Palmitoylation of heat shock protein 90 in mouse sperm / 生理学报

Protein palmitoylation, one of post-translation modifications, refers to the addition of saturated 16-carbon palmitic acid to cysteine residues via the thioester bond. It plays key roles in various functional activities, such as the interaction, stability and location of proteins. Heat shock protein 90 (Hsp90), an important molecular chaperone, has been reported to be involved in sperm capacitation. However, it remains unclear whether protein palmitoylation exists in sperm and whether Hsp90 in sperm is palmitoylated under different physiological conditions. In this study, we examined whether the protein palmitoylation is present in mouse cauda epididymis sperm using acyl-biotin exchange method, predicted the potential palmitoylated sites of Hsp90 by the software CSS-Palm 4.0 and detected the palmitoylated Hsp90 in the mouse sperm from caput epididymis and cauda epididymis by immunoprecipitation. We found that some proteins, approximately 50, 65, 72, 85 and 130 kDa, were palmitoylated in mouse cauda epididymis sperm. Five sites in two Hsp90 isoforms were predicted to be palmitoylated. The results also showed that Hsp90 in mouse sperm was palmitoylated and its palmitoylation level was involved in different physiological conditions: the palmitoylation level of cauda epididymis sperm was higher than that of caput epididymis sperm; and the palmitoylation level after capacitation was much higher than that before capacitation. In conclusion, this study reveals that protein palmitoylation is present in mouse sperm and the palmitoylated Hsp90 is associated with different physiological conditions in sperm.

Establishment and evaluation of methods for determinating cystic fibrosis transmembrane conductance regulator quantitatively / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To establish and evaluate a BA-ELISA method for the quantitative detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein.</p><p><b>METHODS</b>We deliberately selected three tables of CFTR and made the synthetic peptide be expressed in E. coli, then used the antigen to immunize rabbits to obtain the anti-CFTR polyclonal serum. After that, 96 well plates were coated with the purified antibody against CFTR. The antigen CFTR which was extracted from human sperm was detected by anti-CFTR antibody labeled with biotin, horseradish peroxidase conjugated avidin, and the substrate. The concentrations of two kinds of antibodies and the experiment parameters were optimized. Thereby, the double antibody sandwich BA-ELISA method for the quantitative detection of CFTR protein was established. Furthermore, the reproducibility, specificity and so on were evaluated by clinical specimens of sperm.</p><p><b>RESULTS</b>The optimal concentration of coated anti-CFTR IgG was 4 µg/ml, while the biotin labeled anti-CFTR IgG was 10 µg/ml; the optimal blocking buffer was 1% BSA-PBST, the optimal time of the reaction between antigen and antibody was 60 min, the optimal chromogenic time was 15 min, the intra-assay and inter-assay coefficient were 2.16%-9.23% and 2.29%-11.71% respectively; The lowest detectable limit was 0.15 ng/ml; the standard curve had a good linear correlation of R2 = 0.962.</p><p><b>CONCLUSION</b>The BA-ELISA method for the quantitative detection of CTFR protein is successfully established, and it is demonstrated that the method has strong specificity, high sensitivity and good reproducibility. It provides the basis and evidence of the further application of the method.</p>

Establishment of mammalian cell lines for constitutive expression of influenza virus matrix protein 2 / 病毒学报

To establish a mammalian cell line for stable expression of the matrix protein 2 (M2) of influenza virus type A. M2 gene was amplified by PCR from the influenza virus strain A/PR/8/34. The PCR product was cloned into eukaryotic expression vector pcDNA5/FRT. After identification with restriction enzyme digestion, the plasmid was co-transfected with plasmid pOG44 which expressed Flp in Flp-In-CHO cells. The target gene was integrated into chromosome of CHO cells by homologous recombination in vivo. Recombinant CHO-M2 cell lines were selected for hygromycin B resistance. A total of 15 recombinant cell strains with high expression of M2 protein were screened by hygromycin, and the expression of M2 protein was determined by IFA and Western blot. After subculturing for 10 passages, the presence of M2 gene in the CHO-M2 cells was confirmed by PCR, and the expression of M2 protein were proved by IFA and Western blot. We successfully constructed a mammalian cell line which stably expressed M2 protein of influenza virus type A. The cell line will be useful for studies on function of M2 protein and provide tools for novel influenza virus vaccine development.

Expression of neuraminidase gene of influenza virus H1N1 in baculovirus-expression system / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct the recombinant baculovirus with NA gene of Influenza H1N1 virus.</p><p><b>METHODS</b>Full-length NA gene of Influenza virus H1N1 (A/PR/8/34) was amplified by PCR and inserted into pFastBacdual vector to construct the recombinant baculovirus transfer vector pFBD-NA. Recombinant shuttle vectors rBacmid-NA was then obtained after transforming DH10B competent cells containing bacmid plasmids. After transfecting into sf9 cells, recombinant baculovirus rBac-NA was obtained. The rBac-NA genome was extracted and identified by PCR. NA protein expressed by recombinant baculovirus-infected sf9 cells was determined by IFA, Western Bolt and ELISA.</p><p><b>RESULTS</b>PCR results proved that recombinant shuttle vectors rBacmid-NA was successfully constructed. NA protein was detected by IFA and showed strong specific green fluorescence on the surface of infected cells. NA protein was recognized by two polyclonal antibodies specific for NA in Western Blot. ELISA showed specific reaction of recombinant NA protein with mouse polyclonal antibody against influenza virus (PR8), indicating high antigenicity.</p><p><b>CONCLUSION</b>Recombinant baculovirus rBac-NA that expresse NA protein of influenza virus was successfully constructed. This work provides a basis for further study on NA protein function and novel influenza vaccine development.</p>

Construction of recombinant baculovirus co-expressing M1 and HA of influenza A virus / 病毒学报

The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid. The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion. After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid (bacmid) in DH10Bac competent cells, the colonies were identified by antibiotics and blue-white selection. The rBac-mid-M1-HA was verified by PCR and transfected into S f9 cells to produce recombinant baculovirus (rBac-M1-HA). Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot, demonstrating M1 and HA were co-expressed successfully. This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines.

Expression of mucin regulated by recombinant human interleukin-6 in HT-29 cell of colon cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To study the regulation of the mucin protein in colon cancer cell line HT-29 by recombinant human interleukin-6(rIL-6) and to further elucidate the development of colon cancer.</p><p><b>METHODS</b>The HT-29 cells were treated with different concentrations of rIL-6(1, 2, 5, 10 and 20 μg/L), then flow cytometry and RT-PCR were used to detect the expression of mucin1 and mucin2. Transwell invasion assay was used to observe the effect of invasion capability of rIL-6 to HT-29 cells.</p><p><b>RESULTS</b>In colon cancer, the expression of mucin 1 could be promoted by rIL-6 with concentration above 2 μg/L, the expression rates were(12.5±1.6)%, (26.6±2.7)%, (33.9±2.8)% and (58.9±2.5)%, respectively, higher than (8.0±0.8)% in the negative controls (P<0.01), meanwhile, the expression of mucin 2 decreased by rIL-6 with concentration above 2 μg/L, the expression rates were(30.5±2.6)%, (17.0±2.7)%, (11.0±2.0)% and (5.3±1.8)%, respectively, lower than (41.6±3.6)% in negative control(P<0.01). With the increase in rIL-6 concentration, the invasion of HT-29 cells was enhanced.</p><p><b>CONCLUSIONS</b>In colon cancer, the expression of mucin1 can be promoted by rIL-6, while the expression of mucin2 can be inhibited. IL-6 is a promoting effect factor in colon cancer invasion and metastasis.</p>

Subcloning of M1 gene fragment of H5N1 influenza virus and its expression in Escherichia coli / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein.</p><p><b>METHODS</b>M1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1.</p><p><b>RESULTS</b>The recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals.</p><p><b>CONCLUSION</b>These confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.</p>

Construction of vectors expressing M2 and NA genes of H5N1 influenza virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct vectors expressing M2 and NA genes of H5N1 influenza virus.</p><p><b>METHODS</b>Based on the human H5N1 avian influenza virus (A/Anhui/1/2005) isolated in china, M2 and NA genes were amplified by PCR. M2 or NA gene was subcloned into pStar vector to construct recombinant pStar-M2/, pStar-/M2, pStar-NA/and pStar-NA/. Furthermore, both of the M2 and NA genes were subcloned into pStar to construct two genes co-expressing recombinant pStar-M2/NA and pStar-NA/M2. Expression of the genes were detected by IFA after transfection of 293 cells with the recombinant plasmids.</p><p><b>RESULTS</b>Recombinant plasmids were constructed and identified by restriction endonuclease digestion. Expression of the genes cloned into the recombinant plasmids was confirmed by IFA.</p><p><b>CONCLUSION</b>Recombinant plasmids expressing M2 and/or NA genes of H5N1 influenza virus were constructed, which provided basis for development of influenza DNA vaccine.</p>

Construction, expression and immunogenicity analysis of a fusion protein containing M2e of influenza A virus fused to a modified Pseudomonas aeruginosa exotoxin A / 病毒学报

M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.

Construction of recombinant adenovirus co-expressing M1 and HA genes of influenza virus type A / 病毒学报

Based on the human H5N1 influenza virus strain A/Anhui/1/2005, recombinant adenovirus co-expressing M1 and HA genes of H5N1 influenza virus was constructed using an internal ribosome entry site (IRES) sequence to link the two genes. The M1 and HA genes of H5N1 influenza virus were amplified by PCR and subcloned into pStar vector separately. Then the M1-IRES-HA fragment was amplified and subcloned into pShuttle-CMV vector, the shuttle plasmid was then linearized and transformed into BJ5183 bacteria which contained backbone vector pAd-Easy. The recombinant vector pAd-Easy was packaged in 293 cells to get recombinant adenovirus Ad-M1/HA. CPE was observed after 293 cells were transfected by Ad-M1/HA. The co-expression of M1 and HA genes was confirmed by Western-blot and IFA (immunofluorescence assay). The IRES containing recombinant adenovirus allowed functional co-expression of M1 and HA genes and provided the foundation for developing new influenza vaccines with adenoviral vector.

Study on preparation and identification of monoclonal antibodies immunized with H5N1 influenza virus M1 protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies specific for M1 protein of H5N1 subtype human influenza virus, this work may provide new tool in rapid diagnosis and study of type A influenza virus.</p><p><b>METHODS</b>BALB/c mice were immunized with purified recombinant H5N1 (A/Anhui/1/2005)/M1 protein expressed in E. coli. Spleen cells of the immunized mice were fused with sp2/0 cells to produce hybridoma cell lines. ELISA was performed to identify the monoclonal antibody against M1 protein of H5N1. Immunofluorescence assay (IFA) were applied to identify the specificity of these antibodies.</p><p><b>RESULTS</b>Three hybridoma cell lines steadily secreting anti-H5N1/M1 McAb were obtained, and their cross reactivity was confirmed by cross-reaction test and IFA.</p><p><b>CONCLUSION</b>Monoclonal antibodies immunized with H5N1 subtype influenza virus M1 protein are cross-reactive, which can be used to detect different subtype of influenze virus type A.</p>

Construction and immunogenicity study of DNA vaccine expressing human H5N1 influenza virus hemagglutinin / 病毒学报

Based on the first isolated human H5N1 influenza virus strain A/Anhui/1/2005 in China, HA and HA1 genes were amplified and cloned into the eukaryotic expression vector pStar. The recombinant plasmids pStar-HA and pStar-HA1 were transfected into COS7 cells. Western blot and IFA showed that the two recombinant DNA plasmids were successfully expressed in eukaryotic cells. BALB/c mice were immunized with the plasmids DNA by intramuscular injection. Anti-HA specific antibody in peripheral blood of immunized mice was tested by ELISA. The results showed that the recombinant plasmids successfully induced the anti-HA humoral immune response, and there was no significant difference between HA and HA1 as immunogen. This work provides basis for future development of novel avian flu vaccine.

Expression of sTNFR-IgGFc fusion gene in endothelial cell and its application in gene therapy for rheumatoid arthritis / 生物工程学报

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine, acting as a regulator of inflammation and immunity. TNFalpha plays a critical role in the pathogenesis of rheumatoid arthritis. Blocking of TNFa activity suppressed inflammatory tissue damage. In present study, the chimeric gene of soluble TNF receptor and IgG Fc fragment (sTNFR-IgG FC) was cloned into the mammalian cell expression vector pStar. When the plamid pStar/sTNFR-IgGFc-GFP was transfected into endothelial cells, a considerable expression of the sTNFR-IgG Fc fusion protein was detected. Moreover, the product in 100microL expression supernatant could completely antagonize the cytolytic effect of 1ng TNFa on L929 cells, even at 1/64 dilution. Then the plasmid was delivered into CIA-induced rheumatoid arthritis mice by tail vein injection. The expression of sTNFR-IgG Fc was detected in liver by RT-PCR. Animals in treatment group showed reduced symptoms of arthritis and more active. This treatment induced decrease of synovial incrassation and prevented the cartilage destruction of the mice RA model. These results show that tail vein injection is an effective way for gene therapy and sTNFR-IgGFc expression plasmid is potential for the treatment of rheumatoid arthritis.

Screening for new binding proteins which interact with BM2 of influenza B virus with yeast two-hybrid system / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the role of BM2 protein in the life cycle of influenza B virus.</p><p><b>METHODS</b>The authors screened human kidney MATCHMAKER cDNA library for new binding partners of BM2 of influenza B virus by using the yeast two hybrid system with truncated BM2 (26-109 aa) as the bait.</p><p><b>RESULTS</b>Six positive plasmids encoding N-acetylneuraminate pyruvate lyase, angiopoietin 3, zinc finger protein 251, ribosomal protein S20, protein arginine N-methyltransferase 1 variant 1 (PRMT) and transcription factor-like 1 (TCFL1) were obtained.</p><p><b>CONCLUSION</b>The results suggest that BM2 may play an important role in the life cycle of influenza B virus.</p>

Capecitabine combined with TACE for advanced liver cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the clinical efficacy of capecitabine combined with transcatheter arterial chemoembolization (TACE) for advanced liver cancer.</p><p><b>METHODS</b>Forty-nine patients with liver cancer were retrospectively divided into two groups: Treatment group, on the basis of TACE, 23 patients received oral capecitabine at 2500 mg/m(2), twice-daily for 14 days followed by 7-day rest period and repeated in every three week intervals for more than two cycles. Control group, 26 patients received TACE only at 2-month intervals for at least two cycles.</p><p><b>RESULTS</b>In capecitabine and TACE group: there were 1 CR, 14 PR, 5 SD and 3 PD; the overall response rate was 65.2%; the AFP and tumor reduction rates were 68.8% and 73.9%; the median survival time was 11.9 months. In the TACE only group: there were 0 CR, 7 PR, 12 SD and 7 PD; the overall response rate was 26.9%; the AFP and tumor reduction rates were 31.6 % and 30.8%; the median survival time was 8.3 months. The most common side-effects of capecitabine were hand-foot syndrome and diarrhea.</p><p><b>CONCLUSION</b>Capecitabine combined with TACE is safe and effective for advanced liver cancer.</p>

Construction, expression and bioactivity characterization of targeting toxin DT-VEGF / 生物工程学报

Tumor rapid growth depends on neovascularization. Vascular endothelial growth factor, the main mediator during the occurrence and formation of vascularization, has specific receptors whose expression rate shows difference of orders of magnitude between tumor and the normal tissue, so it can be used to transport toxin molecules to the proliferative tumor endothelial and kill cancer cells. In our experiment, we constructed fusion protein DT-VEGF by linking diphtheria toxin's forward 389 amino acids's gene and VEGF165 via a linker. DT-VEGF is expressed in E. coli and purified. Our experiment proves in can kill vascular endothelial cells specifically, and the inhibition of neovascularization of chicken chorionic membrane is also confirmed.

Screening of new binding partners of CIKS with yeast two-hybrid system / 生物工程学报

The NF-kappaB transcription factor plays important regulatory roles in inflammation, apoptosis, immune and stress responses. IkappaB kinase (IKK) composed of two catalytic subunits and a regulator subunit, acts as a key component of NF-kappaB activation pathway through phosphorylation of IkappaB, the inhibitor of NF-kappaB. CIKS (connection to IKK and SAPK), a newly identified cellular protein, is involved in NF-kappaB and JNK activation. Although it has been known that CIKS interacts with IKK complex, and activates both IKK and SAPK when overexpressed; the underling mechanisms are poorly understood. To better understand the physiological roles of CIKS, we have screened human HeLa MATCHMAKER cDNA library for new binding partners of CIKS by using the yeast two-hybrid system with truncated CIKS (151-574) as the bait. The yeast strain AH109 was sequentially transformed with the bait plasmid and the library. The transformants were screened on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal)selective plates. Positive clones were restreaked on SD(-Leu/-Trp / + X-alpha-gal)plates three times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. After 3 screenings on SD(-Leu/-Trp / + X-alpha-gal), the positive clones were further verified on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal) plates. The inserts in AD/library plasmids were amplified by PCR and PCR products were characterized by Hae III digestion to eliminate the duplicates. After screening in selective plates, the positive AD/library plasmids were rescued via transformation of E. coli HB101 and the interactions of CIKS (151-574) with positive AD/library plasmids were further assessed by yeast two-hybrid analysis. Finally, the DNA sequences of the positive AD/library plasmids were determined and BLAST analysis against the databases was performed. The BLAST results indicate that the inserts in the positive plasmids encode RIKEN cDNA 473340F03, PLAC8, CD27BP (Siva-1), CDC5L, SnRNP smB, and DVL2. CDC5L is a key component of the multi-protein complex essential for the formation of pre-mRNA splicing product and is not required for spliceosome assembly. A role for CDC5L in the cell division cycle has been precious suggested as its overexpression of this protein in mammalian cells leads to a shortening G2 phase of the cell cycle, and a negative-dominant mutant of CDC5L lacking the N-terminal activation domain delays the G2 phase cell's entry into the mitosis. It has been reported that SnRNP smB participates in pre-mRNA splicing and CD27BP (Siva-1) binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis and regulates T cell homeostasis. The functions of RIKEN cDNA 473340F03, PLAC8 and DVL2 are unknown. It has been suggested that CIKS functions as a critical component for cross-talk between NF-kappaB and JNK signaling pathways. IKK subunits, which have been demonstrated to interact with CIKS, were not shown up in this experiment. We speculate that the truncated CIKS (151-574) bait may not contain the binding domain that mediates the interaction of IKK subunits with CIKS. Taken together, the above results suggest that CIKS may play a role in cell regulation through interacting with various cellular proteins. Further investigations are required to characterize these interactions.

Effects of long-term oral administration of lanthanum nitrate on the liver of rats / 中华预防医学杂志

<p><b>OBJECTIVE</b>To probe the effects of long-term oral administration of lanthanum nitrate [La(NO(3))(3)] on morphological change in the liver, aftereffect of deposited La in the liver and their mechanism in rats.</p><p><b>METHODS</b>Young Wistar rats were divided into two groups, one fed with 0.1, 0.2, 2.0, 10.0 and 20.0 mg/kg of La(NO(3))(3) for six months and the other for the control. Changes in ratio of liver to body weight were observed after exposure to La(NO(3))(3) at varied doses for six months and one month after six-month exposure, as well as morphology of the liver in the rats with routine histochemistry and transmission electron microscopy (TEM) technique. Content of La in the liver was measured with inductively coupled plasma-mass spectrometry (ICP-MS).</p><p><b>RESULTS</b>Ratio of liver to body weight was significantly higher in the male rats exposed to 20.0 mg/kg of lanthanum for six months than that in the control group. Ratio of liver to body weight restored to normal in the rats exposed to 20.0 mg/kg of La one month after six-month exposure. Infiltration of inflammatory cells in the portal region of the liver, small amount of fat drops in hepatocytic cytoplasm, increased density of mitochondria stroma, lysosome containing highly-electronic-density bodies and dense granules, normal nucleus and slightly deformed nucleus of hepatocytes could be found in the rats exposed to 20.0 mg/kg. Areas of the liver deposited with glycogen after six-month exposure to 20.0 mg/kg of La accounted for (26.1 +/- 1.5)% and (4.1 +/- 1.4)%, respectively for male and female rats, significantly lower than those in the control group [(31.3 +/- 1.4)% and (39.4 +/- 0.9)%, respectively], with a statistical significance and very statistical significance, respectively. There was a little infiltration of inflammatory cells in the portal region of the liver one month after six-month exposure to 20.0 mg/kg of La, and amount of the dense bodies was lower in the rats exposed to La for six months. Liver contents of La in the rats of all experimental groups were lower one month after six-month exposure than those in the rats exposed for six months.</p><p><b>CONCLUSIONS</b>Exposure to a dose of 20.0 mg/kg La(NO(3))(3) for a long term could damage the liver structure to certain extent, but lanthanum deposited in the liver could be eliminated from the body gradually.</p>

Cloning, expression and bio-activity assay of chimeric fusion protein sTNFRII-IgG Fc / 生物工程学报

Tumor necrosis factor (TNF) is a key cytokine in immunology system and is related to many human diseases. In order to inhibit the activity of TNF, cDNA coding for soluble TNF receptor II (sTNFRII) and human IgG Fc were linked using a flexible hinge. This gene was expressed in E. coli as a chimeric protein and purified by metal chelate chromatography. The results show that the fusion protein exists in the physiological form as a dimer, has the ability to bind with TNF and inhibits the cytotoxicity of TNF on L929 cells. Contrasting to monomer sTNFRII, the chimeric protein has an improved bioactivity, and displays potential prospects for research and application.