Construction Of An Expression Vector And Screening A Stable Transfection Cell Line For HSF4b Protein

BackgroundCongenital and infantile cataracts are dominant causes of children blindness in the world. Around 0.3-1.5/1000 of children are suffered from the diseases. The main pathological changes are lens growth retardation, non-transparent lens scar, and chromosomal abnormalities, the main cause of the disease. A variety of gene mutations have been found, such as structural protein coding genes (MIPs, CP49 and alpha-, beta-and gamma-crystallins) and development-related transcription factor (Pax6, FoxE3, Six3, Prox1, Sox2 / 3, Maf, Pitx3, AP-2a and Hsf4) in lens cells. Many clinical and animal experiments have been performed related to the occurrence of congenital cataracts. In 2002, BU and colleagues did a linkage analysis for a congenital cataract family, and identified Hsf4 as a candidate gene for congenital cataract, located it in chromosome 16. The mechanism of cataracts is the mutation of T at 348nt of the gene into C, which causes highly conserved leucine into a proline at 115nt in the DNA binding domain of Hsf4, eventually leading to the occurrence of cataracts. Cataract appeared 3-5 days after birth(P3-5) in Hsf4 gene knockout mice. The results strongly confirmed that the early development of the lens Hsf4 is an important regulation of transcription factor. However, the signaling pathways of Hsf4 transcriptional activity as well as the molecular mechanism of earlier development of lens regulated by Hsf4 remain unclear. Therefore, the biological functions of regulation of Hsf4 during lens development are important for the revealing of pathogenic mechanism of mutant Hsf4 as well as early diagnosis and treatment of Hsf4 mutations-associated congenital cataracts. For this purpose, we built Hsf4 eukaryotic expression vector pcDNA3.0/Hsf4b. However, the transfection of mouse lens epithelial cells (MLEC) with pcDNA3.0/Hsf4b (Hsf4b-/ -) was less efficient which brought problems to the exploration of molecular mechanism of Hsf4b in the regulation of lens development. In vitro establishment of an efficient and stable transfected cell lines expressing Hsf4b would help us to further study the biological function of Hsf4b. ObjectiveWe aim to construct Hsf4b eukaryotic expression vector to facilitate the research on Hsf4b function in vitro, to build Hsf4b recombinant lentiviral vector in order to obtain a continuous and stable cell lines expressing Hsf4b protein and to pave the foundation for further study on the regulation of lens development by Hsf4.MethodsRT-PCR was done with Hsf4b full-length PCR primers and Flag tag at the N-terminal of Hsf4b cDNA, employing the heart cDNA library as PCR template. PCR products were digested with Kpn I and EcoR I, and inserted into pcDNA3.0 plasmid to obtain recombinant plasmid pcDNA-Flag-Hsf4b. The site-directed mutagenesis was performed on the recombinant plasmid. In order to obtain a sustained and stable cell lines expressing Hsf4b protein, Plentiviral-Flag-Hsf4b plasmid was made by ligating pcDNA-Flag-Hsf4b and slow virus expressing lentiviral vector after digestion with XholI and NdeI. This plasmid was co-transfected with viral capsid protein PLP1, PLP2, VSV-G into HEK293T cells. MLEC were infected with the supernatant of the transfected cells with virus and selected by Blascitidin screening. Western blot was used to detect the Plenti/Hsf4b and the corresponding downstream protein Hsp25.ResultsEukaryotic expression vector pcDNA-Flag-Hsf4b was built and transfected into HEK293T cells. The results showed it can express the corresponding protein. However, the efficiency of MLEC transfection was too low to be detected by Western Blot. It was found that the main reason is that transfected cells expressed the corresponding protein and this protein later bound to the CMV in the recombinant plasmid which inhibited the further expression of Hsf4b. The site-directed mutagenesis of CMV on Hsf4b eukaryotic expression vector can remove the inhibition. pcDNA-Flag-Hsf4b mutants was finally screened. Both sequencing and Western blot proved that mutation is successful because the correctly inserted fragment in the MLEC cells have been expressed. Hsf4b lentiviral expression vector was constructed to have a stable and a long time expression of Hsf4b in the MLEC cells. A stable expression cell line was successfully screened which can stably express Hsf4b protein. Using Western blot was used to detect expression of its downstream small heat shock protein Hsp25, which proved that Hsf4b promote Hsp25 expression.Conclusions:Using genetic engineering technology, Hsf4b's cDNA was cloned into the eukaryotic expression vector pcDNA3.0 to construct Hsf4b eukaryotic expression plasmid pcDNA-Flag-Hsf4b. Corresponding protein was successfully expressed in human embryonic kidney cell line 293T. The site-directed mutagenesis of the CMV promoter in Hsf4b eukaryotic expression vector was successfully done to obtain the recombinant plasmid CMVm-Flag-Hsf4b. Hsf4b lentiviral vector Plenti-Flag-Hsf4b was also constructed and successfully transfected into MLEC cells. A stable cell line expressing Hsf4b protein MLEC/Plenti-Hsf4b was made and the expression of small heat shock protein 25 increased in MLEC/Plenti-Hsf4b Cell.