Eukaryotic expression of human IL17-RD-ECD and generation of its monoclonal antibody / 生物工程学报

IL-17 Receptor D (IL-17 RD) is a cytokine receptor that mediates IL-17 signaling and plays an important role in responding to the invasion of extracellular pathogens and many inflammatory and autoimmune diseases such as rheumatoid arthritis. In this study we report the generation of a mouse monoclonal antibody against human IL-17 RD. The recombinant human IL-17RD extracellular domain (hIL-17RD-ECD) was produced in the baculovirus expression system and purified from culture medium of sf9 insect cells. The purified protein was used as a T-dependent antigen to immune Balb/C mice. B cells from the spleen of immunized mice were fused with murine cell SP2/0. Hybridoma cell lines were screened for the production of the monoclonal antibody against hIL-17-RD-ECD using ELISA. A hybridoma cell line 1F8 was found to have a high production of the antibody, which was further confirmed for the specificity by both western blot and ELISA analyses. The monoclonal antibody obtained from hybridoma 1F8 was characterized to be IgG1+Kappa subclass. This study provided a base for the further therapeutic application of the antibody on the autoimmune disease including rheumatoid arthritis.

NOK Interacts With Akt and Enhances Its Activation / 生物化学与生物物理进展

NOK is a newly identified receptor protein-tyrosine kinase (PRTK) molecule that can promote tumorigenesis and metastasis. Previous data showed that NOK could activate the phosphatidylinositol 3'-kinase (PI3K) pathway in stable BaF3 cells. But how does NOK activate PI3K in cells remains unknown. It was showed that NOK physically interacted with the PI3K downstream effector Akt and enhanced its activation in human embryo kidney 293T (HEK293T) cells. Deletion mapping indicated that protein kinase B (Akt) was able to directly contact with the kinase domain of NOK. Inactivating the Akt kinase domain significantly reduced the intermolecular interaction between NOK and Akt, while constitutively active mutant of Akt apparently had a stronger interaction with NOK. NOK did not have an additive effect on insulin-mediated Akt activation. Overall, the results indicate that NOK might complex with Akt and directly activate PI3K/Akt signaling pathway.

c-Cbl Mediated Ubiquitination and Degradation of hSef / 生物化学与生物物理进展

Sef (similar expression to fgf genes) was identified as a feedback antagonist of FGF signaling in zerbrafish, mouse and human. Sefhas been reported to function in different ways, however the regulation of Sef stability remains unknown. The possible role of c-Cbl in the regulation of Sef protein degradation was investigated. Results from coimmunoprecipitation and immunostaining assays reveal that hSef colocalizes and interacts with c-Cbl. Data suggest that the interaction between hSef and c-Cbl results in the ubiquitination and subsequent degradation of the hSef protein. It was proposed that c-Cbl may serve as a modulator to regulate Sef protein stability during FGF signal transduction.

NOK Activates STAT3 Signaling by a JAK2-Dependent Mechanism / 生物化学与生物物理进展

Novel oneogene with kinase-domain (NOK) can activate multiple mitogenic signaling pathways including the janus kinases(JAK) and signal transducer and activator of transcription proteins (STAT). It was showed that NOK specifically and physicallyinteracted with STAT3 in human embryo kidney 293T (HEK293T) cells. In addition, NOK could directly interact with most of the STAT3 subdomains except coiled-coil and C-terminal domains. Removing ectodomain and transmembrane domain of NOK markedly enhanced its intermolecular interaction with STAT3. Also, NOK could co-immtmoprecipitate with JAK2. in vivo. Importantly, co-expression of NOK and JAK2 produced a synergistic effect on NOK-mediated STAT3 activation, while inactivating the kinase domain of JAK2 completely prevented this synergistic effect. Overall, the results indicated that NOK might complex with both STAT3 and JAK2 and activate STAT3 signaling by a JAK2-dependent mechanism.

Construction and characterization of hSef recombinant adenoviral vectors / 生物工程学报

Sef (similar expression to fgf genes) was identified as a feedback antagonist of FGF signaling in zerbrafish, mouse and human. To construct recombinant adenoviral vectors expressing hSef-L and hSef-S, the coding sequences of the two isoforms were amplified and ligated into pAdTrack-CMV, forming shuttle vectors pAdTrack-CMV/hSef-L-Myc and pAdTrack-CMV/hSef-S-Myc. After sequence confirmation, these two shuttle vector plasmids were linearized by Pme I and then co-transformed respectively with the adenoviral genome vector pAdEasy-1 into E. coli BJ5183. The successful recombinants were selected by Kanamycin and confirmed by Pac I digestion. The recombinant vectors Ad-hSef-L-Myc and Ad-hSef-S-Myc were finally digested with Pac I and transfected into HEK293 cells to pack into viral particles. The virus were amplified in 293 cells and used to infect MEF cells. Western blotting analysis was used to demonstrate the expression of hSef-L-Myc and hSef-S-Myc proteins. The inhibitory effects of the adenovirus mediated Sef expression on FGF signaling was further evaluated by Elk luciferase reporter assay. Our results indicated the constructed virus could produce effectively the proteins and then inhibit FGF signaling in MEF cells.

Cloning and expression of human GABARAP and preparation of anti-GABARAP antibodies / 生物工程学报

GABARAP, a microtuble-associated protein, is identified to interact with GABAA receptor. Anchoring of the GABAA receptor to GABARAP helps to cluster the receptor at the synaptic termini and to mediate fast synaptic transmission. GABARAP may mediate interaction of gephyrin with the GABAA receptor to stabilize clusters by forming multimeric structures. Furthermore, GABARAP and gephyrin may play more of a role in receptor sorting and transport to the cell surface than in anchoring to the cytoplasm, because at inhibitory synapses GABARAP appears to associate with transport vesicles rather than the cell surface. The association of GABARAP with NSF (N-ethylmaleimide sensitive factor), a protein involved in intracellular vesicle transport, supports this hypothesis. We cloned cDNA encoding full-length human GABARAP by nested PCR and inserted it into eukaryon expression vector pcDNA6HA and GST fusion protein expression vector pGEX4T2. The recombinant plasmid pGEX4T2-hGABARAP was transformed into E. coli BL21, from which GST-hGABARAP fusion protein was purified after IPTG induction by affinity chromatography with glutathione Sepharose-4B column. The antiserum against GABARAP was generated by immunizing rabbits with the purified GST-hGABARAP and was purified with GST-hGABARAP coupled NHS-activated Sepherose 4 column. The purified polyclonal antibody was effective for Western blotting and immunostaining. The hGABARAP was located both in the cytoplasm and nucleus with an abundant distribution around the peripheral nucleus.

Preparation of anti-hNOK antibodies and expression examination of NOK in the lung cancer tissues / 生物工程学报

Novel Oncogene with Kinase-domain (NOK) is a novel tumor-related gene, coding receptor like protein with a kinase domain. Overexpression of NOK leads to tumorigenesis and metastasis. To further study NOK function in physiological condition, it is necessary to prepare the anti-NOK antibody. In this report, GST fusion protein was adopted to prepare polyclonal antibodies against hNOK. The result showed that the antibodies we generated is with a very high titriation, and can be used for examination of NOK protein by Westernblot. Furthermore, the antibodies were used for immunohistochemistry in lung cancer tissues, and the results demonstrated high expression of hNOK in the tumor tissues. The antibody of hNOK we generated can serve as a diagnostic method for the lung cancer.

A LIF Mutation at The 29th Amino Acid Totally Abolished The Biological Functions / 生物化学与生物物理进展

Leukemia inhibitory factor (LIF) plays important roles in varieties of biological processes. This factor is highly conserved in mammalian animals and only one heterozygous LIF mutation was reported to cause the infertility of women. A LIF mutation was generated and the evidences were provided that the mutation of mature LIF at the 29th amino acid totally abolished its functions, including stimulation of STAT activation assayed by Luciferase reporter gene expression and EMSA experiments. In addition, the mutated LIF failed to inhibit the proliferation of M1 cells. The data indicated that the mutation of LIF did not have a dominant negative effect but lost the biological functions, suggesting that the 29th amino acid is critical for maintaining the activities of LIF.

Effect of cisplatin on ototoxicity and expression of caspase-3 in mouse cochlea / 西安交通大学学报(医学版)

Objective To establish a mice model of cisplatin-induced ototoxicity and to investigate the effect of cisplatin on the expression of caspase-3 in mouse cochlea.Methods Totally 69 Kunming mice were randomly divided into control group,cisplatin 2.5mg/(kg?d) group,cisplatin 3.5mg/(kg?d) group and cisplatin 4.5mg/(kg?d)group.Mice were injected intraperitoneally for 5 days.Auditory brainstem response(ABR) was measured to observe the change of hearing.Envision method of immunohistochemistry was applied to detect the expression of caspase-3 in cochlea.Results The weight and hearing of mice in different dose cisplatin groups were declined significantly as compared with those in control group(P

The cloning and expression of human interleukin-17 receptor-like molecule / 西安交通大学学报(医学版)

Objective To generate an human interleukin-17 receptor-like molecule (IL-17RLM) recombinant plasmid with 6?myc tag and detect its specific expression in eukaryotic cells. Methods Design two specific primers(including the enzyme sites of EcoRⅠand XhoⅠ), reextract hIL-17RLM-L DNA fragment after PCR and insert it into the 6?myc tagged pcDNA3.0 vector, then detect its expression by Western blot after transfecting COS7 cells. Results The 6?myc tagged recombinant plasmid pcDNA3.0- 6?myc /hIL-17RLM-L was generated successfully and its expression can be detected by Western blot in eukaryotic cells. Conclusion The eukaryotic expressing plasmid pcDNA3.0-6?myc /hIL-17RLM-L was generated successfully and its specific expression was realized, which may provide the basis for further research of its biological function.