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@#Objective To express glycoprotein H(gH)of pseudorabies virus(PRV)in mammalian cells and detect its immunogenicity.Methods The gH gene fragment of PRV-XiangA strain was amplified by PCR and inserted into mammalian cell expression vector pIRES-neo3 to construct recombinant expression plasmid pIRES-gH,which was transfected to HEK-293F cells and cultured in suspension for 5 d. The cell culture supernatant was identified by Western blot and purified by nickel ion chromatography column. The purified gH was emulsified with ISA 201 VG adjuvant to immunize 8 female ICR mice,and 8 mice in control group were immunized with the same amount of adjuvant,which was strengthened at 5 and 8weeks after the first dose respectively. The blood samples were collected at 4,7 and 10 weeks after the first dose and detected for the titer of specific antibody and neutralizing antibody in serum of mice;The mice were challenged with PRVXiangA strain(1. 5 × 104TCID50)by nasal drops 2 d after the third blood collection,and observed for the morbidity and mortality daily.Results The recombinant expression plasmid pIRES-gH was constructed correctly as identified by sequencing. The gH protein was successfully expressed and modified by glycosylation in mammalian cells with good reactivity,and about 625 μg purified protein was obtained under 100 mL culture volume. After three times of immunization,mice produced high level of specific antibody and showed the effect of neutralizing PRV,and the titer of neutralizing antibody reached 1∶256. In the challenge test,all the mice in control group became ill and died,while half of the mice in gH immunized group did not get sick with a survival rate of 50%.Conclusion PRV gH was successfully expressed in mammalian cells,and its immune protection was confirmed for the first time,which provided experimental basis for the further research and application of gH,and also provided a new idea for the development of PRV subunit vaccine.
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@#Objective To develop and verify a reporter gene assay(RGA) for the detection of biological activity of human growth hormone(hGH).Methods The biological activity of hGH was evaluated by the expression of luciferase(Luc)activated by hGH binding to hGH receptor(hGHR) on HEK293/GH-Luc cell membrane.The developed detection conditions were as follows:the initial concentration of sample was 1 μg/mL;the cell inoculation amount was(2.45～2.66) × 10~4 cells/well;the sample was of 3-fold serial dilution,with a total of 8 dilutions and the incubation time was 18～24 h.The relative biological activity of the sample was calculated by measuring Luc intensity and comparing it with the national standard by four-parameter fitting.The developed method was verified for specificity,repeatability,intermediate precision,relative accuracy,linear range and durability.Results The excipient components in product and the serum components in culture medium showed no effect on the activity detection results;The geometric coefficient of variation(GCV) of relative titer of one batch of samples in six repeated detections was 6.794%,much lower than 20%.The relative titer GCV detected by two experimenters in different batches of samples at different times were both lower than 20%;The relative deviations of the relative titer determination values of samples at different concentrations were within ±12%,the slope of linear regression equation was 0.982,the linear range was 0.6～1.6 μg/mL,and the coefficient of determination(R~2) was 0.997;The GCV of three batches of stock solutions and one batch of finished products were 4.758%,4.430%,7.294% and 2.771% respectively under the conditions of different cell generation,cell density and sampling location,all of which were less than 20%.Conclusion The developed RGA showed good specificity,repeatability,intermediate precision,relative accuracy,linear range and durability,which met the application requirements and was expected to replace the traditional in vivo biological activity detection methods for the activity evaluation and quality control of hGH.
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Objective:To study the effect of interleukin-11(IL-11),ciliary neurotropic factor (CNTF) and transforming growth factor-β (TGF-β) on the hGH gene promoter activity in rat pituitary MtT/S cells and the interaction with pituitary-specific transcription factor Pit-1.Methods:Stable transformed MtT/S cell line which contains hGH gene promoter -484-30 bp and luciferase reporter gene firstly established,then the concentration of GH in the medium and lysate of MtT/S cells and luciferase activities in MtT/S cells were measured after treatment these cells with the above cytokines,the effects of cytokines on secretion and synthesis of GH,and the promoter activity of the hGH gene were observed.Results:The results showed that IL-11(20 nmol/L),CNTF(10 nmol/L) and TGF-β(5 nmol/L) regulated secretion and synthesis of GH,and the luciferase expression in stable transformed MtT/S cells.IL-11 and CNTF had the stimulatory effect,whereas TGF-β had the inhibitory effect.Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected the regulatory role of these cytokines.Conclusion:IL-11,CNTF and TGF-β regulate the GH production in pituitary MtT/S cell line by regulating the hGH gene promoter activity.Pit-1 may not be involved in these actions.
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La hormona de crecimiento humana (hGH) circula parcialmente unida a su proteína de transporte de alta afinidad (GHBP) la cual resulta del clivaje proteolítico del dominio extracelular del receptor de GH. Recientemente la enzima TACE se identificó como la metaloproteasa responsable del clivaje y liberación de GHBP a circulación. Aunque aún se desconoce la función específica de esta proteína de transporte, distintos trabajos en la literatura demuestran efectos que potencian y efectos inhibitorios sobre la acción de GH. Por otro lado, existen evidencias que demuestran una fuerte relación entre la GHBP y el nivel de receptor de GH en el hígado en situaciones fisiológicas y patológicas. Esto permitió proponer a la determinación de GHBP en suero como un marcador periférico de la abundancia del receptor de GH en los tejidos. La determinación de la concentración de GHBP sería de especial interés para evaluar pacientes con diagnóstico probable de insensibilidad a la acción de GH y orientar el posterior estudio de anormalidades en el gen del receptor de GH. En la presente revisión, también se abordan dificultades metodológicas relacionadas a la medición de GHBP sérica.
Human circulating growth hormone (GH) is partly bound to a high-affinity binding protein (GHBP) which is derived from proteolytical cleavage of the extracellular domain of the GH receptor. Recently, the metalloproteinase TACE has been identified as an important enzyme responsive for inducing GHBP shedding. Although the specific function of GHBP is not fully known, both enhancing and inhibitory roles of this binding protein on GH action have been proposed. Many reports have demonstrated a close relationship between GHBP and the liver GH receptor status in physiological conditions and diseases. Moreover, serum GHBP measurement has been proposed as an useful peripheral index of the GH receptor abundance. Related to the latter, circulating GHBP concentration would be of special interest for the evaluation of GH insensitivity due to GH receptor gene abnormalities. In addition, the present review also focus on methodological problems concerning serum GHBP measurement.
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It is socially fortunate that there is little chance in the civil hospital to experience the victims of high velocity missiles. However it is reasonable thought to educate doctors about the gunshot and explosive injuries who might be mobilized in emergency condition. Authors have experiences to treat the high velocity missile wounds. In order to provide valid data to be an educational material, we clinically analyzed 41cases of those injuries who were treated at the Capital Armed Forces General Hospital from 19xx to 19xx. The results obtained were as follows: 1. In 15 cases there were only soft tissue injuries, The remaining 26 cases had the bone injuries and six of them had two injury sites. 2. In 28 cases the lower extremities were injuried, and they out-numbered the injury of upper extremities. The most frequent site of injuries was the thigh (31.7%). 3. Most common associated injuries were the periphenal nerve injuries, which numbered 10 cases. 4. The early operative treatments were given in 5 out of 32 cases having bone injuries. And the secondary operations, including bone graft and intemal fixation, had to be done in 10 out of the remaining 27 cases due to delayed union or nonunion. 5. There was no infection in cases having only the soft tissue injuries. But the localized osteomyelitis occurred in 4 cases among the cases having bony injuries. 6. Factors affecting the result of high velocity missile wounds were presence of bony involvement, site and extent of injuries, associated thoracoabdominal injuries and presence of peripheral nerve injuries and infection. 7. The evacuation time, chance of early adequate wound management, site of injury and extent of injury were the important factors in deciding the method of treatment. We suggest that the more selective and aggressive measures should be taken in the management of bony injuries.