HT036 Detection, Function Research And Protein Expression

Individuals with severe burn injuries are at risk for developing hypertrophic scar, which is characterized by scar tissue that is red, raised, and rigid. It can result in physical and psychological impairments after burn injuries, and the pathogenesis of hypertrophic scar is one of the most important issues in burn rehabilitation.We previously compared differentially expressed genes between normal skin and hypertrophic scar from 5 patients with microarray containing 4096 human genes and founded a gene, P311, was highly expressed in the early hypertrophic scar[1-2]. P311 protein does not belong to any known family of proteins. Pan, et al. reported that P311 gene could induce a TGF-β1–independent, nonfibrogenic myofibroblast phenotype [3]. Fujitani, et al. found that adenovirus-mediated P311 gene expression could accelerate nerve regeneration of the axotomized facial nerve [4]. In an attempt to explore the potential molecular basis of P311 gene in the formation of hypertrophic scar, we screened the possible candidates, which could bind with P311 protien, from the adult liver cDNA library with a yeast two-hybrid system. We acquired one interactive protein HT036 [5].HT036, however, as a new gene, was first presented into the Genebank by Xu, et al. in 2001, which encoded an intracellular protein. In order to investigate the role of HT036 gene in hypertrophic scar formation, we first compared the expression of HT036 gene between the hypertrophic scar and the skin, and then investigated the effects of HT036 gene on fibroblast transformation. Finally we expressed its encoding protein in Escherichia coli for the further research.Methods:1. Comparison of the expression of HT036 gene between hypertrophic scar and normal skin.Tissue was washed with PBS, and total RNA was extracted with Tripure Reagent. RT-PCR was performed with the TaKaRa RNA PCR Kit. All amplifications shown here represent the products of 30 to cycle within linear range. A primer pair was used to produce aβ-actin amplicion as an internal control. The densities of the amplifications were analyzed by Quantity One.2. The effects of HT036 and P311 genes on fibroblast transformation.DNA representing the entire coding region of human HT036 was amplified using modified primer, digested with EcoRⅠa nd SaLⅠ, and then cloned into pEGFP-N2. The clone was identified by restriction enzymes digestion and sequencing analysis. The plasmids containing HT036 or P311 gene were transfected or cotransfected into MRC-5 cells using LipofectamineTM2000. After 48 hours, the cells were lysed and examized by mouse anti humanα-SMA. GAPDH was used as an internal control. The bands were visualized by chemiluminescence and the densities of the bands were analyzed by Quantity One. The plasmids were also transfected or contranfected into 293 cells as the same method. After 72 hours, the fibrotic factors TGF-β1, MMP-2 and MMP-9 secreted into cell culture medium were examinzed by ELISA method. The standard was diluted into different concentrations, which provided a calibration curve. The optical density was measured at 450 nm. The results were analyzed by Microplate Manager 4.0.3. Inducing expression of human recombinant HT036 protein.We constructed the recombinant prokaryotic expression vector pET30a(+)-HT036 as previous described method. The clone was identified by restriction enzymes digestion and sequencing analysis. The recombinant vector was transformed into Escherichia coli DE3(BL21)pLysS using CaCl2 method. The protein induced by IPTG at 37℃. The expressed protein was identified by anti-his antibody and the optimal inducing-time and inducing-dose of IPTG were also estimated.Results1. The expression difference of HT036 gene between hypertrophic scar and normal skin.Tissue of hypertrophic scar in all 3 patients showed lower HT036 gene expression than normal skin. Comparing with the normal skin, the expression of HT036 gene in hypertrophic scar decreased by 70%,75% and 46% respectively.2. The effects of HT036 gene and P311 gene on fibroblast transformation.Transfection of HT036 gene into MRC-5 cells inhibitedα-SMA expression, while P311 gene stimulated its expression. The relative quantity ofα-SMA in the blank group, HT036 group, P311 group and cotransfected group was 0.13,0.03,0.18 and 0.05 respectively. HT036 gene decreased the amounts of TGF-β1, MMP-2 and MMP-9 in 293 cell culture medium. Compared with P311 group, the secreted amounts decreased by 46.4%,33% and 29% respectively.3. Inducing expression of human recombinant HT036 protein.The recombinant prokaryotic expression vector pET30a(+)-HT036 was successfully constructed. SDS-PAGE showed the recombinant fusion protein of about 29KDa highly expressed in Escherichia coli, which was up to 20% of the total protein. Western blot indicated there was a single specific anti-His band. The best inducing-time was four hours after ITPG administration. However, there was no obvious expression alteration among the different IPTG concentrations.Conclusion1. HT036 gene was low expressed in hypertrophic scar.2. HT036 gene could inhibit fibroblast transformation.3. HT036 protein was successfully expressed.