In modern biology and biotechnology research, recombinant gene expression has been the most popular method to obtain the target protein. In recent years, many foreign genes have been efficiently expressed in Escherichia coli. However, proteins encoded by animal, plant or mesophilic microbial genes often lose activities or become denatured within a few hours at regular growth temperatures for E. coli; some other target proteins are toxic to host cells and therefore difficult to be over-expressed. The new T-vector, pEXC-T, was constructed by combining TA cloning and cold-shock induction to obtain high expression levels with low costs. This paper reports the construction of pEXC-T and optimization of induction techniques for gene expression. Two instable proteins were tested and successfully expressed in soluble form by using pEXC vector. The development of pEXC-T offers a convenient technique for the preparations of recombinant proteins to be used in structure/function studies, or as diagnostic markers and medicinal proteins.
Biotechnology ; Cold Temperature ; Escherichia coli ; genetics ; Gene Expression ; Genetic Vectors ; Plasmids ; genetics ; Recombinant Proteins

Objective To investigate the role of immunophenotyping in distinguishing mycosis fun- goides (MF) from lichen planus and psoriasis.Methods The expression of CD1a,CD4,CD8,ICAM-1, LFA-1,HLA-DR,CD30 and CD7 was measured by ABC immunohistochemical technique in specimens ob- tained from lesional skin of 15 cases of MF,17 cases of lichen planus and 17 cases of psoriasis,and in the skin of 6 healthy controls.Results In the lesional epidermis of MF,the density of cells positive for CD1a, CD30 or ICAM-1,was significantly higher (mononuclear cells,P＜0.001;dendritic cells,P＜0.01) than that in the lesional epidermis of lichen planus,psoriasis and in the skin of healthy controls.The density of cells positive for CD4 or CD8 and of dendritic cells positive for HLA-DR was higher in lesional epidermis of MF than in that of lichen planus.The linear density of CD1a-positive cells (P＜0.01),the percentages of cells positive for ICAM-1 (P＜0.05) or LFA-1 (P＜0.05) were all higher in the lesional dermis of MF than in that of lichen planus.As far as the CD7-positive cell density was concerned,it was higher in the lesional dermis of lichen planus and psoriasis than in that of MF and skin of healthy controls (P＜0.01), while no difference was found between the epidermis of MF and that of lichen planus or psoriasis.Conclu- sion There are differences in the expression of CD1a,CD4,CD8,ICAM-1,LFA-1,HLA-DR,CD30 and CD7 in the lesional skin of MF,lichen planus and psoriasis,which may provide a clue to the pathogenesis of these diseases.

Objective To investigate the expression of haptoglobin(Hp)mRNA and protein in nor-mal human epidermal cells and human keratinocyte cell line-HaCaT cells.Methods In situ hybridization and RT-PCR were used to detect the expression of Hp mRNA in normal human epidermal cells and HaCaT cells.Immunohistochemistry was used to detect the expression of Hp on normal human epidermal cells.Im-munohistochemistry and Western blot were used to detect Hp expression on HaCaT cells.Results There was Hp mRNA expression in normal human keratinocytes and HaCaT cells.There was no Hp mRNA expres-sion in normal human epidermal Langerhans cells.There were some Hp positive dendritic cells in normal human epidermis.There was no obvious Hp protein staining in the HaCaT cells by immunohistochemistry.There was Hp protein band from HaCaT cells by Western blot.Conclusions There is Hp mRNA expression in normal human keratinocytes and HaCaT cells which suggests that normal human keratinocytes and HaCaT cells have the ability to synthesize Hp protein.Normal human epidermal Langerhans cells have no ability to synthesize Hp protein.There is small amount of Hp protein in HaCaT cells.

Objective To detect human papillomavirus(HPV)DNA in peripheral blood of patients with condyloma acuminata(CA).Methods Polymerase chain reaction(PCR)was used to amplify the HPV DNA in skin lesions and peripheral blood of30patients with CA,and20normal blood donors as controls.Re-striction endonucleases Rsa I and Pst I were used for HPV DNA typing.The PCR products from1patient who was HPV positive for both skin lesions and peripheral blood,were cloned and sequenced.Results HPV DNA was identified in all CA skin lesions.HPV DNA was identified in peripheral blood mononuclear cells(PBM-Cs)from8of30(26.7%)patients and in none of20control subjects(P

Objective To investigate the in vitro effect of interferon-γ (IFN-γ) and ATRA on the morphological transition, proliferation of and apoptosis in a human cutaneous squamous cell carcinoma cell line SCL-1. Methods Cultured SCL-1 cells were divided into 6 groups to be treated with ATRA of 1 μmol/L, various concentrations ( 100, 500, 1000 U/ml) of IFN-γ, the combination of ATRA of 1 μmol/L and IFN-y of 1000 U/ml,respectively, or to remain untreated. MTT assay and flow cytometry were performed to evaluate the cell proliferation and apoptosis. The morphological features of apoptotic cells were observed by a transmission electron microscope (TEM) and inverted phase contrast microscope after 1% propidium iodide staining. Results IFN-γ could inhibit the proliferation of SCL-1 cells in a dose-dependent manner, and the most pronounced inhibitory effect was observed at a dose of 1000 U/ml . ATRA and IFN-γ induced an apoptosis in SCL-1 cells, and the early apoptosis rate was 4.84%, 11.96% and 18.71% in SCL-1 cells after treated with ATRA of 1 μmol/L, IFN-γ of 1000 U/ml and their combination, respectively. A series of morphological changes characteristic of apoptosis,such as bipolar changes, were observed in SCL-1 cells treated with ATRA and IFN-γ, with the presence of many early apoptotic cells, which showed a trend towards benign differentiation. Conclusions Within a certain concentration range, IFN-γcan promote the differentiation, but inhibit the proliferation of SCL-1 cells in a dose-dependent manner, and ATRA could enhance the effects of IFN-γ.

Objective To investigate the effects of baicalein and acitretin on the apoptosis in a human cutaneous squamous cell carcinoma cell line, SCL-12. Methods Cultured SCL-12 cells were treated with different concentrations of baicalein (3.125, 6.25, 12.5 μmol/L) and acitretin (2.5, 5.0, 10.0 μ mol/L), alone or in combination, for 48 hours. Subsequently, cell proliferation was detected by MTT assay, and cell apoptosis by ELISA as well as annexin V-FITC and propidium iodide double staining. Real-time quantitative RT-PCR was used to detect the expression of Fas mRNA in SCL-12 cells. Results The cell proliferation of SCL-12 cells was inhibited by baicalein and acitretin alone or in combination. The combination of baicalein and acitretin at the three tested concentrations, except for that of baicalein at 3.125 μmol/L and acitretin at 2.5 μmol/L, more strongly inhibited the proliferation of SCL-12 cells compared with baicalein or acitretin alone, and the inhibitory effect was in a dose-dependent manner. The early apoptosis rate was 9.39% ± 1.52%, 20.86% ± 2.16%,36.85% ± 3.26% in SCL-12 cells treated with baicalein of 3.125 μmol/L, acitretin of 5.0 μmol/L alone and their combination, respectively, significantly higher than that in untreated cells (4.39% ± 0.64%, all P <0.05); the induction of apoptosis in SCL-12 cells by the combination of baicalein and acitretin was stronger than that by baicalein or acitretin alone (F = 138.44, P < 0.05). Baicalein and acitretin alone or in combination significantly increased the mRNA expression of Fas in SCL-12 cells, and the effect of their combination was stronger than that of baicalein or acitretin alone. Conclusions Baicalein and aeitretin could inhibit the growth of and induce the apoptosis in SCL-12 cells, and the effect is enhanced by the combination of baicalein and acitretin, which may be associated with the upregulation of Fas expression in SCL-12 cells.

Objective To investigate the expression of survivin and bcl-2 in human squamous cell carcinoma (SCC) lesions and cell line SCL-1. Methods Tissue samples from 60 patients with SCC and 10 normal human controls were immunohistochemically stained to detect the expressions of survivin and bcl-2.Western blot was used to measure the expressions of bcl-2 and survivin proteins in HaCaT human keratinocytes and SCL-1 human squamous cell carcinoma cells. Results In normal control tissues, there was no expressions of survivin or bcl-2, while in SCC, the expression rates of bcl-2 and survivin were 70% and 60%, respectively,and there was no statistical correlation between the expressions of bcl-2 and survivin (P >0.05). Neither the expression of survivin nor that of bcl-2 was correlated to patients' age, gender or lesional site (all P >0.05). A statistical correlation was observed between the pathological stage in patients and expression of bcl-2 as well as between lymph node metastasis and expression of survivin (both P < 0.05). Western blot analysis revealed a significant increase in the expression of survivin and bcl-2 in SCL-1 cells compared with HaCaT cells. Con-clusion In SCC, survivin and bcl-2 seem to play their roles via different anti-apoptotic pathways.

Objective To investigate the distributions of haptoglobin (Hp) phenotypes and their clinical significance in five skin diseases. Methods Haptoglobin phenotypes were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a discontinuous buffer system, and then confirmed by staining with silver nitrate. Results The distribution of Hp1-1, Hp2-1, Hp 2-2 of Han ethnic group was 9.9 percent, 46.9 percent, 43.2 percent, respectively. Compared with normal controls, the frequency of Hp2-2 in SLE group (59.3%) was markedly high, especially in those with kidney damage, that of Hp2-1 in psoriasis(30.9%) was low, and that of Hp1-1 in eczema was high, especially in those without exudation (27.4%). There was no statistical significance for the distributions of Hp phenotypes in secondary syphilis and condyloma acuminatum. Conclusion Hp phenotypes might be associated with the pathogensis and some clinical manifestations in SLE, psoriasis of eczema.

Fibroblast growth factor 21 (FGF21) is a member of FGF family. It has been demonstrated that FGF21 is an independent, safe and effective regulator of blood glucose levels in vivo. In order to improve the activity of FGF21, we exchanged the beta10-beta12 domain of the human FGF21 with that of the mouse FGF21 to construct a novel FGF21 gene (named hmFGF21), and then subcloned hmFGF21 gene into the SUMO expression vector to create pSUMO-hmFGF21 and transformed it into E. coli Rosetta for expression of the fusion protein SUMO-hmFGF21. Both in vitro and in vivo glucose regulation activity of hmFGF21 was evaluated. The SDS-PAGE result showed that compared with wild-type hFGF21, the soluble expression of hmFGF21 increased about 2-fold. HmFGF21 was more potent in stimulation of glucose uptake in HepG2 cells in vitro. The results of anti-diabetic effect on db/db mice demonstrated that hmFGF21 had better efficacy on controlling the blood glucose of the db/db diabetic animals than wild-type hFGF21. These results suggest that the biological properties of FGF21 are significantly improved by optimization.

Objective To study the relationship of proliferation and activation of T lymphocyte subsets and disease progression in antiretroviral-naive human immunodeficiency virus(HIV)-1-infected individuals.Methods Forty-nine antiretroviral-naive,chronically HIV-1 infected patients and 16 healthy,HIV-1 negative controls were enrolled in this study.The patients were divided into 3 groups according to their CD4+T cell counts:<200×106/L,(200-350)×106/L and>350×106/L.Peripheral blood mononuclear cells(PBMC)were isolated.T cell proliferation index was measured by Ki-67 staining.T cell activation was detected by CD38 staining.The samples were analyzed by flow cytometry.The data were compared by one-way ANOVA.Results The percentage of Ki-67+cells in CIM+T ceils was 7.92%±4.37%in CD4+T cell<200×106/L group,which was significantly higher than those 0.39%d:0.24%in control group,2.61%±2.12%in(200-350)×106/k group and 2.65%±2.13%in>350 X106/L group(F=21.961,P<0.01).The percentage of Ki-67+cells in CD8+T ceils in CD4+T cells<200×106/L group was 2.87%±1.13%,which was also much higher than those in other 3 groups(0.15%±0.90%,1.40%±1.17%,1.22%±0.80%,respectively F=19.203,P<0.01).The Ki-67'CD4'T cells and Ki-67+CD8+T cells were inversely correlated with CD4+T cell counts(r=-0.654,r=-0.539,respectively;P350×106/L group(F=14.333,F=15.412,respectively;P<0.01).The expressions of Ki-67+ on CD4+ and CD8+T cells were positively correlated with CD38 expression(r=0.527,r=0.391,respectively;P=0.002).Conclusions The proliferation and activation of T lymphoeytes are enhanced during HIV/AIDS disease progression.And T eell activation may be the result of persistent immune activation.