4 congenital cataract candidate genes, it occurred a mechanism Hsf4 gene 348 nucleotide T to C, so that the DNA-binding domain of Hsf4 115 highly conserved leucine into proline, and which resulted in the occurrence of cataract. The application of transgenic technology knock addition to mice Hsf4 gene mice 3-5 days after birth (P3-5) appear cataract, the results strongly support Hsf4 regulation lens early developmental transcription factor argument. However, the regulation Hsf4 transcriptional activity of the signal path and the lens early developmental Hsf4 regulation of the molecular mechanism is unclear. The research Hsf4 role in the the lens development process in biology mutation associated with congenital cataract of great significance to reveal a mutant Hsf4 pathogenic mechanism and early diagnosis and treatment with Hsf4. We build the Hsf4 eukaryotic expression vector pcDNA3.0/Hsf4b of however pcDNA3.0/Hsf4b transfected MLEC (Hsf4b-/ -) lens epithelial cells in low transfection efficiency, the development of molecular to explore Hsf4b regulation lens study of the mechanism is more difficult. Vitro establish a high transfection efficiency of cell lines stably expressing Hsf4b we to further study Hsf4b the biological function has important significance. The carrier the purpose build Hsf4b eukaryotic expression, to facilitate research Hsf4b function in vitro. To build Hsf4b the recombinant lentiviral vector is in order to obtain a continuous and to stable expression Hsf4b cell lines. Lay the foundation for the future to continue to study the regulation of lens development in Hsf4 molecular mechanisms. Human heart cDNA library as a template application encompasses Hsf4b the length of the primers for PCR and are added in Hsf4bcDNA the N-terminal Flag tag. The PCR product was digested with Kpn I and EcoR I, linearized pcDNA3.0 plasmid with the two enzymes are connected together to obtain the recombinant plasmid pcDNA-Flag-Hsf4b; do directed mutagenesis on a recombinant plasmid; order to obtain a continuous and stable expression cell lines in Hsf4b, the recombinant plasmid pcDNA-Flag-Hsf4b lentiviral the expression vector Plentiviral with XholI NdeI digested and then connect to get Plentiviral-Flag-Hsf4b plasmid and viral capsid protein PLP1, PLP2, VSV-G turn HEK293T cells transfected into MLEC secreted virus-containing supernatant to infect cells, and to stabilize it by Blascitidin drug screening, with the Western blot lentiviral expression vector Plenti/Hsf4b expression, and detection of the corresponding downstream protein Hsp25 expressed. The results build Hsf4b eukaryotic expression vector pcDNA-Flag-Hsf4b transferred into HEK293T cells showed expression of the corresponding protein, transfected MLEC cells transfection efficiency is very low, Western undetectable, we found its main The reason is that the recombinant plasmid is transfected into cells, the expression of the corresponding protein has bound to the recombinant plasmid CMV inhibited Hsf4b further expression of the related proteins. Will build good Hsf4b eukaryotic expression vector for site-directed mutagenesis on CMV, and this inhibition is discharged, last won the vector pcDNA-Flag-Hsf4b mutant. The sequencing results and the the Western results also prove the mutations success, the expression of the inserted fragments MLEC cells. In order so Hsf4b stable and sustained expression in MLEC cells, constructed Hsf4b lentiviral expression vector and screened by a stable expression cell lines, the Hsf4b cell lines and a Plentiviral (empty vector). Western (Western blot) to detect the expression of downstream small molecule heat shock protein Hsp25, The results show Hsf4b promote expression of Hsp25. The conclusions using genetic engineering techniques, Hsf4b the cDNA was cloned into the eukaryotic expression vector pcDNA3.0 constructed Hsf4b eukaryotic expression plasmid pcDNA-Flag-Hsf4b, protein successfully expressed in human embryonic kidney cell line 293T. CMV promoter plasmid pcDNA-Flag-Hsf4b successful site-directed mutagenesis was recombinant the plasmid CMVm-Flag-Hsf4b. Was constructed the expression vector Hsf4b lentiviral Plenti-Flag-Hsf4b, and successfully transfected MLEC cells. To obtain a stable expression cell strain MLEC/Plenti-Hsf4b Hsf4b protein. The increased expression of the heat shock protein Hsp25 MLEC/Plenti-Hsf4b small molecules in cell lines.

Construction of an Expression Vector and Screening a Stable Transfection Cell Line for HSF4b Protein

Abstract

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